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Sample records for sample proteomic profiling

  1. Sample size determination in clinical proteomic profiling experiments using mass spectrometry for class comparison.

    PubMed

    Cairns, David A; Barrett, Jennifer H; Billingham, Lucinda J; Stanley, Anthea J; Xinarianos, George; Field, John K; Johnson, Phillip J; Selby, Peter J; Banks, Rosamonde E

    2009-01-01

    Mass spectrometric profiling approaches such as MALDI-TOF and SELDI-TOF are increasingly being used in disease marker discovery, particularly in the lower molecular weight proteome. However, little consideration has been given to the issue of sample size in experimental design. The aim of this study was to develop a protocol for the use of sample size calculations in proteomic profiling studies using MS. These sample size calculations can be based on a simple linear mixed model which allows the inclusion of estimates of biological and technical variation inherent in the experiment. The use of a pilot experiment to estimate these components of variance is investigated and is shown to work well when compared with larger studies. Examination of data from a number of studies using different sample types and different chromatographic surfaces shows the need for sample- and preparation-specific sample size calculations.

  2. Analytical validation of serum proteomic profiling for diagnosis of prostate cancer: sources of sample bias.

    PubMed

    McLerran, Dale; Grizzle, William E; Feng, Ziding; Bigbee, William L; Banez, Lionel L; Cazares, Lisa H; Chan, Daniel W; Diaz, Jose; Izbicka, Elzbieta; Kagan, Jacob; Malehorn, David E; Malik, Gunjan; Oelschlager, Denise; Partin, Alan; Randolph, Timothy; Rosenzweig, Nicole; Srivastava, Shiv; Srivastava, Sudhir; Thompson, Ian M; Thornquist, Mark; Troyer, Dean; Yasui, Yutaka; Zhang, Zhen; Zhu, Liu; Semmes, O John

    2008-01-01

    This report and a companion report describe a validation of the ability of serum proteomic profiling via SELDI-TOF mass spectrometry to detect prostatic cancer. Details of this 3-stage process have been described. This report describes the development of the algorithm and results of the blinded test for stage 1. We derived the decision algorithm used in this study from the analysis of serum samples from patients with prostate cancer (n = 181) and benign prostatic hyperplasia (BPH) (n = 143) and normal controls (n = 220). We also derived a validation test set from a separate, geographically diverse set of serum samples from 42 prostate cancer patients and 42 controls without prostate cancer. Aliquots were subjected to randomization and blinded analysis, and data from each laboratory site were subjected to the decision algorithm and decoded. Using the data collected from the validation test set, the decision algorithm was unsuccessful in separating cancer from controls with any predictive utility. Analysis of the experimental data revealed potential sources of bias. The ability of the decision algorithm to successfully differentiate between prostate cancer, BPH, and control samples using data derived from serum protein profiling was compromised by bias.

  3. Proteomic Profiling of Paraffin-Embedded Samples Identifies Metaplasia-Specific and Early-Stage Gastric Cancer Biomarkers

    PubMed Central

    Sousa, Josane F.; Ham, Amy-Joan L.; Whitwell, Corbin; Nam, Ki Taek; Lee, Hyuk-Joon; Yang, Han-Kwang; Kim, Woo Ho; Zhang, Bing; Li, Ming; LaFleur, Bonnie; Liebler, Daniel C.; Goldenring, James R.

    2013-01-01

    Early diagnosis and curative resection are the predominant factors associated with increased survival in patients with gastric cancer. However, most gastric cancer cases are still diagnosed at later stages. Since most pathologic specimens are archived as FFPE samples, the ability to use them to generate expression profiles can greatly improve cancer biomarker discovery. We sought to uncover new biomarkers for stomach preneoplastic metaplasias and neoplastic lesions by generating proteome profiles using FFPE samples. We combined peptide isoelectric focusing and liquid chromatography–tandem mass spectrometry analysis to generate proteomic profiles from FFPE samples of intestinal-type gastric cancer, metaplasia, and normal mucosa. The expression patterns of selected proteins were analyzed by immunostaining first in single tissue sections from normal stomach, metaplasia, and gastric cancer and later in larger tissue array cohorts. We detected 60 proteins up-regulated and 87 proteins down-regulated during the progression from normal mucosa to metaplasia to gastric cancer. Two of the up-regulated proteins, LTF and DMBT1, were validated as specific markers for spasmolytic polypeptide–expressing metaplasia and intestinal metaplasia, respectively. In cancers, significantly lower levels of DMBT1 or LTF correlated with more advanced disease and worse prognosis. Thus, proteomic profiling using FFPE samples has led to the identification of two novel markers for stomach metaplasias and gastric cancer prognosis. PMID:22944598

  4. Proteomic Profiling of Serial Prediagnostic Serum Samples for Early Detection of Colon Cancer in the U.S. Military.

    PubMed

    Shao, Stephanie; Neely, Benjamin A; Kao, Tzu-Cheg; Eckhaus, Janet; Bourgeois, Jolie; Brooks, Jasmin; Jones, Elizabeth E; Drake, Richard R; Zhu, Kangmin

    2017-05-01

    Background: Serum proteomic biomarkers offer a promising approach for early detection of cancer. In this study, we aimed to identify proteomic profiles that could distinguish colon cancer cases from controls using serial prediagnostic serum samples.Methods: This was a nested case-control study of active duty military members. Cases consisted of 264 patients diagnosed with colon cancer between 2001 and 2009. Controls were matched to cases on age, gender, race, serum sample count, and collection date. We identified peaks that discriminated cases from controls using random forest data analysis with a 2/3 training and 1/3 validation dataset. We then included epidemiologic data to see whether further improvement of model performance was obtainable. Proteins that corresponded to discriminatory peaks were identified.Results: Peaks with m/z values of 3,119.32, 2,886.67, 2,939.23, and 5,078.81 were found to discriminate cases from controls with a sensitivity of 69% and a specificity of 67% in the year before diagnosis. When smoking status was included, sensitivity increased to 76% while histories of other cancer and tonsillectomy raised specificity to 76%. Peaks at 2,886.67 and 3,119.32 m/z were identified as histone acetyltransferases while 2,939.24 m/z was a transporting ATPase subunit.Conclusions: Proteomic profiles in the year before cancer diagnosis have the potential to discriminate colon cancer patients from controls, and the addition of epidemiologic information may increase the sensitivity and specificity of discrimination.Impact: Our findings indicate the potential value of using serum prediagnostic proteomic biomarkers in combination with epidemiologic data for early detection of colon cancer. Cancer Epidemiol Biomarkers Prev; 26(5); 711-8. ©2016 AACR. ©2016 American Association for Cancer Research.

  5. Autoantibody Profiling of Glioma Serum Samples to Identify Biomarkers Using Human Proteome Arrays.

    PubMed

    Syed, Parvez; Gupta, Shabarni; Choudhary, Saket; Pandala, Narendra Goud; Atak, Apurva; Richharia, Annie; K P, Manubhai; Zhu, Heng; Epari, Sridhar; Noronha, Santosh B; Moiyadi, Aliasgar; Srivastava, Sanjeeva

    2015-09-15

    The heterogeneity and poor prognosis associated with gliomas, makes biomarker identification imperative. Here, we report autoantibody signatures across various grades of glioma serum samples and sub-categories of glioblastoma multiforme using Human Proteome chips containing ~17000 full-length human proteins. The deduced sets of classifier proteins helped to distinguish Grade II, III and IV samples from the healthy subjects with 88, 89 and 94% sensitivity and 87, 100 and 73% specificity, respectively. Proteins namely, SNX1, EYA1, PQBP1 and IGHG1 showed dysregulation across various grades. Sub-classes of GBM, based on its proximity to the sub-ventricular zone, have been reported to have different prognostic outcomes. To this end, we identified dysregulation of NEDD9, a protein involved in cell migration, with probable prognostic potential. Another subcategory of patients where the IDH1 gene is mutated, are known to have better prognosis as compared to patients carrying the wild type gene. On a comparison of these two cohorts, we found STUB1 and YWHAH proteins dysregulated in Grade II glioma patients. In addition to common pathways associated with tumourigenesis, we found enrichment of immunoregulatory and cytoskeletal remodelling pathways, emphasizing the need to explore biochemical alterations arising due to autoimmune responses in glioma.

  6. Autoantibody Profiling of Glioma Serum Samples to Identify Biomarkers Using Human Proteome Arrays

    PubMed Central

    Syed, Parvez; Gupta, Shabarni; Choudhary, Saket; Pandala, Narendra Goud; Atak, Apurva; Richharia, Annie; KP, Manubhai; Zhu, Heng; Epari, Sridhar; Noronha, Santosh B.; Moiyadi, Aliasgar; Srivastava, Sanjeeva

    2015-01-01

    The heterogeneity and poor prognosis associated with gliomas, makes biomarker identification imperative. Here, we report autoantibody signatures across various grades of glioma serum samples and sub-categories of glioblastoma multiforme using Human Proteome chips containing ~17000 full-length human proteins. The deduced sets of classifier proteins helped to distinguish Grade II, III and IV samples from the healthy subjects with 88, 89 and 94% sensitivity and 87, 100 and 73% specificity, respectively. Proteins namely, SNX1, EYA1, PQBP1 and IGHG1 showed dysregulation across various grades. Sub-classes of GBM, based on its proximity to the sub-ventricular zone, have been reported to have different prognostic outcomes. To this end, we identified dysregulation of NEDD9, a protein involved in cell migration, with probable prognostic potential. Another subcategory of patients where the IDH1 gene is mutated, are known to have better prognosis as compared to patients carrying the wild type gene. On a comparison of these two cohorts, we found STUB1 and YWHAH proteins dysregulated in Grade II glioma patients. In addition to common pathways associated with tumourigenesis, we found enrichment of immunoregulatory and cytoskeletal remodelling pathways, emphasizing the need to explore biochemical alterations arising due to autoimmune responses in glioma. PMID:26370624

  7. Urine sample preparation and fractionation for global proteome profiling by LC-MS.

    PubMed

    Court, Magali; Garin, Jérôme; Masselon, Christophe D

    2015-01-01

    Urine has garnered tremendous interest over the past decade as a potential source of protein biomarkers for various pathologies. However, due to its low protein concentration and the presence of interfering compounds, urine constitutes a challenging analyte in proteomics. In the context of a project aimed at the discovery and evaluation of new candidate biomarkers of bladder cancer in urine, our laboratory has implemented and evaluated an array of preparation techniques for urinary proteome analysis. We present here the protocol that, in our hands, yielded the best overall proteome coverage with the lowest analytical effort. It begins with protein precipitation using trichloroacetic acid, in solution digestion and RP-C18 cartridge desalting of the resulting peptides mixture, and is followed by peptide fractionation by gel-free isoelectric focusing, and nano-LC-MS/MS for database compilation.

  8. Standardization of sample homogenization for mosquito identification using an innovative proteomic tool based on protein profiling.

    PubMed

    Nebbak, Amira; Willcox, Alexandra C; Bitam, Idir; Raoult, Didier; Parola, Philippe; Almeras, Lionel

    2016-12-01

    The rapid spread of vector-borne diseases demands the development of an innovative strategy for arthropod monitoring. The emergence of MALDI-TOF MS as a rapid, low-cost, and accurate tool for arthropod identification is revolutionizing medical entomology. However, as MS spectra from an arthropod can vary according to the body part selected, the sample homogenization method used and the mode and duration of sample storage, standardization of protocols is indispensable prior to the creation and sharing of an MS reference spectra database. In the present study, manual grinding of Anopheles gambiae Giles and Aedes albopictus mosquitoes at the adult and larval (L3) developmental stages was compared to automated homogenization. Settings for each homogenizer were optimized, and glass powder was found to be the best sample disruptor based on its ability to create reproducible and intense MS spectra. In addition, the suitability of common arthropod storage conditions for further MALDI-TOF MS analysis was kinetically evaluated. The conditions that best preserved samples for accurate species identification by MALDI-TOF MS were freezing at -20°C or in liquid nitrogen for up to 6 months. The optimized conditions were objectified based on the reproducibility and stability of species-specific MS profiles. The automation and standardization of mosquito sample preparation methods for MALDI-TOF MS analyses will popularize the use of this innovative tool for the rapid identification of arthropods with medical interest. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. MALDI-TOF-MS Platform for Integrated Proteomic and Peptidomic Profiling of Milk Samples Allows Rapid Detection of Food Adulterations.

    PubMed

    Sassi, Mauro; Arena, Simona; Scaloni, Andrea

    2015-07-15

    Adulteration of ovine, caprine, and buffalo milks with more common bovine material occurs for economic reasons and seasonal availability. Frauds are also associated with the use of powdered milk instead of declared, fresh material. In this context, various analytical methods have been adapted to dairy science applications with the aim to evaluate adulteration of milk samples, although time-consuming, suitable only for speciation or thermal treatment analysis, or useful for a specific fraud type. An integrated MALDI-TOF-MS platform for the combined peptidomic and proteomic profiling of milk samples is here presented, which allows rapid detection of illegal adulterations due to the addition of either nondeclared bovine material to water buffalo, goat, and ovine milks or of powdered bovine milk to the fresh counterpart. Peptide and protein markers of each animal milk were identified after direct analysis of a large number of diluted skimmed and/or enriched diluted skimmed filtrate samples. In parallel, markers of thermal treatment were characterized in different types of commercial milks. Principal components scores of ad hoc prepared species- or thermal treatment-associated adulterated milk samples were subjected to partial least-squares regression, permitting a fast accurate estimate of the fraud extents in test samples at either protein and peptide level. With respect to previous reports on MALDI-TOF-MS protein profiling methodologies for milk speciation, this study extends that approach to the analysis of the thermal treatment and introduces an independent, complementary peptide profiling measurement, which integrates protein data with additional information on peptides, validating final results and ultimately broadening the method applicability.

  10. Translational Targeted Proteomics Profiling of Mitochondrial Energy Metabolic Pathways in Mouse and Human Samples.

    PubMed

    Wolters, Justina C; Ciapaite, Jolita; van Eunen, Karen; Niezen-Koning, Klary E; Matton, Alix; Porte, Robert J; Horvatovich, Peter; Bakker, Barbara M; Bischoff, Rainer; Permentier, Hjalmar P

    2016-09-02

    Absolute measurements of protein abundance are important in the understanding of biological processes and the precise computational modeling of biological pathways. We developed targeted LC-MS/MS assays in the selected reaction monitoring (SRM) mode to quantify over 50 mitochondrial proteins in a single run. The targeted proteins cover the tricarboxylic acid cycle, fatty acid β-oxidation, oxidative phosphorylation, and the detoxification of reactive oxygen species. Assays used isotopically labeled concatemers as internal standards designed to target murine mitochondrial proteins and their human orthologues. Most assays were also suitable to quantify the corresponding protein orthologues in rats. After exclusion of peptides that did not pass the selection criteria, we arrived at SRM assays for 55 mouse, 52 human, and 51 rat proteins. These assays were optimized in isolated mitochondrial fractions from mouse and rat liver and cultured human fibroblasts and in total liver extracts from mouse, rat, and human. The developed proteomics approach is suitable for the quantification of proteins in the mitochondrial energy metabolic pathways in mice, rats, and humans as a basis for translational research. Initial data show that the assays have great potential for elucidating the adaptive response of human patients to mutations in mitochondrial proteins in a clinical setting.

  11. Mass spectrometry-based quantitative proteomic profiling.

    PubMed

    Yan, Wei; Chen, Sharon S

    2005-05-01

    Quantitative proteomics involves the identification and quantitation of protein components in various biological systems. Stable isotope labelling technology, by both metabolic and chemical methods, has been the most commonly used approach for global proteome-wide profiling. Recently, its capability has been extended from labelled pairs to multiple labels, allowing for the simultaneous quantification of multiplex samples. The ion intensity-based quantitative approach has progressively gained more popularity as mass spectrometry performance has improved significantly. Although some success has been reported, it remains difficult comprehensively to characterise the global proteome, due to its enormous complexity and dynamic range. The use of sub-proteome fractionation techniques permits a simplification of the proteome and provides a practical step towards the ultimate dissection of the entire proteome. Further development of the technology for targeting sub-proteomes on a functional basis - such as selecting proteins with differential expression profiles from mass spectrometric analyses, for further mass spectrometric sequencing in an intelligent manner--is expected in the near future.

  12. Dual-color Proteomic Profiling of Complex Samples with a Microarray of 810 Cancer-related Antibodies*

    PubMed Central

    Schröder, Christoph; Jacob, Anette; Tonack, Sarah; Radon, Tomasz P.; Sill, Martin; Zucknick, Manuela; Rüffer, Sven; Costello, Eithne; Neoptolemos, John P.; Crnogorac-Jurcevic, Tatjana; Bauer, Andrea; Fellenberg, Kurt; Hoheisel, Jörg D.

    2010-01-01

    Antibody microarrays have the potential to enable comprehensive proteomic analysis of small amounts of sample material. Here, protocols are presented for the production, quality assessment, and reproducible application of antibody microarrays in a two-color mode with an array of 1,800 features, representing 810 antibodies that were directed at 741 cancer-related proteins. In addition to measures of array quality, we implemented indicators for the accuracy and significance of dual-color detection. Dual-color measurements outperform a single-color approach concerning assay reproducibility and discriminative power. In the analysis of serum samples, depletion of high-abundance proteins did not improve technical assay quality. On the contrary, depletion introduced a strong bias in protein representation. In an initial study, we demonstrated the applicability of the protocols to proteins derived from urine samples. We identified differences between urine samples from pancreatic cancer patients and healthy subjects and between sexes. This study demonstrates that biomedically relevant data can be produced. As demonstrated by the thorough quality analysis, the dual-color antibody array approach proved to be competitive with other proteomic techniques and comparable in performance to transcriptional microarray analyses. PMID:20164060

  13. Proteomic profiling of cancer biomarkers.

    PubMed

    Shau, Hungyi; Chandler, G Scott; Whitelegge, Julian P; Gornbein, Jeffrey A; Faull, Kym F; Chang, Helena R

    2003-07-01

    Early detection and correct diagnosis are essential for effective treatment of cancer and patient survival. Complete sequencing of the human genome, and the genomes of other species, provides valuable tools for discerning the genetic abnormalities in cancer. However, differences between cancerous and normal cells reflect more than variations in genetic sequences and abundance of transcribed RNA. Many cancer biomarkers are manifestation of differences in post-transcriptional splicing and/or post-translational modifications. Thus, proteomic tools are being increasingly utilised in the post-genomic era for discovery of new cancer biomarkers. In this paper we will provide an overview of the biomarker discovery process from the proteomic profiling point of view, with emphasis given to the principles that are involved in the process, including the protein identification strategies, and how surface enhanced laser desorption ionisation mass spectrometry fits into the picture. The aim is to provide a resource for the experimental practitioner seeking awareness of the analytical tools that are now available in contemporary cancer research.

  14. Proteomic profiling of Pachyonychia congenita plantar callus.

    PubMed

    Rice, Robert H; Durbin-Johnson, Blythe P; Salemi, Michelle; Schwartz, Mary E; Rocke, David M; Phinney, Brett S

    2017-08-08

    Callus samples from the ball and the arch of the foot, collected on tape circles, were compared by shotgun proteomic profiling. Pachyonychia congenita subjects were sampled who exhibited a mutation in KRT6A, KRT6B, KRT6C, KRT16 or KRT17, and the proteins were digested and analyzed by tandem mass spectrometry. In comparison with samples from unaffected control subjects, those from subjects with KRT6A or KRT16 mutations displayed the most differences in profile from normal, while those from subjects with KRT6C or KRT17 mutations showed few differences from normal. The profiles from subjects with KRT6B mutations were intermediate in protein profile differences. Degree of departure from the normal profile could be estimated by expression of numerous proteins in callus from the ball of the foot that were consistently different. By contrast, the protein profile from the arch of the foot was hardly affected. The results provide a foundation for noninvasive monitoring of the efficacy of treatments with quantitative assessment of departure from the normal phenotype. Pachyonychia congenita is an orphan disease in which the connection between the basic defect (keratin mutation) and debilitating symptoms (severe plantar pain) is poorly understood. Present work addresses the degree to which the protein profile is altered in the epidermis where the severe pain originates. The results indicate that the mutated keratins differ greatly in the degree to which they elicit perturbations in protein profile. In those cases with markedly altered protein levels, monitoring the callus profile may provide an objective measure of treatment efficacy. Copyright © 2017 Elsevier B.V. All rights reserved.

  15. Proteomic profile of edible bird's nest proteins.

    PubMed

    Liu, Xiaoqing; Lai, Xintian; Zhang, Shiwei; Huang, Xiuli; Lan, Quanxue; Li, Yun; Li, Bifang; Chen, Wei; Zhang, Qinlei; Hong, Dezhi; Yang, Guowu

    2012-12-26

    Edible bird's nest (EBN) is made of the swiftlets' saliva, which has attracted rather more attention owing to its nutritious and medical properties. Although protein constitutes the main composition and plays an important role in EBN, few studies have focused on the proteomic profile of EBN. The purpose of this study was to produce a proteomic map and clarify common EBN proteins. Liquid-phase isoelectric focusing (LIEF) was combined with two-dimensional electrophoresis (2-DE) for comprehensive analysis of EBN proteins. From 20 to 100 protein spots were detected on 2-DE maps of EBN samples from 15 different sources. The proteins were mainly distributed in four taxa (A, B, C, and D) according to their molecular mass. Taxa A and D both contained common proteins and proteins that may be considered another characteristic of EBN. Taxon A was identified using MALDI-TOF-TOF/MS and found to be homologous to acidic mammalian chitinase-like ( Meleagris gallopavo ), which is in glycosyl hydrolase family 18.

  16. Temporal profiles of plasma proteome during childhood development.

    PubMed

    Liu, Chih-Wei; Bramer, Lisa; Webb-Robertson, Bobbie-Jo; Waugh, Kathleen; Rewers, Marian J; Zhang, Qibin

    2017-01-30

    Human blood plasma proteome reflects physiological changes associated with a child's development as well as development of disease states. While age-specific normative values are available for proteins routinely measured in clinical practice, there is paucity of comprehensive longitudinal data regarding changes in human plasma proteome during childhood. We applied TMT-10plex isobaric labeling-based quantitative proteomics to longitudinally profile the plasma proteome in 10 healthy children during their development, each with 9 serial time points from 9months to 15years of age. In total, 1828 protein groups were identified at peptide and protein level false discovery rate of 1% and with at least two razor and unique peptides. The longitudinal expression profiles of 1747 protein groups were statistically modeled and their temporal changes were categorized into 7 different patterns. The patterns and relative abundance of proteins obtained by LC-MS were also verified with ELISA. To our knowledge, this study represents the most comprehensive longitudinal profiling of human plasma proteome to date. The temporal profiles of plasma proteome obtained in this study provide a comprehensive resource and reference for biomarker studies in childhood diseases. Biological significance: A pediatric plasma proteome database with longitudinal expression patterns of 1747 proteins from neonate to adolescence was provided to the research community. 970 plasma proteins had age-dependent expression trends, which demonstrated the importance of longitudinal profiling study to identify the potential biomarkers specific to childhood diseases, and the requirement of strictly age-matched clinical samples in a cross-sectional study in pediatric population.

  17. Plasma proteomic profiling of pediatric osteosarcoma.

    PubMed

    Li, Yiting; Dang, Tu Anh; Man, Tsz-Kwong

    2012-01-01

    The development of a sensitive, specific, and non-invasive approach for cancer detection will facilitate early detection and, hence, improve the outcome of individuals with known cancer predispositions. Proteomic profiling of blood emerges to be a logical choice of such non-invasive or minimal invasive detection. However, plasma biomarker discovery of pediatric cancers lags behind that of adult cancers, suggesting more efforts are needed in this area. In this study, we used surface-enhanced laser desorption/ionization-time of flight mass spectrometry to profile plasma proteome in osteosarcoma patients. Osteosarcoma is a bone cancer that affects many children and young adults. We have shown that the plasma proteome contains a unique cancer signature that can distinguish patients with osteosarcoma from those with a benign bone disease. To improve cancer biomarker discovery in plasma, we have also shown that depletion of two highly abundant plasma proteins increases the detection sensitivity of lower-abundance proteins. The combination of depletion and proteomic profiling may increase the chance of identifying tumor-derived proteins within the plasma of pediatric cancer patients.

  18. Proteomic profiling of skeletal muscle plasticity.

    PubMed

    Ohlendieck, Kay

    2011-10-01

    One of the most striking physiological features of skeletal muscle tissues are their enormous capacity to adapt to changed functional demands. Muscle plasticity has been extensively studied by histological, biochemical, physiological and genetic methods over the last few decades. With the recent emergence of high-throughput and large-scale proteomic techniques, mass spectrometry-based surveys have also been applied to the global analysis of the skeletal muscle protein complement during physiological modifications and pathophysiological alterations. This review outlines and discusses the impact of recent proteomic profiling studies of skeletal muscle transitions, including the effects of chronic electro-stimulation, physical exercise, denervation, disuse atrophy, hypoxia, myotonia, motor neuron disease and age-related fibre type shifting. This includes studies on the human skeletal muscle proteome, animal models of muscle plasticity and major neuromuscular pathologies. The biomedical importance of establishing reliable biomarker signatures for the various molecular and cellular transition phases involved in muscle transformation is critically examined.

  19. Proteomic analysis of tissue samples in translational breast cancer research.

    PubMed

    Gromov, Pavel; Moreira, José M A; Gromova, Irina

    2014-06-01

    In the last decade, many proteomic technologies have been applied, with varying success, to the study of tissue samples of breast carcinoma for protein expression profiling in order to discover protein biomarkers/signatures suitable for: characterization and subtyping of tumors; early diagnosis, and both prognosis and prediction of outcome of chemotherapy. The purpose of this review is to critically appraise what has been achieved to date using proteomic technologies and to bring forward novel strategies - based on the analysis of clinically relevant samples - that promise to accelerate the translation of basic discoveries into the daily breast cancer clinical practice. In particular, we address major issues in experimental design by reviewing the strengths and weaknesses of current proteomic strategies in the context of the analysis of human breast tissue specimens.

  20. In-Depth Profiling of the Peripheral Blood Mononuclear Cells Proteome for Clinical Blood Proteomics

    PubMed Central

    Končarević, Saša; Lößner, Christopher; Pike, Ian; Zucht, Hans-Dieter

    2014-01-01

    Peripheral blood mononuclear cells (PBMCs) are an easy accessible cellular part of the blood organ and, along with platelets, represent the only site of active gene expression in blood. These cells undergo immunophenotypic changes in various diseases and represent a peripheral source of monitoring gene expression and posttranslational modifications relevant to many diseases. Little is known about the source of many blood proteins and we hypothesise that release from PBMCs through active and passive mechanisms may account for a substantial part of the plasma proteome. The use of state-of-the-art proteomic profiling methods in PBMCs will enable minimally invasive monitoring of disease progression or response to treatment and discovery of biomarkers. To achieve this goal, detailed mapping of the PBMC proteome using a sensitive, robust, and quantitative methodological setup is required. We have applied an indepth gel-free proteomics approach using tandem mass tags (TMT), unfractionated and SCX fractionated PBMC samples, and LC-MS/MS with various modulations. This study represents a benchmark in deciphering the PBMC proteome as we provide a deep insight by identifying 4129 proteins and 25503 peptides. The identified proteome defines the scope that enables PBMCs to be characterised as cellular major biomarker pool within the blood organ. PMID:24724028

  1. In-depth profiling of the peripheral blood mononuclear cells proteome for clinical blood proteomics.

    PubMed

    Končarević, Saša; Lößner, Christopher; Kuhn, Karsten; Prinz, Thorsten; Pike, Ian; Zucht, Hans-Dieter

    2014-01-01

    Peripheral blood mononuclear cells (PBMCs) are an easy accessible cellular part of the blood organ and, along with platelets, represent the only site of active gene expression in blood. These cells undergo immunophenotypic changes in various diseases and represent a peripheral source of monitoring gene expression and posttranslational modifications relevant to many diseases. Little is known about the source of many blood proteins and we hypothesise that release from PBMCs through active and passive mechanisms may account for a substantial part of the plasma proteome. The use of state-of-the-art proteomic profiling methods in PBMCs will enable minimally invasive monitoring of disease progression or response to treatment and discovery of biomarkers. To achieve this goal, detailed mapping of the PBMC proteome using a sensitive, robust, and quantitative methodological setup is required. We have applied an indepth gel-free proteomics approach using tandem mass tags (TMT), unfractionated and SCX fractionated PBMC samples, and LC-MS/MS with various modulations. This study represents a benchmark in deciphering the PBMC proteome as we provide a deep insight by identifying 4129 proteins and 25503 peptides. The identified proteome defines the scope that enables PBMCs to be characterised as cellular major biomarker pool within the blood organ.

  2. Shotgun proteome profile of Populus developing xylem

    SciTech Connect

    Kalluri, Udaya C; Hurst, Gregory {Greg} B; Lankford, Patricia K; Ranjan, Priya; Pelletier, Dale A

    2009-01-01

    Understanding the molecular pathways of plant cell wall biosynthesis and remodeling is central to interpreting biological mechanisms underlying plant growth and adaptation as well as leveraging that knowledge towards development of improved bioenergy feedstocks. Here we report the application of shotgun tandem mass spectrometry profiling to the proteome of Populus developing xylem. Additionally, we mined public databases to obtain information in support of subcellular localization, transcript-level expression, and functional categorization of these proteins. Nearly 6000 different proteins were identified from the xylem proteome, with over 4400 proteins identified from one or more unique peptides. In addition to finding protein-level evidence of candidate wall biosynthesis genes from xylem (wood) tissue such as cellulose synthase, phenylalanine ammonia-lyase, and 4-coumarate:CoA ligase, several other potentially new candidate genes in the pathway were discovered. In order to identify low-abundance DNA-regulatory proteins from the developing xylem, a selective nuclear proteome profiling method was developed. Several putative transcription factor and chromatin remodeling proteins were identified using this method, such as LIM and NAC domain transcription factors and CHB3-SWI/SNF-related proteins. Further application of these proteomics methods will enhance understanding not only of cell wall biosynthesis in system biology modeling, but also other plant developmental and physiological pathways.

  3. Shotgun proteome profile of Populus developing xylem.

    PubMed

    Kalluri, Udaya C; Hurst, Gregory B; Lankford, Patricia K; Ranjan, Priya; Pelletier, Dale A

    2009-11-01

    Understanding the molecular pathways of plant cell wall biosynthesis and remodeling is central to interpreting biological mechanisms underlying plant growth and adaptation as well as leveraging that knowledge towards development of improved bioenergy feedstocks. Here, we report the application of shotgun MS/MS profiling to the proteome of Populus developing xylem. Nearly 6000 different proteins were identified from the xylem proteome. To identify low-abundance DNA-regulatory proteins from the developing xylem, a selective nuclear proteome profiling method was developed. Several putative transcription factors and chromatin remodeling proteins were identified using this method, such as NAC domain, CtCP-like and CHB3-SWI/SNF-related proteins. Public databases were mined to obtain information in support of subcellular localization, transcript-level expression and functional categorization of identified proteins. In addition to finding protein-level evidence of candidate cell wall biosynthesis genes from xylem (wood) tissue such as cellulose synthase, sucrose synthase and polygalacturonase, several other potentially new candidate genes in the cell wall biosynthesis pathway were discovered. Further application of such proteomics methods will aid in plant systems biology modeling efforts by enhancing the understanding not only of cell wall biosynthesis but also of other plant developmental and physiological pathways.

  4. Proteomic expression profiling of thyroid neoplasms.

    PubMed

    Braunschweig, Till; Kaserer, Klaus; Chung, Joon-Yong; Bilke, Sven; Krizman, David; Knezevic, Vladimir; Hewitt, Stephen M

    2007-03-01

    Thyroid cancer is the most common endocrine neoplasm with multiple histologic subtypes, each associated with different treatments and outcomes. Differentiating benign neoplasms such as follicular adenomas from malignant entities such as follicular carcinomas and papillary carcinoma can be challenging. To define the proteomic profile of different thyroid tumors, we screened an antibody array of 330 features against five thyroid neoplasms: follicular adenoma, follicular carcinoma, papillary carcinoma, anaplastic carcinoma, and medullary carcinoma as well as normal thyroid epithelium. Eight candidate biomarkers; c-erbB-2, Stat5a, Annexin IV, IL-11, RARα, FGF7, Caspase 9, and phospho-c-myc were identified as differentially expressed on the antibody array, and validated with immunohistochemistry on tissue microarrays, with a total of 144 samples of the same variety of thyroid neoplasms. Analysis revealed c-erbB-2, Annexin IV, and Stat5a have potential clinical utility to differentiate follicular adenoma, follicular carcinoma, and papillary carcinoma from each other. By using an antibody array as a discovery platform and a tissue microarray as a first step in validation on a large number of specimens, we have identified new markers that have potential utility in the diagnosis of thyroid neoplasms. Copyright © 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. Proteome Analysis of Human Perilymph using an Intraoperative Sampling Method.

    PubMed

    Schmitt, Heike Andrea; Pich, Andreas; Schröder, Anke; Scheper, Verena; Lilli, Giorgio; Reuter, Günter; Lenarz, Thomas

    2017-03-10

    The knowledge about the etiology and pathophysiology of sensorineural hearing loss (SNHL) is still very limited. The project aims at the improvement of understanding different types of SNHL by proteome analysis of human perilymph. Sampling of perilymph has been established during inner ear surgeries (cochlear implant and vestibular schwannoma surgeries) and safety of the sampling method was determined by pure tone audiometry. An in-depth shot-gun proteomics approach was performed to identify cochlear proteins and individual proteome in perilymph of patients. This method enables the identification and quantification of protein composition of perilymph. The proteome of 41 collected perilymph samples with volumes of 1-12 µl was analyzed by data dependent acquisition resulting in overall 878 detected protein groups. At least 203 protein groups were solely identified in perilymph, not in reference samples (serum, cerebrospinal fluid), displaying a specific protein pattern for perilymph. Samples were grouped according to age of patients and type of surgery leading to identification of some proteins specific to particular subgroups. Proteins with different abundances between different sample groups were subjected to classification by gene ontology annotations. The identified proteins might be used to develop tools for non-invasive inner ear diagnostics and to elucidate molecular profiles of SNHL.

  6. Proteomic profile of dormant Trichophyton Rubrum conidia

    PubMed Central

    Leng, Wenchuan; Liu, Tao; Li, Rui; Yang, Jian; Wei, Candong; Zhang, Wenliang; Jin, Qi

    2008-01-01

    Background Trichophyton rubrum is the most common dermatophyte causing fungal skin infections in humans. Asexual sporulation is an important means of propagation for T. rubrum, and conidia produced by this way are thought to be the primary cause of human infections. Despite their importance in pathogenesis, the conidia of T. rubrum remain understudied. We intend to intensively investigate the proteome of dormant T. rubrum conidia to characterize its molecular and cellular features and to enhance the development of novel therapeutic strategies. Results The proteome of T. rubrum conidia was analyzed by combining shotgun proteomics with sample prefractionation and multiple enzyme digestion. In total, 1026 proteins were identified. All identified proteins were compared to those in the NCBI non-redundant protein database, the eukaryotic orthologous groups database, and the gene ontology database to obtain functional annotation information. Functional classification revealed that the identified proteins covered nearly all major biological processes. Some proteins were spore specific and related to the survival and dispersal of T. rubrum conidia, and many proteins were important to conidial germination and response to environmental conditions. Conclusion Our results suggest that the proteome of T. rubrum conidia is considerably complex, and that the maintenance of conidial dormancy is an intricate and elaborate process. This data set provides the first global framework for the dormant T. rubrum conidia proteome and is a stepping stone on the way to further study of the molecular mechanisms of T. rubrum conidial germination and the maintenance of conidial dormancy. PMID:18578874

  7. Proteomic profiling of hempseed proteins from Cheungsam.

    PubMed

    Park, Seul-Ki; Seo, Jong-Bok; Lee, Mi-Young

    2012-02-01

    Proteomic profiling of hempseed proteins from a non-drug type of industrial hemp (Cannabis sativa L.), Cheungsam, was conducted using two-dimensional gel electrophoresis and mass spectrometry. A total of 1102 protein spots were resolved on pH 3-10 immobilized pH gradient strips, and 168 unique protein spots were identified. The proteins were categorized based on function, including involvement in energy regulation (23%), metabolism (18%), stress response (16%), unclassified (12%), cytoskeleton (11%), binding function (5%), and protein synthesis (3%). These proteins might have important biological functions in hempseed, such as germination, storage, or development. Copyright © 2011 Elsevier B.V. All rights reserved.

  8. Diagnosis of pancreatic cancer using serum proteomic profiling.

    PubMed

    Bhattacharyya, Sudeepa; Siegel, Eric R; Petersen, Gloria M; Chari, Suresh T; Suva, Larry J; Haun, Randy S

    2004-01-01

    In the United States, mortality rates from pancreatic cancer (PCa) have not changed significantly over the past 50 years. This is due, in part, to the lack of early detection methods for this particularly aggressive form of cancer. The objective of this study was to use high-throughput protein profiling technology to identify biomarkers in the serum proteome for the early detection of resectable PCa. Using surface-enhanced laser desorption/ionization mass spectrometry, protein profiles were generated from sera of 49 PCa patients and 54 unaffected individuals after fractionation on an anion exchange resin. The samples were randomly divided into a training set (69 samples) and test set (34 samples), and two multivariate analysis procedures, classification and regression tree and logistic regression, were used to develop classification models from these spectral data that could distinguish PCa from control serum samples. In the test set, both models correctly classified all of the PCa patient serum samples (100% sensitivity). Using the decision tree algorithm, a specificity of 93.5% was obtained, whereas the logistic regression model produced a specificity of 100%. These results suggest that high-throughput proteomics profiling has the capacity to provide new biomarkers for the early detection and diagnosis of PCa.

  9. Proteomic profiling of lymphocytes in autoimmunity, inflammation and cancer.

    PubMed

    Zhou, Jiebai; Zhu, Zhitu; Bai, Chunxue; Sun, Hongzhi; Wang, Xiangdong

    2014-01-07

    Lymphocytes play important roles in the balance between body defense and noxious agents involved in a number of diseases, e.g. autoimmune diseases, allergic inflammation and cancer. The proteomic analyses have been applied to identify and validate disease-associated and disease-specific biomarkers for therapeutic strategies of diseases. The proteomic profiles of lymphocytes may provide more information to understand their functions and roles in the development of diseases, although proteomic approaches in lymphocytes are still limited. The present review overviewed the proteomics-based studies on lymphocytes to headlight the proteomic profiles of lymphocytes in diseases, such as autoimmune diseases, allergic inflammation and cancer, with a special focus on lung diseases. We will explore the potential significance of diagnostic biomarkers and therapeutic targets from the current status in proteomic studies of lymphocytes and discuss the value of the currently available proteomic methodologies in the lymphocytes research.

  10. Proteomic profiling of lymphocytes in autoimmunity, inflammation and cancer

    PubMed Central

    2014-01-01

    Lymphocytes play important roles in the balance between body defense and noxious agents involved in a number of diseases, e.g. autoimmune diseases, allergic inflammation and cancer. The proteomic analyses have been applied to identify and validate disease-associated and disease-specific biomarkers for therapeutic strategies of diseases. The proteomic profiles of lymphocytes may provide more information to understand their functions and roles in the development of diseases, although proteomic approaches in lymphocytes are still limited. The present review overviewed the proteomics-based studies on lymphocytes to headlight the proteomic profiles of lymphocytes in diseases, such as autoimmune diseases, allergic inflammation and cancer, with a special focus on lung diseases. We will explore the potential significance of diagnostic biomarkers and therapeutic targets from the current status in proteomic studies of lymphocytes and discuss the value of the currently available proteomic methodologies in the lymphocytes research. PMID:24397796

  11. The proteomic profile of a mouse model of proliferative vitreoretinopathy.

    PubMed

    Márkus, Bernadett; Pató, Zsuzsanna; Sarang, Zsolt; Albert, Réka; Tőzsér, József; Petrovski, Goran; Csősz, Éva

    2017-08-01

    Proliferative vitreoretinopathy (PVR) develops as a complication of retinal detachment surgery and represents a devastating condition leading to serious vision loss. A good animal model that permits extensive functional studies and drug testing is crucial in finding better therapeutic modalities for PVR. A previously established mouse model, using dispase injection, was analyzed from the proteomic point of view, examining global protein profile changes by 2D electrophoresis, image analysis and HPLC-tandem mass spectrometry-based protein identification. The easy applicability of the mouse model was used to study the role of transglutaminase 2 (TG2) in PVR formation by proteomic examination of dispase-induced TG2 knockout vitreous samples. Our data demonstrate that, despite the altered appearance of crystallin proteins, the lack of TG2 did not prevent the development of PVR.

  12. Proteomic profiling of the rat hypothalamus

    PubMed Central

    2012-01-01

    Background The hypothalamus plays a pivotal role in numerous mechanisms highly relevant to the maintenance of body homeostasis, such as the control of food intake and energy expenditure. Impairment of these mechanisms has been associated with the metabolic disturbances involved in the pathogenesis of obesity. Since rodent species constitute important models for metabolism studies and the rat hypothalamus is poorly characterized by proteomic strategies, we performed experiments aimed at constructing a two-dimensional gel electrophoresis (2-DE) profile of rat hypothalamus proteins. Results As a first step, we established the best conditions for tissue collection and protein extraction, quantification and separation. The extraction buffer composition selected for proteome characterization of rat hypothalamus was urea 7 M, thiourea 2 M, CHAPS 4%, Triton X-100 0.5%, followed by a precipitation step with chloroform/methanol. Two-dimensional (2-D) gels of hypothalamic extracts from four-month-old rats were analyzed; the protein spots were digested and identified by using tandem mass spectrometry and database query using the protein search engine MASCOT. Eighty-six hypothalamic proteins were identified, the majority of which were classified as participating in metabolic processes, consistent with the finding of a large number of proteins with catalytic activity. Genes encoding proteins identified in this study have been related to obesity development. Conclusion The present results indicate that the 2-DE technique will be useful for nutritional studies focusing on hypothalamic proteins. The data presented herein will serve as a reference database for studies testing the effects of dietary manipulations on hypothalamic proteome. We trust that these experiments will lead to important knowledge on protein targets of nutritional variables potentially able to affect the complex central nervous system control of energy homeostasis. PMID:22519962

  13. Urine sample preparation for proteomic analysis.

    PubMed

    Olszowy, Pawel; Buszewski, Boguslaw

    2014-10-01

    Sample preparation for both environmental and more importantly biological matrices is a bottleneck of all kinds of analytical processes. In the case of proteomic analysis this element is even more important due to the amount of cross-reactions that should be taken into consideration. The incorporation of new post-translational modifications, protein hydrolysis, or even its degradation is possible as side effects of proteins sample processing. If protocols are evaluated appropriately, then identification of such proteins does not bring difficulties. However, if structural changes are provided without sufficient attention then protein sequence coverage will be reduced or even identification of such proteins could be impossible. This review summarizes obstacles and achievements in protein sample preparation of urine for proteome analysis using different tools for mass spectrometry analysis. The main aim is to present comprehensively the idea of urine application as a valuable matrix. This article is dedicated to sample preparation and application of urine mainly in novel cancer biomarkers discovery. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  14. Preparation of urine samples for proteomic analysis.

    PubMed

    Pieper, Rembert

    2008-01-01

    Reproducible procedures for the preparation of protein samples isolated from human urine are essential for meaningful proteomic analyses. Key applications are the discovery of novel proteins or their modifications in the human urine as well as protein biomarker discovery for diseases and drug treatments. The methodology presented here features experimental steps aimed at limiting protein losses because of organic solvent precipitation, effective separation of proteins from other compounds in the human urine and molecular weight-based enrichment of proteins in two distinct fractions. Urinary proteins are separated from cellular debris in the urine via centrifugation, concentrated with 5-kDa-cutoff membrane concentration devices and separated via size exclusion chromatography into fractions with a higher and a lower molecular weight than 30 kDa, respectively. A successive optional affinity removal step for highly abundant plasma proteins is described. Finally, buffer exchange steps useful for specific downstream proteomic analysis experiments of urinary proteins are presented, such as 2-dimensional gel electrophoresis, differential protein or peptide labeling and digestion with trypsin for LC-MS/MS analysis.

  15. Proteome Profiling of Human Cutaneous Leishmaniasis Lesion

    PubMed Central

    da Silva Santos, Claire; Attarha, Sanaz; Saini, Ravi Kanth; Boaventura, Viviane; Costa, Jackson; Khouri, Ricardo; Barral-Netto, Manoel; Brodskyn, Cláudia Ida; Souchelnytskyi, Serhiy

    2015-01-01

    In this study, we used proteomics and biological network analysis to evaluate the potential biological processes and components present in the identified proteins of biopsies from cutaneous leishmaniasis (CL) patients infected by Leishmania braziliensis in comparison with normal skin. We identified 59 proteins differently expressed in samples from infected and normal skin. Biological network analysis employing identified proteins showed the presence of networks that may be involved in the cell death mediated by cytotoxic T lymphocytes. After immunohistochemical analyses, the expression of caspase-9, caspase-3, and granzyme B was validated in the tissue and positively correlated with the lesion size in CL patients. In conclusion, this work identified differentially expressed proteins in the inflammatory site of CL, revealed enhanced expression of caspase-9, and highlighted mechanisms associated with the progression of tissue damage observed in lesions. PMID:25207817

  16. Proteomic profiling predicts drug response to novel targeted anticancer therapeutics.

    PubMed

    Lin, Fan; Li, Zilin; Hua, Yunfen; Lim, Yoon Pin

    2016-01-01

    Most recently approved anti-cancer drugs by the US FDA are targeted therapeutic agents and this represents an important trend for future anticancer therapy. Unlike conventional chemotherapy that rarely considers individual differences, it is crucial for targeted therapies to identify the beneficial subgroup of patients for the treatment. Currently, genomics and transcriptomics are the major 'omic' analytics used in studies of drug response prediction. However, proteomic profiling excels both in its advantages of directly detecting an instantaneous dynamic of the whole proteome, which contains most current diagnostic markers and therapeutic targets. Moreover, proteomic profiling improves understanding of the mechanism for drug resistance and helps finding optimal combination therapy. This article reviews the recent success of applications of proteomic analytics in predicting the response to targeted anticancer therapeutics, and discusses the potential avenues and pitfalls of proteomic platforms and techniques used most in the field.

  17. Proteomic profiling of the epileptic dentate gyrus

    PubMed Central

    Li, Aiqing; Choi, Yun-Sik; Dziema, Heather; Cao, Ruifeng; Cho, Hee-Yeon; Jung, Yeon Joo; Obrietan, Karl

    2010-01-01

    The development of epilepsy is often associated with marked changes in central nervous system cell structure and function. Along these lines, reactive gliosis and granule cell axonal sprouting within the dentate gyrus of the hippocampus are commonly observed in individuals with temporal lobe epilepsy. Here we used the pilocarpine model of temporal lobe epilepsy in mice to screen the proteome and phosphoproteome of the dentate gyrus to identify molecular events that are altered as part of the pathogenic process. Using a two-dimensional gel electrophoresis-based approach, followed by liquid chromatography-tandem mass spectrometry, 24 differentially expressed proteins, including 9 phosphoproteins, were identified. Functionally, these proteins were organized into several classes, including synaptic physiology, cell structure, cell stress, metabolism and energetics. The altered expression of three proteins involved in synaptic physiology, actin, profilin 1 and α-synuclein, was validated by secondary methods. Interestingly, marked changes in protein expression were detected in the supragranular cell region, an area where robust mossy fibers sprouting occurs. Together, these data provide new molecular insights into the altered protein profile of the epileptogenic dentate gyrus and point to potential pathophysiologic mechanisms underlying epileptogenesis. PMID:20608933

  18. Emerging Affinity-Based Proteomic Technologies for Large-Scale Plasma Profiling in Cardiovascular Disease.

    PubMed

    Smith, J Gustav; Gerszten, Robert E

    2017-04-25

    Plasma biomarkers that reflect molecular states of the cardiovascular system are central for clinical decision making. Routinely used plasma biomarkers include troponins, natriuretic peptides, and lipoprotein particles, yet interrogate only a modest subset of pathways relevant to cardiovascular disease. Systematic profiling of a larger portion of circulating plasma proteins (the plasma proteome) will provide opportunities for unbiased discovery of novel markers to improve diagnostic or predictive accuracy. In addition, proteomic profiling may inform pathophysiological understanding and point to novel therapeutic targets. Obstacles for comprehensive proteomic profiling include the immense size and structural heterogeneity of the proteome, and the broad range of abundance levels, as well. Proteome-wide, untargeted profiling can be performed in tissues and cells with tandem mass spectrometry. However, applications to plasma are limited by the need for complex preanalytical sample preparation stages limiting sample throughput. Multiplexing of targeted methods based on capture and detection of specific proteins are therefore receiving increasing attention in plasma proteomics. Immunoaffinity assays are the workhorse for measuring individual proteins but have been limited for proteomic applications by long development times, cross-reactivity preventing multiplexing, specificity issues, and incomplete sensitivity to detect proteins in the lower range of the abundance spectrum (below picograms per milliliter). Emerging technologies to address these issues include nucleotide-labeled immunoassays and aptamer reagents that can be automated for efficient multiplexing of thousands of proteins at high sample throughput, coupling of affinity capture methods to mass spectrometry for improved specificity, and ultrasensitive detection systems to measure low-abundance proteins. In addition, proteomics can now be integrated with modern genomics tools to comprehensively relate

  19. Detection of Bladder Cancer Using Proteomic Profiling of Urine Sediments

    PubMed Central

    Majewski, Tadeusz; Spiess, Philippe E.; Bondaruk, Jolanta; Black, Peter; Clarke, Charlotte; Benedict, William; Dinney, Colin P.; Grossman, Herbert Barton; Tang, Kuang S.; Czerniak, Bogdan

    2012-01-01

    We used protein expression profiles to develop a classification rule for the detection and prognostic assessment of bladder cancer in voided urine samples. Using the Ciphergen PBS II ProteinChip Reader, we analyzed the protein profiles of 18 pairs of samples of bladder tumor and adjacent urothelium tissue, a training set of 85 voided urine samples (32 controls and 53 bladder cancer), and a blinded testing set of 68 voided urine samples (33 controls and 35 bladder cancer). Using t-tests, we identified 473 peaks showing significant differential expression across different categories of paired bladder tumor and adjacent urothelial samples compared to normal urothelium. Then the intensities of those 473 peaks were examined in a training set of voided urine samples. Using this approach, we identified 41 protein peaks that were differentially expressed in both sets of samples. The expression pattern of the 41 protein peaks was used to classify the voided urine samples as malignant or benign. This approach yielded a sensitivity and specificity of 59% and 90%, respectively, on the training set and 80% and 100%, respectively, on the testing set. The proteomic classification rule performed with similar accuracy in low- and high-grade bladder carcinomas. In addition, we used hierarchical clustering with all 473 protein peaks on 65 benign voided urine samples, 88 samples from patients with clinically evident bladder cancer, and 127 samples from patients with a history of bladder cancer to classify the samples into Cluster A or B. The tumors in Cluster B were characterized by clinically aggressive behavior with significantly shorter metastasis-free and disease-specific survival. PMID:22879988

  20. Detection of bladder cancer using proteomic profiling of urine sediments.

    PubMed

    Majewski, Tadeusz; Spiess, Philippe E; Bondaruk, Jolanta; Black, Peter; Clarke, Charlotte; Benedict, William; Dinney, Colin P; Grossman, Herbert Barton; Tang, Kuang S; Czerniak, Bogdan

    2012-01-01

    We used protein expression profiles to develop a classification rule for the detection and prognostic assessment of bladder cancer in voided urine samples. Using the Ciphergen PBS II ProteinChip Reader, we analyzed the protein profiles of 18 pairs of samples of bladder tumor and adjacent urothelium tissue, a training set of 85 voided urine samples (32 controls and 53 bladder cancer), and a blinded testing set of 68 voided urine samples (33 controls and 35 bladder cancer). Using t-tests, we identified 473 peaks showing significant differential expression across different categories of paired bladder tumor and adjacent urothelial samples compared to normal urothelium. Then the intensities of those 473 peaks were examined in a training set of voided urine samples. Using this approach, we identified 41 protein peaks that were differentially expressed in both sets of samples. The expression pattern of the 41 protein peaks was used to classify the voided urine samples as malignant or benign. This approach yielded a sensitivity and specificity of 59% and 90%, respectively, on the training set and 80% and 100%, respectively, on the testing set. The proteomic classification rule performed with similar accuracy in low- and high-grade bladder carcinomas. In addition, we used hierarchical clustering with all 473 protein peaks on 65 benign voided urine samples, 88 samples from patients with clinically evident bladder cancer, and 127 samples from patients with a history of bladder cancer to classify the samples into Cluster A or B. The tumors in Cluster B were characterized by clinically aggressive behavior with significantly shorter metastasis-free and disease-specific survival.

  1. Quantitative Profiling of Single Formalin Fixed Tumour Sections: proteomics for translational research

    PubMed Central

    Hughes, Christopher S.; McConechy, Melissa K.; Cochrane, Dawn R.; Nazeran, Tayyebeh; Karnezis, Anthony N.; Huntsman, David G.; Morin, Gregg B.

    2016-01-01

    Although re-sequencing of gene panels and mRNA expression profiling are now firmly established in clinical laboratories, in-depth proteome analysis has remained a niche technology, better suited for studying model systems rather than challenging materials such as clinical trial samples. To address this limitation, we have developed a novel and optimized platform called SP3-Clinical Tissue Proteomics (SP3-CTP) for in-depth proteome profiling of practical quantities of tumour tissues, including formalin fixed and paraffin embedded (FFPE). Using single 10 μm scrolls of clinical tumour blocks, we performed in-depth quantitative analyses of individual sections from ovarian tumours covering the high-grade serous, clear cell, and endometrioid histotypes. This examination enabled the generation of a novel high-resolution proteome map of ovarian cancer histotypes from clinical tissues. Comparison of the obtained proteome data with large-scale genome and transcriptome analyses validated the observed proteome biology for previously validated hallmarks of this disease, and also identified novel protein features. A tissue microarray analysis validated cystathionine gamma-lyase (CTH) as a novel clear cell carcinoma feature with potential clinical relevance. In addition to providing a milestone in the understanding of ovarian cancer biology, these results show that in-depth proteomic analysis of clinically annotated FFPE materials can be effectively used as a biomarker discovery tool and perhaps ultimately as a diagnostic approach. PMID:27713570

  2. Quantitative Profiling of Single Formalin Fixed Tumour Sections: proteomics for translational research.

    PubMed

    Hughes, Christopher S; McConechy, Melissa K; Cochrane, Dawn R; Nazeran, Tayyebeh; Karnezis, Anthony N; Huntsman, David G; Morin, Gregg B

    2016-10-07

    Although re-sequencing of gene panels and mRNA expression profiling are now firmly established in clinical laboratories, in-depth proteome analysis has remained a niche technology, better suited for studying model systems rather than challenging materials such as clinical trial samples. To address this limitation, we have developed a novel and optimized platform called SP3-Clinical Tissue Proteomics (SP3-CTP) for in-depth proteome profiling of practical quantities of tumour tissues, including formalin fixed and paraffin embedded (FFPE). Using single 10 μm scrolls of clinical tumour blocks, we performed in-depth quantitative analyses of individual sections from ovarian tumours covering the high-grade serous, clear cell, and endometrioid histotypes. This examination enabled the generation of a novel high-resolution proteome map of ovarian cancer histotypes from clinical tissues. Comparison of the obtained proteome data with large-scale genome and transcriptome analyses validated the observed proteome biology for previously validated hallmarks of this disease, and also identified novel protein features. A tissue microarray analysis validated cystathionine gamma-lyase (CTH) as a novel clear cell carcinoma feature with potential clinical relevance. In addition to providing a milestone in the understanding of ovarian cancer biology, these results show that in-depth proteomic analysis of clinically annotated FFPE materials can be effectively used as a biomarker discovery tool and perhaps ultimately as a diagnostic approach.

  3. Derivative component analysis for mass spectral serum proteomic profiles

    PubMed Central

    2014-01-01

    Background As a promising way to transform medicine, mass spectrometry based proteomics technologies have seen a great progress in identifying disease biomarkers for clinical diagnosis and prognosis. However, there is a lack of effective feature selection methods that are able to capture essential data behaviors to achieve clinical level disease diagnosis. Moreover, it faces a challenge from data reproducibility, which means that no two independent studies have been found to produce same proteomic patterns. Such reproducibility issue causes the identified biomarker patterns to lose repeatability and prevents it from real clinical usage. Methods In this work, we propose a novel machine-learning algorithm: derivative component analysis (DCA) for high-dimensional mass spectral proteomic profiles. As an implicit feature selection algorithm, derivative component analysis examines input proteomics data in a multi-resolution approach by seeking its derivatives to capture latent data characteristics and conduct de-noising. We further demonstrate DCA's advantages in disease diagnosis by viewing input proteomics data as a profile biomarker via integrating it with support vector machines to tackle the reproducibility issue, besides comparing it with state-of-the-art peers. Results Our results show that high-dimensional proteomics data are actually linearly separable under proposed derivative component analysis (DCA). As a novel multi-resolution feature selection algorithm, DCA not only overcomes the weakness of the traditional methods in subtle data behavior discovery, but also suggests an effective resolution to overcoming proteomics data's reproducibility problem and provides new techniques and insights in translational bioinformatics and machine learning. The DCA-based profile biomarker diagnosis makes clinical level diagnostic performances reproducible across different proteomic data, which is more robust and systematic than the existing biomarker discovery based

  4. Comparative profiling of the sperm proteome.

    PubMed

    Holland, Ashling; Ohlendieck, Kay

    2015-02-01

    The highly complex and species-selective mechanism of fertilization is a central theme of developmental biology. Gametogenesis, sperm activation, and egg-sperm recognition are fundamental biological processes, warranting detailed studies into the molecular composition of gametes. Biological MS has been instrumental for the comprehensive itemizing of gamete proteomes. The protein constellation of sperm cells and its subcellular structures has been established for a variety of animal species. Spermatogenesis and the crucial activation of sperm cells as a prerequisite of successful fertilization and physiological adaptations to external stressors was investigated using proteomics, as well as the underlying mechanisms of male infertility with respect to proteome-wide alterations. This review outlines recent achievements of sperm proteomics and exemplifies the usefulness of gel-based surveys by outlining the comparative analysis of abnormal spermatozoa in globozoospermia. Besides label-free MS techniques and cell-based labeling methodology, high-resolution fluorescence 2DE has been shown to be highly suitable as a proteomic biomarker discovery tool in sperm protein research. The appropriateness of novel protein markers for improving our understanding of normal spermatogenesis and sperm activation versus the molecular pathogenesis of male infertility will be discussed. New biomarker candidates might be useful to improve diagnostic, prognostic, and therapeutic aspects of infertility.

  5. Trends in sample preparation for classical and second generation proteomics.

    PubMed

    Cañas, Benito; Piñeiro, Carmen; Calvo, Enrique; López-Ferrer, Daniel; Gallardo, Jose Manuel

    2007-06-15

    Sample preparation is a fundamental step in the proteomics workflow. However, it is not easy to find compiled information updating this subject. In this paper, the strategies and protocols for protein extraction and identification, following either classical or second generation proteomics methodologies, are reviewed. Procedures for: tissue disruption, cell lysis, sample pre-fractionation, protein separation by 2-DE, protein digestion, mass spectrometry analysis, multidimensional peptide separations and quantification of protein expression level are described.

  6. Proteomic Profiling of Rat Thyroarytenoid Muscle

    ERIC Educational Resources Information Center

    Welham, Nathan V.; Marriott, Gerard; Bless, Diane M.

    2006-01-01

    Purpose: Proteomic methodologies offer promise in elucidating the systemwide cellular and molecular processes that characterize normal and diseased thyroarytenoid (TA) muscle. This study examined methodological issues central to the application of 2-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (2D SDS-PAGE) to the study of…

  7. Proteomic Profiling of Rat Thyroarytenoid Muscle

    ERIC Educational Resources Information Center

    Welham, Nathan V.; Marriott, Gerard; Bless, Diane M.

    2006-01-01

    Purpose: Proteomic methodologies offer promise in elucidating the systemwide cellular and molecular processes that characterize normal and diseased thyroarytenoid (TA) muscle. This study examined methodological issues central to the application of 2-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (2D SDS-PAGE) to the study of…

  8. The Urine Proteome Profile Is Different in Neuromyelitis Optica Compared to Multiple Sclerosis: A Clinical Proteome Study

    PubMed Central

    Kristensen, Lars P.; Burton, Mark; Csepany, Tunde; Simo, Magdolna; Dioszeghy, Peter; Sejbaek, Tobias; Grebing, Manuela; Heegaard, Niels H. H.; Illes, Zsolt

    2015-01-01

    Objectives Inflammatory demyelinating diseases of the CNS comprise a broad spectrum of diseases like neuromyelitis optica (NMO), NMO spectrum disorders (NMO-SD) and multiple sclerosis (MS). Despite clear classification criteria, differentiation can be difficult. We hypothesized that the urine proteome may differentiate NMO from MS. Methods The proteins in urine samples from anti-aquaporin 4 (AQP4) seropositive NMO/NMO-SD patients (n = 32), patients with MS (n = 46) and healthy subjects (HS, n = 31) were examined by quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS) after trypsin digestion and iTRAQ labelling. Immunoglobulins (Ig) in the urine were validated by nephelometry in an independent cohort (n = 9–10 pr. groups). Results The analysis identified a total of 1112 different proteins of which 333 were shared by all 109 subjects. Cluster analysis revealed differences in the urine proteome of NMO/NMO-SD compared to HS and MS. Principal component analysis also suggested that the NMO/NMO-SD proteome profile was useful for classification. Multivariate regression analysis revealed a 3-protein profile for the NMO/NMO-SD versus HS discrimination, a 6-protein profile for NMO/NMO-SD versus MS discrimination and an 11-protein profile for MS versus HS discrimination. All protein panels yielded highly significant ROC curves (AUC in all cases >0.85, p≤0.0002). Nephelometry confirmed the presence of increased Ig-light chains in the urine of patients with NMO/NMO-SD. Conclusion The urine proteome profile of patients with NMO/NMO-SD is different from MS and HS. This may reflect differences in the pathogenesis of NMO/NMO-SD versus MS and suggests that urine may be a potential source of biomarkers differentiating NMO/NMO-SD from MS. PMID:26460890

  9. Proteomic profiling in schizophrenia: enabling stratification for more effective treatment

    PubMed Central

    2013-01-01

    Schizophrenia is a heterogeneous psychiatric disorder characterized by an array of clinical manifestations. Although the best known manifestations include serious effects on mood and behavior, patients can also display co-morbidities, including immune system or metabolic abnormalities. Thorough characterization of these conditions using proteomic profiling methods has increased our knowledge of these molecular differences and has helped to unravel the complexity and heterogeneity of this debilitating condition. This could lead to patient stratification through characterization of biochemically different subtypes of the disease. In addition, proteomic methods have recently been used for molecular characterization of the mechanism of action of antipsychotic medications in both preclinical models and patients. This has resulted in identification of molecular panels that show some promise for prediction of response or for monitoring treatment outcome. This review describes how proteomic profiling methods can impact the future of schizophrenia diagnosis and therapeutics, and facilitate personalized medicine approaches for more effective treatment management of schizophrenia patients. PMID:23531373

  10. Ultra-Fast Sample Preparation for High-Throughput Proteomics

    SciTech Connect

    Lopez-Ferrer, Daniel; Hixson, Kim K.; Belov, Mikhail E.; Smith, Richard D.

    2011-06-21

    Sample preparation oftentimes can be the Achilles Heel of any analytical process and in the field of proteomics, preparing samples for mass spectrometric analysis is no exception. Current goals, concerning proteomic sample preparation on a large scale, include efforts toward improving reproducibility, reducing the time of processing and ultimately the automation of the entire workflow. This chapter reviews an array of recent approaches applied to bottom-up proteomics sample preparation to reduce the processing time down from hours to minutes. The current state-of-the-art in the field uses different energy inputs like microwave, ultrasound or pressure to perform the four basic steps in sample preparation: protein extraction, denaturation, reduction and alkylation, and digestion. No single energy input for enhancement of proteome sample preparation has become the universal gold standard. Instead, a combination of different energy inputs tend to produce the best results. This chapter further describes the future trends in the field such as the hyphenation of sample preparation with downstream detection and analysis systems. Finally, a detailed protocol describing the combined use of both pressure cycling technology and ultrasonic energy inputs to hasten proteomic sample preparation is presented.

  11. The proteomic profile of hair damage.

    PubMed

    Sinclair, R; Flagler, M J; Jones, L; Rufaut, N; Davis, M G

    2012-06-01

    Monilethrix is a congenital hair shaft disorder with associated fragility. Many of the changes seen in monilethrix hair on light microscopy and scanning electron microscopy are also seen in hair weathering and cosmetic damage to hair. We used monilethrix as a model to investigate the relationship between hair protein structure and hair strength and resistance to cosmetic insult. We applied proteomic techniques to identify novel peptide damage markers for chemical oxidative damage to hair. The findings suggest that specific sites in the protein structure of hair are targeted during oxidative damage from bleaching, a unique insight into how chemical damage compromises the structural integrity of the hair shaft at the molecular level. Applying proteomics to the study of congenital and acquired hair shaft disorders can deliver new insights into hair damage and novel strategies to strengthen hair. © 2012 The Authors. BJD © 2012 British Association of Dermatologists.

  12. Environmental proteomics: applications of proteome profiling in environmental microbiology and biotechnology.

    PubMed

    Lacerda, Carla M R; Reardon, Kenneth F

    2009-01-01

    In this review, we present the use of proteomics to advance knowledge in the field of environmental biotechnology, including studies of bacterial physiology, metabolism and ecology. Bacteria are widely applied in environmental biotechnology for their ability to catalyze dehalogenation, methanogenesis, denitrification and sulfate reduction, among others. Their tolerance to radiation and toxic compounds is also of importance. Proteomics has an important role in helping uncover the pathways behind these cellular processes. Environmental samples are often highly complex, which makes proteome studies in this field especially challenging. Some of these challenges are the lack of genome sequences for the vast majority of environmental bacteria, difficulties in isolating bacteria and proteins from certain environments, and the presence of complex microbial communities. Despite these challenges, proteomics offers a unique dynamic view into cellular function. We present examples of environmental proteomics of model organisms, and then discuss metaproteomics (microbial community proteomics), which has the potential to provide insights into the function of a community without isolating organisms. Finally, the environmental proteomics literature is summarized as it pertains to the specific application areas of wastewater treatment, metabolic engineering, microbial ecology and environmental stress responses.

  13. An effective plasma membrane proteomics approach for small tissue samples

    PubMed Central

    Smolders, Katrien; Lombaert, Nathalie; Valkenborg, Dirk; Baggerman, Geert; Arckens, Lutgarde

    2015-01-01

    Advancing the quest for new drug targets demands the development of innovative plasma membrane proteome research strategies applicable to small, functionally defined tissue samples. Biotinylation of acute tissue slices and streptavidin pull-down followed by shotgun proteomics allowed the selective extraction and identification of >1,600 proteins of which >60% are associated with the plasma membrane, including (G-protein coupled) receptors, ion channels and transporters, and this from mm3-scale tissue. PMID:26047021

  14. Large-Scale and Deep Quantitative Proteome Profiling Using Isobaric Labeling Coupled with Two-Dimensional LC-MS/MS.

    PubMed

    Gritsenko, Marina A; Xu, Zhe; Liu, Tao; Smith, Richard D

    2016-01-01

    Comprehensive, quantitative information on abundances of proteins and their posttranslational modifications (PTMs) can potentially provide novel biological insights into diseases pathogenesis and therapeutic intervention. Herein, we introduce a quantitative strategy utilizing isobaric stable isotope-labeling techniques combined with two-dimensional liquid chromatography-tandem mass spectrometry (2D-LC-MS/MS) for large-scale, deep quantitative proteome profiling of biological samples or clinical specimens such as tumor tissues. The workflow includes isobaric labeling of tryptic peptides for multiplexed and accurate quantitative analysis, basic reversed-phase LC fractionation and concatenation for reduced sample complexity, and nano-LC coupled to high resolution and high mass accuracy MS analysis for high confidence identification and quantification of proteins. This proteomic analysis strategy has been successfully applied for in-depth quantitative proteomic analysis of tumor samples and can also be used for integrated proteome and PTM characterization, as well as comprehensive quantitative proteomic analysis across samples from large clinical cohorts.

  15. Spatially-Resolved Proteomics: Rapid Quantitative Analysis of Laser Capture Microdissected Alveolar Tissue Samples

    SciTech Connect

    Clair, Geremy; Piehowski, Paul D.; Nicola, Teodora; Kitzmiller, Joseph A.; Huang, Eric L.; Zink, Erika M.; Sontag, Ryan L.; Orton, Daniel J.; Moore, Ronald J.; Carson, James P.; Smith, Richard D.; Whitsett, Jeffrey A.; Corley, Richard A.; Ambalavanan, Namasivayam; Ansong, Charles

    2016-12-22

    Global proteomics approaches allow characterization of whole tissue lysates to an impressive depth. However, it is now increasingly recognized that to better understand the complexity of multicellular organisms, global protein profiling of specific spatially defined regions/substructures of tissues (i.e. spatially-resolved proteomics) is essential. Laser capture microdissection (LCM) enables microscopic isolation of defined regions of tissues preserving crucial spatial information. However, current proteomics workflows entail several manual sample preparation steps and are challenged by the microscopic mass-limited samples generated by LCM, and that impact measurement robustness, quantification, and throughput. Here, we coupled LCM with a fully automated sample preparation workflow that with a single manual step allows: protein extraction, tryptic digestion, peptide cleanup and LC-MS/MS analysis of proteomes from microdissected tissues. Benchmarking against the current state of the art in ultrasensitive global proteomic analysis, our approach demonstrated significant improvements in quantification and throughput. Using our LCM-SNaPP proteomics approach, we characterized to a depth of more than 3,400 proteins, the ontogeny of protein changes during normal lung development in laser capture microdissected alveolar tissue containing ~4,000 cells per sample. Importantly, the data revealed quantitative changes for 350 low abundance transcription factors and signaling molecules, confirming earlier transcript-level observations and defining seven modules of coordinated transcription factor/signaling molecule expression patterns, suggesting that a complex network of temporal regulatory control directs normal lung development with epigenetic regulation fine-tuning pre-natal developmental processes. Our LCM-proteomics approach facilitates efficient, spatially-resolved, ultrasensitive global proteomics analyses in high-throughput that will be enabling for several clinical and

  16. Proteomic profiling of human plasma for cancer biomarker discovery.

    PubMed

    Huang, Zhao; Ma, Linguang; Huang, Canhua; Li, Qifu; Nice, Edouard C

    2017-03-01

    Over the past decades, substantial advances have been made in both the early diagnosis and accurate prognosis of many cancers because of the impressive development of novel proteomic strategies. However, it remains difficult to standardize proteomic approaches. In addition, the heterogeneity of proteins in distinct tissues results in incomplete population of the whole proteome, which inevitably limits its clinical practice. As one of the most complex proteomes in the human body, the plasma proteome contains secreted proteins originating from multiple organs and tissues, making it a favorable matrix for comprehensive biomarker discovery. Here, we will discuss the roles of plasma proteome profiling in cancer biomarker discovery and validation, and highlight both the inherent advantages and disadvantages. Although several hurdles lay ahead, further advances in this technology will greatly increase our understanding of cancer biology, reveal new biomarkers and biomarker panels, and open a new avenue for more efficient early diagnosis and surveillance of cancer, leading toward personalized medicine. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. Proteomics: Challenges, Techniques and Possibilities to Overcome Biological Sample Complexity

    PubMed Central

    Chandramouli, Kondethimmanahalli; Qian, Pei-Yuan

    2009-01-01

    Proteomics is the large-scale study of the structure and function of proteins in complex biological sample. Such an approach has the potential value to understand the complex nature of the organism. Current proteomic tools allow large-scale, high-throughput analyses for the detection, identification, and functional investigation of proteome. Advances in protein fractionation and labeling techniques have improved protein identification to include the least abundant proteins. In addition, proteomics has been complemented by the analysis of posttranslational modifications and techniques for the quantitative comparison of different proteomes. However, the major limitation of proteomic investigations remains the complexity of biological structures and physiological processes, rendering the path of exploration paved with various difficulties and pitfalls. The quantity of data that is acquired with new techniques places new challenges on data processing and analysis. This article provides a brief overview of currently available proteomic techniques and their applications, followed by detailed description of advantages and technical challenges. Some solutions to circumvent technical difficulties are proposed. PMID:20948568

  18. Proteomic profile of Piper tuberculatum (Piperaceae).

    PubMed

    Cotinguiba, F; López, S N; Budzinski, I G F; Labate, C A; Kato, M J; Furlan, M

    2017-07-10

    Piper tuberculatum (Piperaceae) is a species that accumulates especially amides as secondary metabolites and several biological activities was previously reported. In this article, we report a proteomic study of P. tuberculatum. Bidimensional electrophoresis (2D SDS-PAGE) and mass spectrometry (ESI-Q-TOF) were used in this study. Over a hundred spots and various peptides were identified in this species and the putative functions of these peptides related to defense mechanism as biotic and abiotic stress were assigned. The information presented extend the range of molecular information of P. tuberculatum.

  19. Proteomic profiling of Tectona grandis L. leaf.

    PubMed

    Quiala, Elisa; Cañal, María Jesús; Rodríguez, Roberto; Yagüe, Norma; Chávez, Maité; Barbón, Raúl; Valledor, Luis

    2012-04-01

    Tectona grandis L. (teak) is one of the premier hardwood timbers in the world, ranking at present in the top five tropical hardwood species in terms of worldwide plantation area. Characterization of the proteins present in teak leaves will provide a basis for the development of new tools aimed at assisting tree selection, the monitoring of plant propagation, and the certification of clonal and phenotypic identities. In this paper, we describe the extraction, separation, and identification of leaf proteins from T. grandis using a TCA/acetone protocol, 2DE, and MALDI-TOF. After TCA/acetone protein extraction of leaves, 998 well-resolved spots were detected in Coomassie-stained gels within the 10-114 kDa relative molecular mass (Mr) range at a pH ranging from 3 to 11. A total of 120 spots were digested and subjected to MS. Of these, 100 nonredundant protein species were successfully identified. Functional classification of the identified proteins revealed that proteins involved in photosynthesis, protein translation, and energy production were the most abundant. This work is the first high-throughput attempt to study the T. grandis leaf proteome and represents a stepping stone for further differential expression proteomic studies related to growth, development, biomass production, and culture-associated physiological responses. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  20. Alternative profiling platform based on MELDI and its applicability in clinical proteomics.

    PubMed

    Najam-ul-Haq, Muhammad; Rainer, Matthias; Trojer, Lukas; Feuerstein, Isabel; Vallant, Rainer Markus; Huck, Christian W; Bakry, Rania; Bonn, Günther Karl

    2007-08-01

    The presence of numerous proteomics data and their results in literature reveal the importance and influence of proteins and peptides on human cell cycle. For instance, the proteomic profiling of biological samples, such as serum, plasma or cells, and their organelles, carried out by surface-enhanced laser desorption/ionization mass spectrometry, has led to the discovery of numerous key proteins involved in many biological disease processes. However, questions still remain regarding the reproducibility, bioinformatic artifacts and cross-validations of such experimental set-ups. The authors have developed a material-based approach, termed material-enhanced laser desorption/ionization mass spectrometry (MELDI-MS), to facilitate and improve the robustness of large-scale proteomic experiments. MELDI-MS includes a fully automated protein-profiling platform, from sample preparation and analysis to data processing involving state-of-the-art methods, which can be further improved. Multiplexed protein pattern analysis, based on material morphology, physical characteristics and chemical functionalities provides a multitude of protein patterns and allows prostate cancer samples to be distinguished from non-prostate cancer samples. Furthermore, MELDI-MS enables not only the analysis of protein signatures, but also the identification of potential discriminating peaks via capillary liquid chromatography mass spectrometry. The optimized MELDI approach offers a complete proteomics platform with improved sensitivity, selectivity and short sample preparation times.

  1. The proteome of sickle cell disease: insights from exploratory proteomic profiling

    PubMed Central

    Yuditskaya, Susan; Suffredini, Anthony F; J Kato, Gregory

    2011-01-01

    The expanding realm of exploratory proteomics has added a unique dimension to the study of the complex pathophysiology involved in sickle cell disease. A review of proteomic studies published on sickle cell erythrocytes and plasma shows trends of upregulation of antioxidant proteins, an increase in cytoskeletal defects, an increase in protein repair and turnover components, a decrease in lipid raft proteins and apolipoprotein dysregulation. Many of these findings are consistent with the pathophysiology of sickle cell disease, including high oxidant burden, resulting in damage to cytoskeletal and other proteins, and erythrocyte rigidity. More unexpected findings, such as a decrease in lipid raft components and apolipoprotein dysregulation, offer previously unexplored targets for future investigation and potential therapeutic intervention. Exploratory proteomic profiling is a valuable source of hypothesis generation for the cellular and molecular pathophysiology of sickle cell disease. PMID:21142886

  2. Leaf proteome profiling of transgenic mint infected with Alternaria alternata.

    PubMed

    Sinha, Ragini; Bhattacharyya, Dipto; Majumdar, Aparupa Bose; Datta, Riddhi; Hazra, Saptarshi; Chattopadhyay, Sharmila

    2013-11-20

    The genus Mentha has been widely used in food, flavor, culinary, cosmetic and pharmaceutical industries. Substantial damage to this crop happened regularly due to environmental stresses like metal toxicity and pathogen attack. Here, an approach has been taken to raise transgenic mint over-expressing γ-glutamyl-cysteine synthetase (γ-ECS), the rate-limiting enzyme of GSH biosynthesis, resulted enhanced GSH content and its in planta expression confers significant tolerance towards abiotic/biotic stresses viz. metal toxicity - Cd, Zn as well as against infection of Alternaria alternata and Rhizoctonia solani. A differential proteomic analysis through 2-DE and MALDI TOF-TOF MSMS was performed to focus on the altered abundance of functionally important protein species in control and infected transgenic mint. Results showed a significant variation in the protein profile of the infected transgenic plant as compared to the wild/control transgenic counterpart. In addition to protein species related to stress and defense, redox regulation, transcription factors and energy & metabolism, protein species related to signaling and gene regulation as well as cell division also showed differential accumulation in infected transgenic. Hence, proteomics can be used as a tool to decipher the mechanism of action of GSH in providing tolerance against a necrotrophic fungus, A. alternata in transgenic mint. The reported work describes a comparative proteomics of non-model unsequenced plants like Mentha. There is a comparative protein profile between transgenic and its wild counterparts under control and infected condition. The work has an impact in crop proteomics and also tries to explain the application of proteomic approach to decipher the mechanism by which a foreign metabolite mediates stress tolerance in plant under control and infected condition. This article is part of a Special Issue entitled: Translational Plant Proteomics. Copyright © 2013 Elsevier B.V. All rights reserved.

  3. Glaucoma related Proteomic Alterations in Human Retina Samples

    PubMed Central

    Funke, Sebastian; Perumal, Natarajan; Beck, Sabine; Gabel-Scheurich, Silke; Schmelter, Carsten; Teister, Julia; Gerbig, Claudia; Gramlich, Oliver W.; Pfeiffer, Norbert; Grus, Franz H.

    2016-01-01

    Glaucoma related proteomic changes have been documented in cell and animal models. However, proteomic studies investigating on human retina samples are still rare. In the present work, retina samples of glaucoma and non-glaucoma control donors have been examined by a state-of-the-art mass spectrometry (MS) workflow to uncover glaucoma related proteomic changes. More than 600 proteins could be identified with high confidence (FDR < 1%) in human retina samples. Distinct proteomic changes have been observed in 10% of proteins encircling mitochondrial and nucleus species. Numerous proteins showed a significant glaucoma related level change (p < 0.05) or distinct tendency of alteration (p < 0.1). Candidates were documented to be involved in cellular development, stress and cell death. Increase of stress related proteins and decrease of new glaucoma related candidates, ADP/ATP translocase 3 (ANT3), PC4 and SRFS1-interacting protein 1 (DFS70) and methyl-CpG-binding protein 2 (MeCp2) could be documented by MS. Moreover, candidates could be validated by Accurate Inclusion Mass Screening (AIMS) and immunostaining and supported for the retinal ganglion cell layer (GCL) by laser capture microdissection (LCM) in porcine and human eye cryosections. The workflow allowed a detailed view into the human retina proteome highlighting new molecular players ANT3, DFS70 and MeCp2 associated to glaucoma. PMID:27425789

  4. A Sampling of the Yeast Proteome

    PubMed Central

    Futcher, B.; Latter, G. I.; Monardo, P.; McLaughlin, C. S.; Garrels, J. I.

    1999-01-01

    In this study, we examined yeast proteins by two-dimensional (2D) gel electrophoresis and gathered quantitative information from about 1,400 spots. We found that there is an enormous range of protein abundance and, for identified spots, a good correlation between protein abundance, mRNA abundance, and codon bias. For each molecule of well-translated mRNA, there were about 4,000 molecules of protein. The relative abundance of proteins was measured in glucose and ethanol media. Protein turnover was examined and found to be insignificant for abundant proteins. Some phosphoproteins were identified. The behavior of proteins in differential centrifugation experiments was examined. Such experiments with 2D gels can give a global view of the yeast proteome. PMID:10523624

  5. Serum proteomic profiling of major depressive disorder.

    PubMed

    Bot, M; Chan, M K; Jansen, R; Lamers, F; Vogelzangs, N; Steiner, J; Leweke, F M; Rothermundt, M; Cooper, J; Bahn, S; Penninx, B W J H

    2015-07-14

    Much has still to be learned about the molecular mechanisms of depression. This study aims to gain insight into contributing mechanisms by identifying serum proteins related to major depressive disorder (MDD) in a large psychiatric cohort study. Our sample consisted of 1589 participants of the Netherlands Study of Depression and Anxiety, comprising 687 individuals with current MDD (cMDD), 482 individuals with remitted MDD (rMDD) and 420 controls. We studied the relationship between MDD status and the levels of 171 serum proteins detected on a multi-analyte profiling platform using adjusted linear regression models. Pooled analyses of two independent validation cohorts (totaling 78 MDD cases and 156 controls) was carried out to validate our top markers. Twenty-eight analytes differed significantly between cMDD cases and controls (P < 0.05), whereas 10 partly overlapping markers differed significantly between rMDD cases and controls. Antidepressant medication use and comorbid anxiety status did not substantially impact on these findings. Sixteen of the cMDD-related markers had been assayed in the pooled validation cohorts, of which seven were associated with MDD. The analytes prominently associated with cMDD related to diverse cell communication and signal transduction processes (pancreatic polypeptide, macrophage migration inhibitory factor, ENRAGE, interleukin-1 receptor antagonist and tenascin-C), immune response (growth-regulated alpha protein) and protein metabolism (von Willebrand factor). Several proteins were implicated in depression. Changes were more prominent in cMDD, suggesting that molecular alterations in serum are associated with acute depression symptomatology. These findings may help to establish serum-based biomarkers of depression and could improve our understanding of its pathophysiology.

  6. The proteomic profile of whole and glandular saliva in healthy pain-free subjects

    PubMed Central

    Jasim, Hajer; Olausson, Patrik; Hedenberg-Magnusson, Britt; Ernberg, Malin; Ghafouri, Bijar

    2016-01-01

    Determination of the variability in the salivary proteome is a prerequisite for the development of saliva as a diagnostic and prognostic tool in particular physiological states. In this context, it is important that technical variability induced by sample collection and processing is kept at minimum to be able to reproducibly assess variability in states of health and disease. In the current study, the proteome profile in unstimulated and stimulated whole, parotid and sublingual saliva was investigated using two-dimensional gel electrophoresis. Saliva samples were structurally collected from ten examined and characterized healthy individuals during the exactly same conditions. The results demonstrated that different collection methods provide clear differences in the snapshot of the salivary proteome and also in the relative amount of specific proteins. The variable nature of the salivary proteome suggests that different approaches may have to be adopted when studying its composition or its possible role as an indicator for particular physiological states. The results emphasize the importance of consistency when collecting saliva samples for proteomic analysis. PMID:27976689

  7. Proteome Profiling Outperforms Transcriptome Profiling for Coexpression Based Gene Function Prediction

    SciTech Connect

    Wang, Jing; Ma, Zihao; Carr, Steven A.; Mertins, Philipp; Zhang, Hui; Zhang, Zhen; Chan, Daniel W.; Ellis, Matthew J. C.; Townsend, R. Reid; Smith, Richard D.; McDermott, Jason E.; Chen, Xian; Paulovich, Amanda G.; Boja, Emily S.; Mesri, Mehdi; Kinsinger, Christopher R.; Rodriguez, Henry; Rodland, Karin D.; Liebler, Daniel C.; Zhang, Bing

    2016-11-11

    Coexpression of mRNAs under multiple conditions is commonly used to infer cofunctionality of their gene products despite well-known limitations of this “guilt-by-association” (GBA) approach. Recent advancements in mass spectrometry-based proteomic technologies have enabled global expression profiling at the protein level; however, whether proteome profiling data can outperform transcriptome profiling data for coexpression based gene function prediction has not been systematically investigated. Here, we address this question by constructing and analyzing mRNA and protein coexpression networks for three cancer types with matched mRNA and protein profiling data from The Cancer Genome Atlas (TCGA) and the Clinical Proteomic Tumor Analysis Consortium (CPTAC). Our analyses revealed a marked difference in wiring between the mRNA and protein coexpression networks. Whereas protein coexpression was driven primarily by functional similarity between coexpressed genes, mRNA coexpression was driven by both cofunction and chromosomal colocalization of the genes. Functionally coherent mRNA modules were more likely to have their edges preserved in corresponding protein networks than functionally incoherent mRNA modules. Proteomic data strengthened the link between gene expression and function for at least 75% of Gene Ontology (GO) biological processes and 90% of KEGG pathways. A web application Gene2Net (http://cptac.gene2net.org) developed based on the three protein coexpression networks revealed novel gene-function relationships, such as linking ERBB2 (HER2) to lipid biosynthetic process in breast cancer, identifying PLG as a new gene involved in complement activation, and identifying AEBP1 as a new epithelial-mesenchymal transition (EMT) marker. Our results demonstrate that proteome profiling outperforms transcriptome profiling for coexpression based gene function prediction. Proteomics should be integrated if not preferred in gene function and human disease studies

  8. High-resolution proteomic profiling of spider venom: expanding the toxin diversity of Phoneutria nigriventer venom.

    PubMed

    Liberato, Tarcísio; Troncone, Lanfranco Ranieri Paolo; Yamashiro, Edson T; Serrano, Solange M T; Zelanis, André

    2016-03-01

    Here we present a proteomic characterization of Phoneutria nigriventer venom. A shotgun proteomic approach allowed the identification, for the first time, of O-glycosyl hydrolases (chitinases) in P. nigriventer venom. The electrophoretic profiles under nonreducing and reducing conditions, and protein identification by mass spectrometry, indicated the presence of oligomeric toxin structures in the venom. Complementary proteomic approaches allowed for a qualitative and semi-quantitative profiling of P. nigriventer venom complexity, expanding its known venom proteome diversity.

  9. Toward Plasma Proteome Profiling with Ion Mobility-Mass Spectrometry

    SciTech Connect

    Valentine, Stephen J.; Plasencia, Manolo D.; Liu, Xiaoyun; Krishnan, Meera; Naylor, Stephen; Udseth, Harold R.; Smith, Richard D.; Clemmer, David E.

    2006-11-01

    Differential, functional, and mapping proteomic analyses of complex biological mixtures suffer from a lack of component resolution. Here we describe the application of ion mobility-mass spectrometry (IMSMS) to this problem. With this approach, components that are separated by liquid chromatography are dispersed based on differences in their mobilities through a buffer gas prior to being analyzed by MS. The inclusion of the gas-phase dispersion provides more than an order of magnitude enhancement in component resolution at no cost to data acquisition time. Additionally, the mobility separation often removes high-abundance species from spectral regions containing low-abundance species, effectively increasing measurement sensitivity and dynamic range. Finally, collision-induced dissociation of all ions can be recorded in a single experimental sequence while conventional MS methods sequentially select precursors. The approach is demonstrated in a single, rapid (3.3 h) analysis of a plasma digest sample where abundant proteins have not been removed. Protein database searches have yielded 731 high confidence peptide assignments corresponding to 438 unique proteins. Results have been compiled into an initial analytical map to be used -after further augmentation and refinement- for comparative plasma profiling studies.

  10. Proteomic and peptidomic profiling of Brazilian artisanal 'Coalho' cheese.

    PubMed

    Silva, Roberto A; Bezerra, Vilma S; Pimentel, Maria do Carmo B; Porto, Ana Lúcia F; Cavalcanti, Maria Taciana H; Filho, José Luiz L

    2016-10-01

    Artisanal 'Coalho' cheese is a product typically popular in the Brazilian north-eastern region. Production of this cheese represents about 9.2% of the internal crude product of Pernambuco State. Several peptides are generated from hydrolysis of αS1 -, αS2 -, β-, and κ-caseins during manufacture of this cheese. The commercial importance of Brazilian artisanal 'Coalho' cheese justifies the examination of both the protein and peptide profiles of cheeses from six cities of the semi-arid region of Pernambuco State, Brazil. SDS-PAGE of the aqueous extracts of 'Coalho' cheeses (WSP) showed bands of lactoferrin, β-lactoglobulin, β-lactoglobulin (dimer), α-lactoalbumin, bovine serum albumin, α-casein, β-casein, κ-casein and para-κ-casein. A total of 57 to 72 peptides were confirmed by mass spectra in the different samples of 'Coalho' cheese which 32 known peptides (11 from αS1 -casein, three from αS2 -casein, 15 from β-casein and three from κ-casein), comprising seven caseinphosphopeptides. Among the unidentified peptides, three showed high intensity peaks in all 'Coalho' cheeses studied (with molecular weights of 1597, 1725/1726, 2778/2779 Da). The proteomic studies revealed peptides that may represent molecular markers or fingerprints for investigating the quality control and regional characterisation of these 'Coalho' cheeses. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

  11. High throughput profile-profile based fold recognition for the entire human proteome.

    PubMed

    McGuffin, Liam J; Smith, Richard T; Bryson, Kevin; Sørensen, Søren-Aksel; Jones, David T

    2006-06-07

    In order to maintain the most comprehensive structural annotation databases we must carry out regular updates for each proteome using the latest profile-profile fold recognition methods. The ability to carry out these updates on demand is necessary to keep pace with the regular updates of sequence and structure databases. Providing the highest quality structural models requires the most intensive profile-profile fold recognition methods running with the very latest available sequence databases and fold libraries. However, running these methods on such a regular basis for every sequenced proteome requires large amounts of processing power. In this paper we describe and benchmark the JYDE (Job Yield Distribution Environment) system, which is a meta-scheduler designed to work above cluster schedulers, such as Sun Grid Engine (SGE) or Condor. We demonstrate the ability of JYDE to distribute the load of genomic-scale fold recognition across multiple independent Grid domains. We use the most recent profile-profile version of our mGenTHREADER software in order to annotate the latest version of the Human proteome against the latest sequence and structure databases in as short a time as possible. We show that our JYDE system is able to scale to large numbers of intensive fold recognition jobs running across several independent computer clusters. Using our JYDE system we have been able to annotate 99.9% of the protein sequences within the Human proteome in less than 24 hours, by harnessing over 500 CPUs from 3 independent Grid domains. This study clearly demonstrates the feasibility of carrying out on demand high quality structural annotations for the proteomes of major eukaryotic organisms. Specifically, we have shown that it is now possible to provide complete regular updates of profile-profile based fold recognition models for entire eukaryotic proteomes, through the use of Grid middleware such as JYDE.

  12. Pathway analysis of kidney cancer using proteomics and metabolic profiling

    PubMed Central

    Perroud, Bertrand; Lee, Jinoo; Valkova, Nelly; Dhirapong, Amy; Lin, Pei-Yin; Fiehn, Oliver; Kültz, Dietmar; Weiss, Robert H

    2006-01-01

    Background Renal cell carcinoma (RCC) is the sixth leading cause of cancer death and is responsible for 11,000 deaths per year in the US. Approximately one-third of patients present with disease which is already metastatic and for which there is currently no adequate treatment, and no biofluid screening tests exist for RCC. In this study, we have undertaken a comprehensive proteomic analysis and subsequently a pathway and network approach to identify biological processes involved in clear cell RCC (ccRCC). We have used these data to investigate urinary markers of RCC which could be applied to high-risk patients, or to those being followed for recurrence, for early diagnosis and treatment, thereby substantially reducing mortality of this disease. Results Using 2-dimensional electrophoresis and mass spectrometric analysis, we identified 31 proteins which were differentially expressed with a high degree of significance in ccRCC as compared to adjacent non-malignant tissue, and we confirmed some of these by immunoblotting, immunohistochemistry, and comparison to published transcriptomic data. When evaluated by several pathway and biological process analysis programs, these proteins are demonstrated to be involved with a high degree of confidence (p values < 2.0 E-05) in glycolysis, propanoate metabolism, pyruvate metabolism, urea cycle and arginine/proline metabolism, as well as in the non-metabolic p53 and FAS pathways. In a pilot study using random urine samples from both ccRCC and control patients, we performed metabolic profiling and found that only sorbitol, a component of an alternative glycolysis pathway, is significantly elevated at 5.4-fold in RCC patients as compared to controls. Conclusion Extensive pathway and network analysis allowed for the discovery of highly significant pathways from a set of clear cell RCC samples. Knowledge of activation of these processes will lead to novel assays identifying their proteomic and/or metabolomic signatures in biofluids

  13. Trends in proteomic analysis of human vitreous humor samples.

    PubMed

    Rocha, Ana S; Santos, Fátima M; Monteiro, João P; Castro-de-Sousa, João P; Queiroz, João A; Tomaz, Cândida T; Passarinha, Luís A

    2014-09-01

    Proteomic analysis of human vitreous humor (VH) may elucidate the pathogenesis of retinal ocular diseases and may provide information for the development of potential therapeutic targets due to its pivotal location near lens and retina. The discovery of whole VH proteome involves a complex analysis of thousands of proteins simultaneously. Therefore, in proteomic studies the protein fractionation is important for reducing sample complexity, facilitating the access to the low-abundant proteins, and recognizing them as biotargets for clinical research. Although several separation methods have been used, gel-based proteomics are the most popular and versatile ones applied for global protein separation. However, chromatographic methods and its combination with other separation techniques are now beginning to be used as promising set-ups for VH protein identification. This review attempts to offer an overview of the techniques currently used with VH, exploring its methodological demands, exposing its advantages, and helping the reader to plan future experiences. Moreover, this review shows the relevance of VH proteomic analysis as a tool for the study of the mechanisms underlying some ocular diseases and for the development of new therapeutic approaches.

  14. Fertility-related proteomic profiling bull spermatozoa separated by percoll.

    PubMed

    Park, Yoo-Jin; Kwon, Woo-Sung; Oh, Shin-Ae; Pang, Myung-Geol

    2012-08-03

    Infertility or subfertility of bovine spermatozoa may lead to disintegration of the breeding system and large economic losses. Recently, proteomics have identified candidates for the sperm fertility biomarkers, but no definite studies have clearly identified the relationship between the proteome and sperm fertility after proteomic study. Therefore, to determine the clinical value of the protein markers identified by proteomic study, we first compared the protein expression profiles of spermatozoa from high and low fertility bulls using 2-dimensional electrophoresis. We then investigated the relationship between protein expression and the fertility of individual bulls as assessed by Western blot analysis. Five proteins, enolase 1 (ENO1), ATP synthase H+ transporting mitochondrial F1 complex beta subunit, apoptosis-stimulating of p53 protein 2, alpha-2-HS-glycoprotein, and phospholipid hydroperoxide glutathione peroxide, were more highly represented in high fertility bulls, whereas three proteins, voltage dependent anion channel 2 (VDAC2), ropporin-1, and ubiquinol-cytochrome-c reductase complex core protein 2 (UQCRC2), were more highly represented in low fertility bulls. Among those proteins, ENO1, VDAC2, and UQCRC2 were significantly correlated with individual fertility. Therefore, these results suggest that concurrent comparisons between protein expression and other fertility assays may represent a good in vitro assay to determine sperm fertility.

  15. Activity-based probes for the proteomic profiling of metalloproteases

    PubMed Central

    Saghatelian, Alan; Jessani, Nadim; Joseph, Arul; Humphrey, Mark; Cravatt, Benjamin F.

    2004-01-01

    Metalloproteases (MPs) are a large and diverse class of enzymes implicated in numerous physiological and pathological processes, including tissue remodeling, peptide hormone processing, and cancer. MPs are tightly regulated by multiple posttranslational mechanisms in vivo, hindering their functional analysis by conventional genomic and proteomic methods. Here we describe a general strategy for creating activity-based proteomic probes for MPs by coupling a zinc-chelating hydroxamate to a benzophenone photocrosslinker, which promote selective binding and modification of MP active sites, respectively. These probes labeled active MPs but not their zymogen or inhibitor-bound counterparts and were used to identify members of this enzyme class up-regulated in invasive cancer cells and to evaluate the selectivity of MP inhibitors in whole proteomes. Interestingly, the matrix metalloproteinase inhibitor GM6001 (ilomastat), which is currently in clinical development, was found to also target the neprilysin, aminopeptidase, and dipeptidylpeptidase clans of MPs. These results demonstrate that MPs can display overlapping inhibitor sensitivities despite lacking sequence homology and stress the need to evaluate MP inhibitors broadly across this enzyme class to develop agents with suitable target selectivities in vivo. Activity-based profiling offers a powerful means for conducting such screens, as this approach can be carried out directly in whole proteomes, thereby facilitating the discovery of disease-associated MPs concurrently with inhibitors that selectively target these proteins. PMID:15220480

  16. Proteome Profiling Outperforms Transcriptome Profiling for Coexpression Based Gene Function Prediction.

    PubMed

    Wang, Jing; Ma, Zihao; Carr, Steven A; Mertins, Philipp; Zhang, Hui; Zhang, Zhen; Chan, Daniel W; Ellis, Matthew J C; Townsend, R Reid; Smith, Richard D; McDermott, Jason E; Chen, Xian; Paulovich, Amanda G; Boja, Emily S; Mesri, Mehdi; Kinsinger, Christopher R; Rodriguez, Henry; Rodland, Karin D; Liebler, Daniel C; Zhang, Bing

    2017-01-01

    Coexpression of mRNAs under multiple conditions is commonly used to infer cofunctionality of their gene products despite well-known limitations of this "guilt-by-association" (GBA) approach. Recent advancements in mass spectrometry-based proteomic technologies have enabled global expression profiling at the protein level; however, whether proteome profiling data can outperform transcriptome profiling data for coexpression based gene function prediction has not been systematically investigated. Here, we address this question by constructing and analyzing mRNA and protein coexpression networks for three cancer types with matched mRNA and protein profiling data from The Cancer Genome Atlas (TCGA) and the Clinical Proteomic Tumor Analysis Consortium (CPTAC). Our analyses revealed a marked difference in wiring between the mRNA and protein coexpression networks. Whereas protein coexpression was driven primarily by functional similarity between coexpressed genes, mRNA coexpression was driven by both cofunction and chromosomal colocalization of the genes. Functionally coherent mRNA modules were more likely to have their edges preserved in corresponding protein networks than functionally incoherent mRNA modules. Proteomic data strengthened the link between gene expression and function for at least 75% of Gene Ontology (GO) biological processes and 90% of KEGG pathways. A web application Gene2Net (http://cptac.gene2net.org) developed based on the three protein coexpression networks revealed novel gene-function relationships, such as linking ERBB2 (HER2) to lipid biosynthetic process in breast cancer, identifying PLG as a new gene involved in complement activation, and identifying AEBP1 as a new epithelial-mesenchymal transition (EMT) marker. Our results demonstrate that proteome profiling outperforms transcriptome profiling for coexpression based gene function prediction. Proteomics should be integrated if not preferred in gene function and human disease studies. © 2017 by

  17. Proteome Profiling Outperforms Transcriptome Profiling for Coexpression Based Gene Function Prediction*

    PubMed Central

    Wang, Jing; Ma, Zihao; Carr, Steven A.; Mertins, Philipp; Zhang, Hui; Zhang, Zhen; Chan, Daniel W.; Ellis, Matthew J. C.; Townsend, R. Reid; Smith, Richard D.; McDermott, Jason E.; Chen, Xian; Paulovich, Amanda G.; Boja, Emily S.; Mesri, Mehdi; Kinsinger, Christopher R.; Rodriguez, Henry; Rodland, Karin D.; Liebler, Daniel C.; Zhang, Bing

    2017-01-01

    Coexpression of mRNAs under multiple conditions is commonly used to infer cofunctionality of their gene products despite well-known limitations of this “guilt-by-association” (GBA) approach. Recent advancements in mass spectrometry-based proteomic technologies have enabled global expression profiling at the protein level; however, whether proteome profiling data can outperform transcriptome profiling data for coexpression based gene function prediction has not been systematically investigated. Here, we address this question by constructing and analyzing mRNA and protein coexpression networks for three cancer types with matched mRNA and protein profiling data from The Cancer Genome Atlas (TCGA) and the Clinical Proteomic Tumor Analysis Consortium (CPTAC). Our analyses revealed a marked difference in wiring between the mRNA and protein coexpression networks. Whereas protein coexpression was driven primarily by functional similarity between coexpressed genes, mRNA coexpression was driven by both cofunction and chromosomal colocalization of the genes. Functionally coherent mRNA modules were more likely to have their edges preserved in corresponding protein networks than functionally incoherent mRNA modules. Proteomic data strengthened the link between gene expression and function for at least 75% of Gene Ontology (GO) biological processes and 90% of KEGG pathways. A web application Gene2Net (http://cptac.gene2net.org) developed based on the three protein coexpression networks revealed novel gene-function relationships, such as linking ERBB2 (HER2) to lipid biosynthetic process in breast cancer, identifying PLG as a new gene involved in complement activation, and identifying AEBP1 as a new epithelial-mesenchymal transition (EMT) marker. Our results demonstrate that proteome profiling outperforms transcriptome profiling for coexpression based gene function prediction. Proteomics should be integrated if not preferred in gene function and human disease studies. PMID

  18. Proteome profiling of breast cancer biopsies reveals a wound healing signature of cancer-associated fibroblasts.

    PubMed

    Groessl, Michael; Slany, Astrid; Bileck, Andrea; Gloessmann, Kerstin; Kreutz, Dominique; Jaeger, Walter; Pfeiler, Georg; Gerner, Christopher

    2014-11-07

    Breast cancer is still the most common type of cancer in women; an important role in carcinogenesis is actually attributed to cancer-associated fibroblasts. In this study, we investigated whether it is possible to assess the functional state of cancer-associated fibroblasts through tumor tissue proteome profiling. Tissue proteomics was performed on tumor-central, tumor-near, and tumor-distant biopsy sections from breast adenocarcinoma patients, which allowed us to identify 2074 proteins. Data were interpreted referring to reference proteome profiles generated from primary human mammary fibroblasts comprising 4095 proteins. These cells were analyzed in quiescent cell state as well as after in vitro treatment with TGFβ or IL-1β, stimulating wound healing or inflammatory processes, respectively. Representative for cancer cells, we investigated the mammary carcinoma cell line ZR-75-1, identifying 5212 proteins. All mass analysis data have been made fully accessible via ProteomeXchange, DOI PXD001311 and PXD001323-8. Comparison of tissue proteomics data with all of those reference profiles revealed predominance of cancer cell-derived proteins within the tumor and fibroblast-derived proteins in the tumor-distant tissue sections. Remarkably, proteins characteristic for acute inflammation were hardly identified in the tissue samples. In contrast, several proteins found by us to be induced by TGFβ in mammary fibroblasts, including fibulin-5, SLC2A1, and MUC18, were positively identified in all tissue samples, with relatively higher abundance in tumor neighboring tissue sections. These findings indicate a predominance of cancer-associated fibroblasts with wound healing activities localized around tumors.

  19. Construction of protein profile classification model and screening of proteomic signature of acute leukemia

    PubMed Central

    Xu, Yun; Zhuo, Jiacai; Duan, Yonggang; Shi, Benhang; Chen, Xuhong; Zhang, Xiaoli; Xiao, Liang; Lou, Jin; Huang, Ruihong; Zhang, Qiongli; Du, Xin; Li, Ming; Wang, Daping; Shi, Dunyun

    2014-01-01

    The French-American-British (FAB) and WHO classifications provide important guidelines for the diagnosis, treatment, and prognostic prediction of acute leukemia, but are incapable of accurately differentiating all subtypes, and not well correlated with the clinical outcomes. In this study, we performed the protein profiling of the bone marrow mononuclear cells from the patients with acute leukemia and the health volunteers (control) by surface enhanced laser desorption/ionization-time of flight mass spectrometry (SELDI_TOF_MS). The patients with acute leukemia were analyzed as unitary by the profiling that were grouped into acute promyelocytic leukemia (APL), acute myeloid leukemia-granulocytic (AML-Gran), acute myeloid leukemia-monocytic (AML-Mon) acute lymphocytic leukemia (ALL), and control. Based on 109 proteomic signatures, the classification models of acute leukemia were constructed to screen the predictors by the improvement of the proteomic signatures and to detect their expression characteristics. According to the improvement and the expression characteristics of the predictors, the proteomic signatures (M3829, M1593, M2121, M2536, M1016) characterized successively each group (CON, APL, AML-Gra, AML-Mon, ALL) were screened as target molecules for identification. Meanwhile, the proteomic-based class of determinant samples could be made by the classification models. The credibility of the proteomic-based classification passed the evaluation of Biomarker Patterns Software 5.0 (BPS 5.0) scoring and validated application in clinical practice. The results suggested that the proteomic signatures characterized by different blasts were potential for developing new treatment and monitoring approaches of leukemia blasts. Moreover, the classification model was potential in serving as new diagnose approach of leukemia. PMID:25337199

  20. DeepPep: Deep proteome inference from peptide profiles

    PubMed Central

    2017-01-01

    Protein inference, the identification of the protein set that is the origin of a given peptide profile, is a fundamental challenge in proteomics. We present DeepPep, a deep-convolutional neural network framework that predicts the protein set from a proteomics mixture, given the sequence universe of possible proteins and a target peptide profile. In its core, DeepPep quantifies the change in probabilistic score of peptide-spectrum matches in the presence or absence of a specific protein, hence selecting as candidate proteins with the largest impact to the peptide profile. Application of the method across datasets argues for its competitive predictive ability (AUC of 0.80±0.18, AUPR of 0.84±0.28) in inferring proteins without need of peptide detectability on which the most competitive methods rely. We find that the convolutional neural network architecture outperforms the traditional artificial neural network architectures without convolution layers in protein inference. We expect that similar deep learning architectures that allow learning nonlinear patterns can be further extended to problems in metagenome profiling and cell type inference. The source code of DeepPep and the benchmark datasets used in this study are available at https://deeppep.github.io/DeepPep/. PMID:28873403

  1. Proteomic profiling of high risk medulloblastoma reveals functional biology

    PubMed Central

    Staal, Jerome A.; Lau, Ling San; Zhang, Huizhen; Ingram, Wendy J.; Hallahan, Andrew R.; Northcott, Paul A.; Pfister, Stefan M.; Wechsler-Reya, Robert J.; Rusert, Jessica M.; Taylor, Michael D.; Cho, Yoon-Jae; Packer, Roger J.; Brown, Kristy J.; Rood, Brian R.

    2015-01-01

    Genomic characterization of medulloblastoma has improved molecular risk classification but struggles to define functional biological processes, particularly for the most aggressive subgroups. We present here a novel proteomic approach to this problem using a reference library of stable isotope labeled medulloblastoma-specific proteins as a spike-in standard for accurate quantification of the tumor proteome. Utilizing high-resolution mass spectrometry, we quantified the tumor proteome of group 3 medulloblastoma cells and demonstrate that high-risk MYC amplified tumors can be segregated based on protein expression patterns. We cross-validated the differentially expressed protein candidates using an independent transcriptomic data set and further confirmed them in a separate cohort of medulloblastoma tissue samples to identify the most robust proteogenomic differences. Interestingly, highly expressed proteins associated with MYC-amplified tumors were significantly related to glycolytic metabolic pathways via alternative splicing of pyruvate kinase (PKM) by heterogeneous ribonucleoproteins (HNRNPs). Furthermore, when maintained under hypoxic conditions, these MYC-amplified tumors demonstrated increased viability compared to non-amplified tumors within the same subgroup. Taken together, these findings highlight the power of proteomics as an integrative platform to help prioritize genetic and molecular drivers of cancer biology and behavior. PMID:25970789

  2. Proteomic profiling of high risk medulloblastoma reveals functional biology.

    PubMed

    Staal, Jerome A; Lau, Ling San; Zhang, Huizhen; Ingram, Wendy J; Hallahan, Andrew R; Northcott, Paul A; Pfister, Stefan M; Wechsler-Reya, Robert J; Rusert, Jessica M; Taylor, Michael D; Cho, Yoon-Jae; Packer, Roger J; Brown, Kristy J; Rood, Brian R

    2015-06-10

    Genomic characterization of medulloblastoma has improved molecular risk classification but struggles to define functional biological processes, particularly for the most aggressive subgroups. We present here a novel proteomic approach to this problem using a reference library of stable isotope labeled medulloblastoma-specific proteins as a spike-in standard for accurate quantification of the tumor proteome. Utilizing high-resolution mass spectrometry, we quantified the tumor proteome of group 3 medulloblastoma cells and demonstrate that high-risk MYC amplified tumors can be segregated based on protein expression patterns. We cross-validated the differentially expressed protein candidates using an independent transcriptomic data set and further confirmed them in a separate cohort of medulloblastoma tissue samples to identify the most robust proteogenomic differences. Interestingly, highly expressed proteins associated with MYC-amplified tumors were significantly related to glycolytic metabolic pathways via alternative splicing of pyruvate kinase (PKM) by heterogeneous ribonucleoproteins (HNRNPs). Furthermore, when maintained under hypoxic conditions, these MYC-amplified tumors demonstrated increased viability compared to non-amplified tumors within the same subgroup. Taken together, these findings highlight the power of proteomics as an integrative platform to help prioritize genetic and molecular drivers of cancer biology and behavior.

  3. In-depth proteomic profiling of the uveal melanoma secretome

    PubMed Central

    Prendergast, Samuel; Simpson, Deborah; Hammond, Dean E.; Madigan, Michele C.; Beynon, Robert J.; Coupland, Sarah E.

    2016-01-01

    Uveal melanoma (UM), the most common primary intraocular tumour in adults, is characterised by a high frequency of metastases to the liver, typically with a fatal outcome. Proteins secreted from cancer cells (‘secretome’) are biologically important molecules thought to contribute to tumour progression. We examined the UM secretome by applying a label-free nanoLCMS/MS proteomic approach to profile proteins secreted into culture media by primary UM tumours with a high− (HR; n = 11) or low− (LR; n = 4) metastatic risk, compared to normal choroidal melanocytes (NCM) from unaffected post-mortem eyes. Across the three groups, 1843 proteins were identified at a 1% false discovery rate; 758 of these by at least 3 unique peptides, and quantified. The majority (539/758, 71%) of proteins were classified as secreted either by classical (144, 19%), non-classical (43, 6%) or exosomal (352, 46%) mechanisms. Bioinformatic analyzes showed that the secretome composition reflects biological differences and similarities of the samples. Ingenuity® pathway analysis of the secreted protein dataset identified abundant proteins involved in cell proliferation-, growth- and movement. Hepatic fibrosis/hepatic stellate cell activation and the mTORC1-S6K signalling axis were among the most differentially regulated biological processes in UM as compared with NCM. Further analysis of proteins upregulated ≥ 2 in HR-UM only, identified exosomal proteins involved in extracellular matrix remodelling and cancer cell migration/invasion; as well as classically secreted proteins, possibly representing novel biomarkers of metastatic disease. In conclusion, UM secretome analysis identifies novel proteins and pathways that may contribute to metastatic development at distant sites, particularly in the liver. PMID:27391064

  4. Chemical proteomics: terra incognita for novel drug target profiling

    PubMed Central

    Huang, Fuqiang; Zhang, Boya; Zhou, Shengtao; Zhao, Xia; Bian, Ce; Wei, Yuquan

    2012-01-01

    The growing demand for new therapeutic strategies in the medical and pharmaceutic fields has resulted in a pressing need for novel druggable targets. Paradoxically, however, the targets of certain drugs that are already widely used in clinical practice have largely not been annotated. Because the pharmacologic effects of a drug can only be appreciated when its interactions with cellular components are clearly delineated, an integrated deconvolution of drug-target interactions for each drug is necessary. The emerging field of chemical proteomics represents a powerful mass spectrometry (MS)-based affinity chromatography approach for identifying proteome-wide small molecule-protein interactions and mapping these interactions to signaling and metabolic pathways. This technique could comprehensively characterize drug targets, profile the toxicity of known drugs, and identify possible off-target activities. With the use of this technique, candidate drug molecules could be optimized, and predictable side effects might consequently be avoided. Herein, we provide a holistic overview of the major chemical proteomic approaches and highlight recent advances in this area as well as its potential applications in drug discovery. PMID:22640626

  5. High Resolution Quantitative Synaptic Proteome Profiling of Mouse Brain Regions After Auditory Discrimination Learning

    PubMed Central

    Kolodziej, Angela; Smalla, Karl-Heinz; Richter, Sandra; Engler, Alexander; Pielot, Rainer; Dieterich, Daniela C.; Tischmeyer, Wolfgang; Naumann, Michael; Kähne, Thilo

    2016-01-01

    The molecular synaptic mechanisms underlying auditory learning and memory remain largely unknown. Here, the workflow of a proteomic study on auditory discrimination learning in mice is described. In this learning paradigm, mice are trained in a shuttle box Go/NoGo-task to discriminate between rising and falling frequency-modulated tones in order to avoid a mild electric foot-shock. The protocol involves the enrichment of synaptosomes from four brain areas, namely the auditory cortex, frontal cortex, hippocampus, and striatum, at different stages of training. Synaptic protein expression patterns obtained from trained mice are compared to naïve controls using a proteomic approach. To achieve sufficient analytical depth, samples are fractionated in three different ways prior to mass spectrometry, namely 1D SDS-PAGE/in-gel digestion, in-solution digestion and phospho-peptide enrichment. High-resolution proteomic analysis on a mass spectrometer and label-free quantification are used to examine synaptic protein profiles in phospho-peptide-depleted and phospho-peptide-enriched fractions of synaptosomal protein samples. A commercial software package is utilized to reveal proteins and phospho-peptides with significantly regulated relative synaptic abundance levels (trained/naïve controls). Common and differential regulation modes for the synaptic proteome in the investigated brain regions of mice after training were observed. Subsequently, meta-analyses utilizing several databases are employed to identify underlying cellular functions and biological pathways. PMID:28060347

  6. High Resolution Quantitative Synaptic Proteome Profiling of Mouse Brain Regions After Auditory Discrimination Learning.

    PubMed

    Kolodziej, Angela; Smalla, Karl-Heinz; Richter, Sandra; Engler, Alexander; Pielot, Rainer; Dieterich, Daniela C; Tischmeyer, Wolfgang; Naumann, Michael; Kähne, Thilo

    2016-12-15

    The molecular synaptic mechanisms underlying auditory learning and memory remain largely unknown. Here, the workflow of a proteomic study on auditory discrimination learning in mice is described. In this learning paradigm, mice are trained in a shuttle box Go/NoGo-task to discriminate between rising and falling frequency-modulated tones in order to avoid a mild electric foot-shock. The protocol involves the enrichment of synaptosomes from four brain areas, namely the auditory cortex, frontal cortex, hippocampus, and striatum, at different stages of training. Synaptic protein expression patterns obtained from trained mice are compared to naïve controls using a proteomic approach. To achieve sufficient analytical depth, samples are fractionated in three different ways prior to mass spectrometry, namely 1D SDS-PAGE/in-gel digestion, in-solution digestion and phospho-peptide enrichment. High-resolution proteomic analysis on a mass spectrometer and label-free quantification are used to examine synaptic protein profiles in phospho-peptide-depleted and phospho-peptide-enriched fractions of synaptosomal protein samples. A commercial software package is utilized to reveal proteins and phospho-peptides with significantly regulated relative synaptic abundance levels (trained/naïve controls). Common and differential regulation modes for the synaptic proteome in the investigated brain regions of mice after training were observed. Subsequently, meta-analyses utilizing several databases are employed to identify underlying cellular functions and biological pathways.

  7. Urine sample preparation in 96-well filter plates for quantitative clinical proteomics.

    PubMed

    Yu, Yanbao; Suh, Moo-Jin; Sikorski, Patricia; Kwon, Keehwan; Nelson, Karen E; Pieper, Rembert

    2014-06-03

    Urine is an important, noninvasively collected body fluid source for the diagnosis and prognosis of human diseases. Liquid chromatography mass spectrometry (LC-MS) based shotgun proteomics has evolved as a sensitive and informative technique to discover candidate disease biomarkers from urine specimens. Filter-aided sample preparation (FASP) generates peptide samples from protein mixtures of cell lysate or body fluid origin. Here, we describe a FASP method adapted to 96-well filter plates, named 96FASP. Soluble urine concentrates containing ~10 μg of total protein were processed by 96FASP and LC-MS resulting in 700-900 protein identifications at a 1% false discovery rate (FDR). The experimental repeatability, as assessed by label-free quantification and Pearson correlation analysis for shared proteins among replicates, was high (R ≥ 0.97). Application to urinary pellet lysates which is of particular interest in the context of urinary tract infection analysis was also demonstrated. On average, 1700 proteins (±398) were identified in five experiments. In a pilot study using 96FASP for analysis of eight soluble urine samples, we demonstrated that protein profiles of technical replicates invariably clustered; the protein profiles for distinct urine donors were very different from each other. Robust, highly parallel methods to generate peptide mixtures from urine and other body fluids are critical to increase cost-effectiveness in clinical proteomics projects. This 96FASP method has potential to become a gold standard for high-throughput quantitative clinical proteomics.

  8. Urine Sample Preparation in 96-Well Filter Plates for Quantitative Clinical Proteomics

    PubMed Central

    2015-01-01

    Urine is an important, noninvasively collected body fluid source for the diagnosis and prognosis of human diseases. Liquid chromatography mass spectrometry (LC-MS) based shotgun proteomics has evolved as a sensitive and informative technique to discover candidate disease biomarkers from urine specimens. Filter-aided sample preparation (FASP) generates peptide samples from protein mixtures of cell lysate or body fluid origin. Here, we describe a FASP method adapted to 96-well filter plates, named 96FASP. Soluble urine concentrates containing ∼10 μg of total protein were processed by 96FASP and LC-MS resulting in 700–900 protein identifications at a 1% false discovery rate (FDR). The experimental repeatability, as assessed by label-free quantification and Pearson correlation analysis for shared proteins among replicates, was high (R ≥ 0.97). Application to urinary pellet lysates which is of particular interest in the context of urinary tract infection analysis was also demonstrated. On average, 1700 proteins (±398) were identified in five experiments. In a pilot study using 96FASP for analysis of eight soluble urine samples, we demonstrated that protein profiles of technical replicates invariably clustered; the protein profiles for distinct urine donors were very different from each other. Robust, highly parallel methods to generate peptide mixtures from urine and other body fluids are critical to increase cost-effectiveness in clinical proteomics projects. This 96FASP method has potential to become a gold standard for high-throughput quantitative clinical proteomics. PMID:24797144

  9. Liquid MALDI MS Analysis of Complex Peptide and Proteome Samples.

    PubMed

    Wiangnon, Kanjana; Cramer, Rainer

    2016-09-02

    Matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) is well-known to be a powerful technique for the analysis of biological samples. By using glycerol-based liquid support matrices (LSMs) instead of conventional MALDI matrices the power of this technique can be extended further. In this study, we exploited LSMs for the identification of complex samples, that is, the Lactobacillus proteome and a bovine serum albumin (BSA) digest. Liquid and solid MALDI samples were manually and robotically prepared by coupling a nanoflow high-performance liquid chromatography (nanoHPLC) system to an automated MALDI sample spotting device. MS and MS/MS data were successfully acquired at the femtomole level using TOF/TOF as well as Q-TOF instrumentation and used for protein identification searching sequence databases. For the BSA digest analysis, liquid MALDI samples resulted in peptide mass fingerprints, which led to a higher confidence in protein identification compared with solid (crystalline) MALDI samples; however, postsource decay (PSD) MS/MS analysis of both the proteome of Lactobacillus plantarum WCFS1 cells and BSA digest showed that further optimization of the formation and detection of peptide fragment ions is still needed for liquid MALDI samples, as the MS/MS ion search score was lower than that for the solid MALDI samples, reflecting the poorer quality of the liquid MALDI-PSD spectra, which can be attributed to the differences in PSD parameters and their optimization that is currently achievable.

  10. Proteomic Profiling Of Two-Dimensional Gel Electrophoresis Protein Expression Data

    NASA Astrophysics Data System (ADS)

    Ahmad, Norhaiza; Zhang, J.; Brown, P. J.; James, D. C.; Birch, J. R.; Racher, A. J.; Smales, C. M.

    2008-01-01

    We have undertaken two-dimensional gel electrophoresis (2-DE) proteomic profiling on a series of cell lines with different recombinant antibody production rates. Due to the nature of 2-DE proteomic investigations there will always be `process variability' factors in any data set collected in this way. Some of this variation will arise during sample preparation, gel running and staining, while further variation will arise from the gel analysis procedure. Therefore, in order to identify all significant changes in protein expression between biological samples when analysed by 2-DE, the system precision or `error', and how this correlates to protein abundance, must be known. Only then can the system be considered robust and investigators accurately and confidently report all observable statistically significant changes in protein expression. We introduce an expression variability test to identify protein spots whose expression correlates with increased antibody production. The results have highlighted a small number of candidate proteins for further investigation.

  11. Proteomic profiling change during the early development of silicosis disease

    PubMed Central

    Miao, Rongming; Ding, Bangmei; Zhang, Yingyi; Xia, Qian; Li, Yong

    2016-01-01

    Background Silicosis is one of several severe occupational diseases for which effective diagnostic tools during early development are currently unavailable. In this study we focused on proteomic profiling during the early stages of silicosis to investigate the pathophysiology and identify the proteins involved. Methods Two-dimensional (2D) gel electrophoresis and MALDI-TOF-MS were used to assess the proteomic differences between healthy individuals (HI), dust-exposed workers without silicosis (DEW) and silicosis patients (SP). Proteins abundances that differed by a factor of two-fold or greater were subjected to more detailed analysis, and enzyme linked to immunosorbent assay (ELISA) was employed to correlate with protein expression data. Results Compared with HI, 42 proteins were more abundant and 8 were less abundant in DEW, and these were also differentially accumulated in SP. Closer inspection revealed that serine protease granzyme A, alpha-1-B-glycoprotein (A1BG) and the T4 surface glycoprotein precursor (TSGP) were among the up-regulated proteins in DEW and SP. Significant changes in serine proteases, glycoproteins and proto-oncogenes may be associated with the response to cytotoxicity and infectious pathogens by activation of T cells, positive regulation of extracellular matrix structural constituents and immune response, and fibroblast proliferation. Up-regulation of cytokines included TNFs, interferon beta precursor, interleukin 6, atypical chemokine receptor 2, TNFR13BV, and mutant IL-17F may be involved in the increased and persistent immune response and fibrosis that occurred during silicosis development. Conclusions Granzymes, glycoproteins, cytokines and immune factors were dramatically involved in the immune response, metabolism, signal regulation and fibrosis during the early development of silicosis. Proteomic profiling has expanded our understanding of the pathogenesis of silicosis, and identified a number of targets that may be potential

  12. Proteomic profiling change during the early development of silicosis disease.

    PubMed

    Miao, Rongming; Ding, Bangmei; Zhang, Yingyi; Xia, Qian; Li, Yong; Zhu, Baoli

    2016-03-01

    Silicosis is one of several severe occupational diseases for which effective diagnostic tools during early development are currently unavailable. In this study we focused on proteomic profiling during the early stages of silicosis to investigate the pathophysiology and identify the proteins involved. Two-dimensional (2D) gel electrophoresis and MALDI-TOF-MS were used to assess the proteomic differences between healthy individuals (HI), dust-exposed workers without silicosis (DEW) and silicosis patients (SP). Proteins abundances that differed by a factor of two-fold or greater were subjected to more detailed analysis, and enzyme linked to immunosorbent assay (ELISA) was employed to correlate with protein expression data. Compared with HI, 42 proteins were more abundant and 8 were less abundant in DEW, and these were also differentially accumulated in SP. Closer inspection revealed that serine protease granzyme A, alpha-1-B-glycoprotein (A1BG) and the T4 surface glycoprotein precursor (TSGP) were among the up-regulated proteins in DEW and SP. Significant changes in serine proteases, glycoproteins and proto-oncogenes may be associated with the response to cytotoxicity and infectious pathogens by activation of T cells, positive regulation of extracellular matrix structural constituents and immune response, and fibroblast proliferation. Up-regulation of cytokines included TNFs, interferon beta precursor, interleukin 6, atypical chemokine receptor 2, TNFR13BV, and mutant IL-17F may be involved in the increased and persistent immune response and fibrosis that occurred during silicosis development. Granzymes, glycoproteins, cytokines and immune factors were dramatically involved in the immune response, metabolism, signal regulation and fibrosis during the early development of silicosis. Proteomic profiling has expanded our understanding of the pathogenesis of silicosis, and identified a number of targets that may be potential biomarkers for early diagnosis of this

  13. Quantitative reactivity profiling predicts functional cysteines in proteomes

    PubMed Central

    Weerapana, Eranthie; Wang, Chu; Simon, Gabriel M.; Richter, Florian; Khare, Sagar; Dillon, Myles B.D.; Bachovchin, Daniel A.; Mowen, Kerri; Baker, David; Cravatt, Benjamin F.

    2010-01-01

    Cysteine is the most intrinsically nucleophilic amino acid in proteins, where its reactivity is tuned to perform diverse biochemical functions. The absence of a consensus sequence that defines functional cysteines in proteins has hindered their discovery and characterization. Here, we describe a proteomics method to quantitatively profile the intrinsic reactivity of cysteine residues en masse directly in native biological systems. Hyperreactivity was a rare feature among cysteines and found to specify a wide range of activities, including nucleophilic and reductive catalysis and sites of oxidative modification. Hyperreactive cysteines were identified in several proteins of uncharacterized function, including a residue conserved across eukaryotic phylogeny that we show is required for yeast viability and involved in iron-sulfur protein biogenesis. Finally, we demonstrate that quantitative reactivity profiling can also form the basis for screening and functional assignment of cysteines in computationally designed proteins, where it discriminated catalytically active from inactive cysteine hydrolase designs. PMID:21085121

  14. Proteomic Profiling of Human Liver Biopsies: Hepatitis C Virus-Induced Fibrosis and Mitochondrial Dysfunction

    SciTech Connect

    Diamond, Deborah L.; Jacobs, Jon M.; Paeper, Bryan; Proll, Sean; Gritsenko, Marina A.; Carithers, Jr., Robert L.; Larson , Anne M.; Yeh, Matthew M.; Camp, David G.; Smith, Richard D.; Katze, Michael G.

    2007-09-01

    Liver biopsies from HCV-infected patients offer the unique opportunity to study human liver biology and disease in vivo. However, the low protein yields associated with these small samples present a significant challenge for proteomic analysis. In this study we describe the application of an ultra-sensitive proteomics platform for performing robust quantitative proteomic studies on microgram amounts of HCV-infected human liver tissue from 15 patients at different stages of fibrosis. A high quality liver protein data base containing 5,920 unique protein identifications supported high throughput quantitative studies using 16O:18O stable isotope labeling in combination with the accurate mass and time (AMT) tag approach. A total of 1,641 liver biopsy proteins were quantified and ANOVA identified 210 proteins exhibiting statistically significant differences associated with fibrosis stage. Hierarchical clustering revealed that biopsies representative of later fibrosis stages (e.g. Batts-Ludwig stages 3-4) exhibited a distinct protein expression profile indicating an apparent down-regulation of many proteins when compared to samples from earlier fibrosis stages (e.g. Batts-Ludwig stages 0-2). Functional analysis of these signature proteins suggests that impairment of key mitochondrial processes including fatty acid oxidation and oxidative phosphorylation, and response to oxidative stress and reactive oxygen species occurs during advanced stage 3-4 fibrosis. In conclusion, the results reported here represent a significant advancement in clinical proteomics providing to our knowledge, the first demonstration of global proteomic alterations accompanying liver disease progression in patients chronically infected with HCV. Our findings contribute to a generally emerging theme associating oxidative stress and hepatic mitochondrial dysfunction with HCV pathogenesis.

  15. Proteomic profile response of Paracoccidioides lutzii to the antifungal argentilactone.

    PubMed

    Prado, Renata S; Bailão, Alexandre M; Silva, Lívia C; de Oliveira, Cecília M A; Marques, Monique F; Silva, Luciano P; Silveira-Lacerda, Elisângela P; Lima, Aliny P; Soares, Célia M; Pereira, Maristela

    2015-01-01

    The dimorphic fungi Paracoccidioides spp. are the etiological agents of paracoccidioidomycosis (PCM), a mycosis of high incidence in Brazil. The toxicity of drug treatment and the emergence of resistant organisms have led to research for new candidates for drugs. In this study, we demonstrate that the natural product argentilactone was not cytotoxic or genotoxic to MRC5 cells at the IC50 concentration to the fungus. We also verified the proteomic profile of Paracoccidioides lutzii after incubation with argentilactone using a label free quantitative proteome nanoUPLC-MS(E). The results of this study indicated that the fungus has a global metabolic adaptation in the presence of argentilactone. Enzymes of important pathways, such as glycolysis, the Krebs cycle and the glyoxylate cycle, were repressed, which drove the metabolism to the methylcytrate cycle and beta-oxidation. Proteins involved in cell rescue, defense and stress response were induced. In this study, alternative metabolic pathways adopted by the fungi were elucidated, helping to elucidate the course of action of the compound studied.

  16. Proteomic profile response of Paracoccidioides lutzii to the antifungal argentilactone

    PubMed Central

    Prado, Renata S.; Bailão, Alexandre M.; Silva, Lívia C.; de Oliveira, Cecília M. A.; Marques, Monique F.; Silva, Luciano P.; Silveira-Lacerda, Elisângela P.; Lima, Aliny P.; Soares, Célia M.; Pereira, Maristela

    2015-01-01

    The dimorphic fungi Paracoccidioides spp. are the etiological agents of paracoccidioidomycosis (PCM), a mycosis of high incidence in Brazil. The toxicity of drug treatment and the emergence of resistant organisms have led to research for new candidates for drugs. In this study, we demonstrate that the natural product argentilactone was not cytotoxic or genotoxic to MRC5 cells at the IC50 concentration to the fungus. We also verified the proteomic profile of Paracoccidioides lutzii after incubation with argentilactone using a label free quantitative proteome nanoUPLC-MSE. The results of this study indicated that the fungus has a global metabolic adaptation in the presence of argentilactone. Enzymes of important pathways, such as glycolysis, the Krebs cycle and the glyoxylate cycle, were repressed, which drove the metabolism to the methylcytrate cycle and beta-oxidation. Proteins involved in cell rescue, defense and stress response were induced. In this study, alternative metabolic pathways adopted by the fungi were elucidated, helping to elucidate the course of action of the compound studied. PMID:26150808

  17. Proteomic profiling reveals that collismycin A is an iron chelator

    PubMed Central

    Kawatani, Makoto; Muroi, Makoto; Wada, Akira; Inoue, Gyo; Futamura, Yushi; Aono, Harumi; Shimizu, Kenshirou; Shimizu, Takeshi; Igarashi, Yasuhiro; Takahashi-Ando, Naoko; Osada, Hiroyuki

    2016-01-01

    Collismycin A (CMA), a microbial product, has anti-proliferative activity against cancer cells, but the mechanism of its action remains unknown. Here, we report the identification of the molecular target of CMA by ChemProteoBase, a proteome-based approach for drug target identification. ChemProteoBase profiling showed that CMA is closely clustered with di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone, an iron chelator. CMA bound to both Fe(II) and Fe(III) ions and formed a 2:1 chelator-iron complex with a redox-inactive center. CMA-induced cell growth inhibition was completely canceled by Fe(II) and Fe(III) ions, but not by other metal ions such as Zn(II) or Cu(II). Proteomic and transcriptomic analyses showed that CMA affects the glycolytic pathway due to the accumulation of HIF-1α. These results suggest that CMA acts as a specific iron chelator, leading to the inhibition of cancer cell growth. PMID:27922079

  18. Proteomic profile of mouse fibroblasts exposed to pure magnesium extract.

    PubMed

    Zhen, Zhen; Luthringer, Bérengère; Yang, Li; Xi, Tingfei; Zheng, Yufeng; Feyerabend, Frank; Willumeit, Regine; Lai, Chen; Ge, Zigang

    2016-12-01

    Magnesium and its alloys gain wide attention as degradable biomaterials. In order to reveal the molecular mechanism of the influence of biodegradable magnesium on cells, proteomics analysis was performed in this work. After mouse fibroblasts (L929) were cultured with or without Mg degradation products (Mg-extract) for 8, 24, and 48h, changes in protein expression profiles were obtained using isobaric tags for relative and absolute quantitation (iTRAQ) coupled two dimensional liquid chromatography-tandem mass spectrometry (2D LC MS/MS). A total of 867 proteins were identified (relying on at least two peptides). Compared to the control group, 205, 282, and 217 regulated proteins were identified at 8, 24, and 48h, respectively. 65 common proteins were up or down- regulated within all the three time points, which were involved in various physiological and metabolic activities. Consistent with viability, proliferation, and cell cycle analysis, stimulated energy metabolism as well as protein synthesis pathways were discussed, indicating a possible effect of Mg-extract on L929 proliferation. Furthermore, endocytosis and focal adhesion processes were also discussed. This proteomics study uncovers early cellular mechanisms triggered by Mg degradation products and highlights the cytocompatibility of biodegradable metallic materials for biomedical applications such as stents or orthopaedic implants. Copyright © 2016. Published by Elsevier B.V.

  19. A framework for personalized medicine: prediction of drug sensitivity in cancer by proteomic profiling

    PubMed Central

    2012-01-01

    Background The goal of personalized medicine is to provide patients optimal drug screening and treatment based on individual genomic or proteomic profiles. Reverse-Phase Protein Array (RPPA) technology offers proteomic information of cancer patients which may be directly related to drug sensitivity. For cancer patients with different drug sensitivity, the proteomic profiling reveals important pathophysiologic information which can be used to predict chemotherapy responses. Results The goal of this paper is to present a framework for personalized medicine using both RPPA and drug sensitivity (drug resistance or intolerance). In the proposed personalized medicine system, the prediction of drug sensitivity is obtained by a proposed augmented naive Bayesian classifier (ANBC) whose edges between attributes are augmented in the network structure of naive Bayesian classifier. For discriminative structure learning of ANBC, local classification rate (LCR) is used to score augmented edges, and greedy search algorithm is used to find the discriminative structure that maximizes classification rate (CR). Once a classifier is trained by RPPA and drug sensitivity using cancer patient samples, the classifier is able to predict the drug sensitivity given RPPA information from a patient. Conclusion In this paper we proposed a framework for personalized medicine where a patient is profiled by RPPA and drug sensitivity is predicted by ANBC and LCR. Experimental results with lung cancer data demonstrate that RPPA can be used to profile patients for drug sensitivity prediction by Bayesian network classifier, and the proposed ANBC for personalized cancer medicine achieves better prediction accuracy than naive Bayes classifier in small sample size data on average and outperforms other the state-of-the-art classifier methods in terms of classification accuracy. PMID:22759571

  20. Analysis of Biostimulated Microbial Communities from Two Field Experiments Reveals Temporal and Spatial Differences in Proteome Profiles

    SciTech Connect

    Callister, Stephen J; Wilkins, Mike; Nicora, Carrie D.; Williams, Ken; Banfield, Jillian F.; Verberkmoes, Nathan C; Hettich, Robert {Bob} L; N'Guessan, A. Lucie; Mouser, Paula J; Elifantz, Hila; Smith, Richard D.; Lovley, Derek; Lipton, Mary S; Long, Phil

    2010-01-01

    Stimulated by an acetate-amendment field experiment conducted in 2007, anaerobic microbial populations in the aquifer at the Rifle Integrated Field Research Challenge site in Colorado reduced mobile U(VI) to insoluble U(IV). During this experiment, planktonic biomass was sampled at various time points to quantitatively evaluate proteomes. In 2008, an acetateamended field experiment was again conducted in a similar manner to the 2007 experiment. As there was no comprehensive metagenome sequence available for use in proteomics analysis, we systematically evaluated 12 different organism genome sequences to generate sets of aggregate genomes, or pseudo-metagenomes , for supplying relative quantitative peptide and protein identifications. Proteomics results support previous observations of the dominance of Geobacteraceae during biostimulation using acetate as sole electron donor, and revealed a shift from an early stage of iron reduction to a late stage of iron reduction. Additionally,ashift from iron reduction to sulfate reduction was indicated by changes in the contribution of proteome information contributed by different organism genome sequences within the aggregate set. In addition, the comparison of proteome measurements made between the 2007 field experiment and 2008 field experiment revealed differences in proteome profiles. These differences may be the result of alterations in abundance and population structure within the planktonic biomass samples collected for analysis.

  1. Analysis of biostimulated microbial communities from two field experiments reveals temporal and spatial differences in proteome profiles

    SciTech Connect

    Callister, S.J.; Wilkins, M.J.; Nicora, C.D.; Williams, K.H.; Banfield, J.F.; VerBerkmoes, N.C.; Hettich, R.L.; NGuessan, A.L.; Mouser, P.J.; Elifantz, H.; Smith, R.D.; Lovley, D.R.; Lipton, M.S.; Long, P.E.

    2010-07-15

    Stimulated by an acetate-amendment field experiment conducted in 2007, anaerobic microbial populations in the aquifer at the Rifle Integrated Field Research Challenge site in Colorado reduced mobile U(VI) to insoluble U(IV). During this experiment, planktonic biomass was sampled at various time points to quantitatively evaluate proteomes. In 2008, an acetate-amended field experiment was again conducted in a similar manner to the 2007 experiment. As there was no comprehensive metagenome sequence available for use in proteomics analysis, we systematically evaluated 12 different organism genome sequences to generate sets of aggregate genomes, or “pseudo-metagenomes”, for supplying relative quantitative peptide and protein identifications. Proteomics results support previous observations of the dominance of Geobacteraceae during biostimulation using acetate as sole electron donor, and revealed a shift from an early stage of iron reduction to a late stage of iron reduction. Additionally, a shift from iron reduction to sulfate reduction was indicated by changes in the contribution of proteome information contributed by different organism genome sequences within the aggregate set. In addition, the comparison of proteome measurements made between the 2007 field experiment and 2008 field experiment revealed differences in proteome profiles. These differences may be the result of alterations in abundance and population structure within the planktonic biomass samples collected for analysis.

  2. Urinary proteomic profiling in severe obesity and obstructive sleep apnoea with CPAP treatment.

    PubMed

    Seetho, Ian W; Ramírez-Torres, Adela; Albalat, Amaya; Mullen, William; Mischak, Harald; Parker, Robert J; Craig, Sonya; Duffy, Nick; Hardy, Kevin J; Burniston, Jatin G; Wilding, John Ph

    2015-01-01

    Obstructive sleep apnoea (OSA) is common in obesity and is associated with cardiovascular and metabolic complications. Continuous positive airway pressure (CPAP) in OSA may lead to physiological changes reflected in the urinary proteome. The aim of this study was to characterise the urinary proteome in severely obese adult subjects with OSA who were receiving CPAP compared with severely obese subjects without OSA. Severely obese subjects with and without OSA were recruited. Subjects with OSA were receiving CPAP. Body composition and blood pressure measurements were recorded. Urinary samples were analysed by Capillary Electrophoresis-Mass Spectrometry (CE-MS). Twenty-seven subjects with OSA-on-CPAP (age 49±7years, BMI 43±7 kg/m(2)) and 25 controls without OSA (age 52±9years, BMI 39±4 kg/m(2)) were studied. Age and BMI were not significantly different between groups. Mean CPAP use for OSA patients was 14.5±1.0 months. Metabolic syndrome was present in 14(52%) of those with OSA compared with 6(24%) of controls (p=0.039). A urinary proteome comprising 15 peptides was identified showing differential expression between the groups (p<0.01). Although correction for multiple testing did not reach significance, sequences were determined for 8 peptides demonstrating origins from collagens, fibrinogen beta chain and T-cadherin that may be associated with underlying cardiovascular disease mechanisms in OSA. The urinary proteome is compared in OSA with CPAP and without OSA in severe obesity. The effects of CPAP on OSA may lead to changes in the urinary peptides but further research work is needed to investigate the potential role for urinary proteomics in characterising urinary peptide profiles in OSA.

  3. Systems-wide temporal proteomic profiling in glucose-starved Bacillus subtilis

    PubMed Central

    Otto, Andreas; Bernhardt, Jörg; Meyer, Hanna; Schaffer, Marc; Herbst, Florian-A.; Siebourg, Juliane; Mäder, Ulrike; Lalk, Michael; Hecker, Michael; Becher, Dörte

    2010-01-01

    Functional genomics of the Gram-positive model organism Bacillus subtilis reveals valuable insights into basic concepts of cell physiology. In this study, we monitor temporal changes in the proteome, transcriptome and extracellular metabolome of B. subtilis caused by glucose starvation. For proteomic profiling, a combination of in vivo metabolic labelling and shotgun mass spectrometric analysis was carried out for five different proteomic subfractions (cytosolic, integral membrane, membrane, surface and extracellular proteome fraction), leading to the identification of ∼52% of the predicted proteome of B. subtilis. Quantitative proteomic and corresponding transcriptomic data were analysed with Voronoi treemaps linking functional classification and relative expression changes of gene products according to their fate in the stationary phase. The obtained data comprise the first comprehensive profiling of changes in the membrane subfraction and allow in-depth analysis of major physiological processes, including monitoring of protein degradation. PMID:21266987

  4. Proteomic analysis of hair shafts from monozygotic twins: Expression profiles and genetically variant peptides.

    PubMed

    Wu, Pei-Wen; Mason, Katelyn E; Durbin-Johnson, Blythe P; Salemi, Michelle; Phinney, Brett S; Rocke, David M; Parker, Glendon J; Rice, Robert H

    2017-07-01

    Forensic association of hair shaft evidence with individuals is currently assessed by comparing mitochondrial DNA haplotypes of reference and casework samples, primarily for exclusionary purposes. Present work tests and validates more recent proteomic approaches to extract quantitative transcriptional and genetic information from hair samples of monozygotic twin pairs, which would be predicted to partition away from unrelated individuals if the datasets contain identifying information. Protein expression profiles and polymorphic, genetically variant hair peptides were generated from ten pairs of monozygotic twins. Profiling using the protein tryptic digests revealed that samples from identical twins had typically an order of magnitude fewer protein expression differences than unrelated individuals. The data did not indicate that the degree of difference within twin pairs increased with age. In parallel, data from the digests were used to detect genetically variant peptides that result from common nonsynonymous single nucleotide polymorphisms in genes expressed in the hair follicle. Compilation of the variants permitted sorting of the samples by hierarchical clustering, permitting accurate matching of twin pairs. The results demonstrate that genetic differences are detectable by proteomic methods and provide a framework for developing quantitative statistical estimates of personal identification that increase the value of hair shaft evidence. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. Large-Scale and Deep Quantitative Proteome Profiling Using Isobaric Labeling Coupled with Two-Dimensional LC-MS/MS

    SciTech Connect

    Gritsenko, Marina A.; Xu, Zhe; Liu, Tao; Smith, Richard D.

    2016-02-12

    Comprehensive, quantitative information on abundances of proteins and their post-translational modifications (PTMs) can potentially provide novel biological insights into diseases pathogenesis and therapeutic intervention. Herein, we introduce a quantitative strategy utilizing isobaric stable isotope-labelling techniques combined with two-dimensional liquid chromatography-tandem mass spectrometry (2D-LC-MS/MS) for large-scale, deep quantitative proteome profiling of biological samples or clinical specimens such as tumor tissues. The workflow includes isobaric labeling of tryptic peptides for multiplexed and accurate quantitative analysis, basic reversed-phase LC fractionation and concatenation for reduced sample complexity, and nano-LC coupled to high resolution and high mass accuracy MS analysis for high confidence identification and quantification of proteins. This proteomic analysis strategy has been successfully applied for in-depth quantitative proteomic analysis of tumor samples, and can also be used for integrated proteome and PTM characterization, as well as comprehensive quantitative proteomic analysis across samples from large clinical cohorts.

  6. A Method for Microalgae Proteomics Analysis Based on Modified Filter-Aided Sample Preparation.

    PubMed

    Li, Song; Cao, Xupeng; Wang, Yan; Zhu, Zhen; Zhang, Haowei; Xue, Song; Tian, Jing

    2017-04-11

    With the fast development of microalgal biofuel researches, the proteomics studies of microalgae increased quickly. A filter-aided sample preparation (FASP) method is widely used proteomics sample preparation method since 2009. Here, a method of microalgae proteomics analysis based on modified filter-aided sample preparation (mFASP) was described to meet the characteristics of microalgae cells and eliminate the error caused by over-alkylation. Using Chlamydomonas reinhardtii as the model, the prepared sample was tested by standard LC-MS/MS and compared with the previous reports. The results showed mFASP is suitable for most of occasions of microalgae proteomics studies.

  7. [Direct proteome profiling of human blood serum in the experiment with 5-day dry immersion].

    PubMed

    Pastushkova, L Kh; Pakharukova, N A; Trifonova, O P; Dobrokhotov, I V; Valeeva, O A; Larina, I M

    2011-01-01

    Purpose of the investigation was to determine changes in blood plasma proteome in healthy human subjects (n = 14, 19 to 26 y.o.) in an experiment with dry immersion (DI). Plasma samples were drawn 7 and 2 days before the exposure, on DI days 2, 3 and 5, and on days 1, 3, 7 and 15 after the experiment. Previous to direct MALDI-TOF mass-spectrometric profiling, serum samples were pre-fractionated and enriched with magnetic particles MB WCX (WCX--a weak cation exchanger) on ClinProt (Bruker Daltonics). In each spectrum, 175 MS-peaks were detected on average within the mass range from 1000 to 17,000 Da with the signal/noise ratio = 5. Student's criterion (p < 0.05) was used to define reliable differences between DI and baseline samples from 48 peaks (27.4 % of all the proteome profile peaks). On DI days 2 and 3, growth of peak areas was observed in fragments of complement system proteins C3 and C4, high-molecular kininogen and fibrinogen that can be attributed to organism adaptation to conditions of the experiment. Significant increases of the peak area of apolipoprotein CI (reduced form with segregated threonine and proline) and C4 enzymes of the complement system, and fibrinogen on the first day after the experiment can be related to changes in motor activities of the subjects.

  8. Current clinical application of genomic and proteomic profiling in non-small-cell lung cancer.

    PubMed

    Tanvetyanon, Tawee; Creelan, Benjamin C; Chiappori, Alberto A

    2014-01-01

    Genomic or proteomic profiling of cancer can be broadly defined as a systematic grouping of cancer based on its genetic or protein makeup. In the management of non-small-cell lung cancer (NSCLC), genomic and proteomic profiling applications have become useful in early disease detection, diagnosis, treatment, and prognostication. We reviewed the recent literature on the applications of genomic and proteomic profiling in NSCLC. Important applications were summarized into those already adopted as standard care and those still under investigation. For genomic profiling, testing for EGFR mutation and ALK rearrangement has become routine for adenocarcinoma. Multiplex assay and malignancy-risk gene signature are both important applications in development. A test to predict outcome after treatment with an epidermal growth factor rector/tyrosine kinase inhibitor and a screening blood test for lung cancer are being investigated for use in proteomic profiling. Genomic profiling is routine in patients with NSCLC, and proteomic profiling shows promise. Additional genomic and proteomic profiling applications may also prove to be useful contributions in the care of these patients.

  9. Proteomic profiling of developing cotton fibers from wild and domesticated Gossypium barbadense.

    PubMed

    Hu, Guanjing; Koh, Jin; Yoo, Mi-Jeong; Grupp, Kara; Chen, Sixue; Wendel, Jonathan F

    2013-10-01

    Pima cotton (Gossypium barbadense) is widely cultivated because of its long, strong seed trichomes ('fibers') used for premium textiles. These agronomically advanced fibers were derived following domestication and thousands of years of human-mediated crop improvement. To gain an insight into fiber development and evolution, we conducted comparative proteomic and transcriptomic profiling of developing fiber from an elite cultivar and a wild accession. Analyses using isobaric tag for relative and absolute quantification (iTRAQ) LC-MS/MS technology identified 1317 proteins in fiber. Of these, 205 were differentially expressed across developmental stages, and 190 showed differential expression between wild and cultivated forms, 14.4% of the proteome sampled. Human selection may have shifted the timing of developmental modules, such that some occur earlier in domesticated than in wild cotton. A novel approach was used to detect possible biased expression of homoeologous copies of proteins. Results indicate a significant partitioning of duplicate gene expression at the protein level, but an approximately equal degree of bias for each of the two constituent genomes of allopolyploid cotton. Our results demonstrate the power of complementary transcriptomic and proteomic approaches for the study of the domestication process. They also provide a rich database for mining for functional analyses of cotton improvement or evolution.

  10. Proteomic Profiling of Mycobacterium tuberculosis Identifies Nutrient-starvation-responsive Toxin–antitoxin Systems*

    PubMed Central

    Albrethsen, Jakob; Agner, Jeppe; Piersma, Sander R.; Højrup, Peter; Pham, Thang V.; Weldingh, Karin; Jimenez, Connie R.; Andersen, Peter; Rosenkrands, Ida

    2013-01-01

    In order to successfully enter the latent stage, Mycobacterium tuberculosis must adapt to conditions such as nutrient limitation and hypoxia. In vitro models that mimic latent infection are valuable tools for describing the changes in metabolism that occur when the bacterium exists in a non-growing form. We used two complementary proteomic approaches, label-free LC-MS/MS analysis and two-dimensional difference gel electrophoresis, to determine the proteome profile of extracellular proteins from M. tuberculosis cultured under nutrient starvation. Through the label-free LC-MS/MS analysis of fractionated samples, 1176 proteins were identified from culture filtrates of log phase and nutrient-starved cultures, and the protein levels of 230 proteins were increased in nutrient-starved culture filtrates, whereas those of 208 proteins were decreased. By means of Gene Ontology clustering analysis, significant differences in the overall metabolism during nutrient starvation were detected. Notably, members of the toxin–antitoxin systems were present in larger quantities in nutrient-starved cultures, supporting a role for these global modules as M. tuberculosis switches its metabolism into dormancy. Decreased abundance of proteins involved in amino acid and protein synthesis was apparent, as well as changes in the lipid metabolism. Further analysis of the dataset identified increased abundance of lipoproteins and decreased abundance of ESAT-6 family proteins. Results from the two-dimensional difference gel electrophoresis proteomics demonstrated overall agreement with the LC-MS/MS data and added complementary insights about protein degradation and modification. PMID:23345537

  11. Effects of a Terrified-Sound Stress on Serum Proteomic Profiling in Mice.

    PubMed

    Yang, Juan; Zhang, Xin; Xiong, Xiaofan; Wu, Qiuhua; Zhao, Lingyu; Liu, Liying; Qin, Yannan; Song, Tusheng; Huang, Chen

    2015-10-01

    The serum proteomic profiles of mice exposed to terrified-sound-induced stress and after stress release were investigated. Serum samples from 32 mice were divided into four groups (n = 8 each) and analyzed using matrix-assisted laser desorption and ionization time-of-flight mass spectrometry techniques (MALDI-TOF MS) combined with magnetic bead-based weak cation-exchange chromatography. ClinProTools software identified several distinct markers that differed between the stressed and control groups and between the stress released and stressed released controls. Of 33 m/z peaks that differed among the four groups, 17 were significantly different (P < 0.05). Five peaks (m/z: 2793.37, 2924.86, 1979.90, 3492.49, 3880.24) showed significant differences in expression after exposure to terrified-sound stress and returned to control levels after stress release. These were sequence identified as peptide regions of dimethylaniline monooxygenase, myosin-9, uncharacterized protein in Rattus norvegicus, apolipoprotein C-I, and plasma serine protease inhibitor (Serpina 5). Our study provides the first evidence of significant changes in serum proteomic profiles in mice exposed to terrified-sound stress, which suggests that protein expression profiles are affected by the stress. Normal expression levels were restored after stress release, suggesting the activation of self-adjustment mechanisms for the recovery of protein expression levels altered by this stress.

  12. A unique proteomic profile on surface IgM ligation in unmutated chronic lymphocytic leukemia

    PubMed Central

    Perrot, Aurore; Pionneau, Cédric; Nadaud, Sophie; Davi, Frédéric; Leblond, Véronique; Jacob, Frédéric; Merle-Béral, Hélène; Herbrecht, Raoul; Béné, Marie-Christine; Gribben, John G.; Vallat, Laurent

    2011-01-01

    Chronic lymphocytic leukemia (CLL) is characterized by a highly variable clinical course with 2 extreme subsets: indolent, ZAP70− and mutated immunoglobulin heavy chain gene (M-CLL); and aggressive, ZAP70+ and unmutated immunoglobulin heavy chain (UM-CLL). Given the long-term suspicion of antigenic stimulation as a primum movens in the disease, the role of the B-cell receptor has been extensively studied in various experimental settings; albeit scarcely in a comparative dynamic proteomic approach. Here we use a quantitative 2-dimensional fluorescence difference gel electrophoresis technology to compare 48 proteomic profiles of the 2 CLL subsets before and after anti-IgM ligation. Differentially expressed proteins were subsequently identified by mass spectrometry. We show that unstimulated M- and UM-CLL cells display distinct proteomic profiles. Furthermore, anti-IgM stimulation induces a specific proteomic response, more pronounced in the more aggressive CLL. Statistical analyses demonstrate several significant protein variations according to stimulation conditions. Finally, we identify an intermediate form of M-CLL cells, with an indolent profile (ZAP70−) but sharing aggressive proteomic profiles alike UM-CLL cells. Collectively, this first quantitative and dynamic proteome analysis of CLL further dissects the complex molecular pathway after B-cell receptor stimulation and depicts distinct proteomic profiles, which could lead to novel molecular stratification of the disease. PMID:21602524

  13. Proteomic Challenges: Sample Preparation Techniques for Microgram-Quantity Protein Analysis from Biological Samples

    PubMed Central

    Feist, Peter; Hummon, Amanda B.

    2015-01-01

    Proteins regulate many cellular functions and analyzing the presence and abundance of proteins in biological samples are central focuses in proteomics. The discovery and validation of biomarkers, pathways, and drug targets for various diseases can be accomplished using mass spectrometry-based proteomics. However, with mass-limited samples like tumor biopsies, it can be challenging to obtain sufficient amounts of proteins to generate high-quality mass spectrometric data. Techniques developed for macroscale quantities recover sufficient amounts of protein from milligram quantities of starting material, but sample losses become crippling with these techniques when only microgram amounts of material are available. To combat this challenge, proteomicists have developed micro-scale techniques that are compatible with decreased sample size (100 μg or lower) and still enable excellent proteome coverage. Extraction, contaminant removal, protein quantitation, and sample handling techniques for the microgram protein range are reviewed here, with an emphasis on liquid chromatography and bottom-up mass spectrometry-compatible techniques. Also, a range of biological specimens, including mammalian tissues and model cell culture systems, are discussed. PMID:25664860

  14. Three-dimensional electrophoresis for quantitative profiling of complex proteomes.

    PubMed

    Mauro, Sergio; Colignon, Bertrand; Dieu, Marc; Delaive, Edouard; Raes, Martine

    2015-01-01

    Quantitative 2D-gel-dependent proteomics became feasible with 2D fluorescence difference gel electrophoresis (2D-DIGE), and this technique has gained wide acceptance because it has eliminated the gel to gel variations and greatly facilitated the quantitative comparisons across gels for many different experimental conditions. However, the co-migration of several proteins in the same spot is still a major limitation which detracts from the accuracy of comparative quantification and prevents unambiguous post-translational modifications (PTMs) detection.A protocol based on traditional polyacrylamide gel IEF sample fractionation, and followed by two consecutive SDS-PAGE electrophoreses alleviates co-migration limitations. The use of two different buffer systems for SDS-PAGE is central to the proposed approach.

  15. Advancing clinicopathologic diagnosis of high-risk neuroblastoma using computerized image analysis and proteomic profiling.

    PubMed

    Niazi, M Khalid Khan; Chung, Jonathan H; Heaton-Johnson, Katherine J; Martinez, Daniel; Castellanos, Raquel; Irwin, Meredith; Master, Stephen R; Pawel, Bruce; Gurcan, Metin N; Weiser, Daniel

    2016-07-21

    A subset of patients with neuroblastoma are at extremely high risk for treatment failure, though they are not identifiable at diagnosis and therefore have the highest mortality with conventional treatment approaches. Despite tremendous understanding of clinical and biological features that correlate with prognosis, neuroblastoma at ultra-high risk for treatment failure remains a diagnostic challenge. As a first step towards improving prognostic risk stratification within the high-risk group of patients, we determined the feasibility of using computerized image analysis and proteomic profiling on single slides from diagnostic tissue specimens. After expert pathologist review of tumor sections to ensure quality and representative material input, we evaluated multiple regions of single slides as well as multiple sections from different patients' tumors using computational histologic analysis and semi-quantitative proteomic profiling. We found that both approaches determined that intertumor heterogeneity was greater than intratumor heterogeneity. Unbiased clustering of samples was greatest within a tumor, suggesting a single section can be representative of the tumor as a whole. There is expected heterogeneity between tumor samples from different individuals with a high degree of similarity among specimens derived from the same patient. Both techniques are novel to supplement pathologist review of neuroblastoma for refined risk stratification, particularly since we demonstrate these results using only a single slide derived from what is usually a scarce tissue resource. Due to limitations of traditional approaches for upfront stratification, integration of new modalities with data derived from one section of tumor hold promise as tools to improve outcomes.

  16. Advancing Clinicopathologic Diagnosis of High-risk Neuroblastoma Using Computerized Image Analysis and Proteomic Profiling.

    PubMed

    Niazi, M Khalid Khan; Chung, Jonathan H; Heaton-Johnson, Katherine J; Martinez, Daniel; Castellanos, Raquel; Irwin, Meredith S; Master, Stephen R; Pawel, Bruce R; Gurcan, Metin N; Weiser, Daniel A

    2017-01-01

    A subset of patients with neuroblastoma are at extremely high risk for treatment failure, though they are not identifiable at diagnosis and therefore have the highest mortality with conventional treatment approaches. Despite tremendous understanding of clinical and biological features that correlate with prognosis, neuroblastoma at ultra-high risk for treatment failure remains a diagnostic challenge. As a first step towards improving prognostic risk stratification within the high-risk group of patients, we determined the feasibility of using computerized image analysis and proteomic profiling on single slides from diagnostic tissue specimens. After expert pathologist review of tumor sections to ensure quality and representative material input, we evaluated multiple regions of single slides as well as multiple sections from different patients' tumors using computational histologic analysis and semiquantitative proteomic profiling. We found that both approaches determined that intertumor heterogeneity was greater than intratumor heterogeneity. Unbiased clustering of samples was greatest within a tumor, suggesting a single section can be representative of the tumor as a whole. There is expected heterogeneity between tumor samples from different individuals with a high degree of similarity among specimens derived from the same patient. Both techniques are novel to supplement pathologist review of neuroblastoma for refined risk stratification, particularly since we demonstrate these results using only a single slide derived from what is usually a scarce tissue resource. Due to limitations of traditional approaches for upfront stratification, integration of new modalities with data derived from one section of tumor hold promise as tools to improve outcomes.

  17. Integrative Proteomics and Phosphoproteomics Profiling Reveals Dynamic Signaling Networks and Bioenergetics Pathways Underlying T Cell Activation.

    PubMed

    Tan, Haiyan; Yang, Kai; Li, Yuxin; Shaw, Timothy I; Wang, Yanyan; Blanco, Daniel Bastardo; Wang, Xusheng; Cho, Ji-Hoon; Wang, Hong; Rankin, Sherri; Guy, Cliff; Peng, Junmin; Chi, Hongbo

    2017-03-21

    The molecular circuits by which antigens activate quiescent T cells remain poorly understood. We combined temporal profiling of the whole proteome and phosphoproteome via multiplexed isobaric labeling proteomics technology, computational pipelines for integrating multi-omics datasets, and functional perturbation to systemically reconstruct regulatory networks underlying T cell activation. T cell receptors activated the T cell proteome and phosphoproteome with discrete kinetics, marked by early dynamics of phosphorylation and delayed ribosome biogenesis and mitochondrial activation. Systems biology analyses identified multiple functional modules, active kinases, transcription factors and connectivity between them, and mitochondrial pathways including mitoribosomes and complex IV. Genetic perturbation revealed physiological roles for mitochondrial enzyme COX10-mediated oxidative phosphorylation in T cell quiescence exit. Our multi-layer proteomics profiling, integrative network analysis, and functional studies define landscapes of the T cell proteome and phosphoproteome and reveal signaling and bioenergetics pathways that mediate lymphocyte exit from quiescence.

  18. Discovery of serum biomarkers of ovarian cancer using complementary proteomic profiling strategies

    PubMed Central

    Arslan‐Low, Elif; Kabir, Musarat; Worthington, Jenny; Camuzeaux, Stephane; Sinclair, John; Szaub, Joanna; Afrough, Babak; Podust, Vladimir N.; Fourkala, Evangelia‐Ourania; Cubizolles, Myriam; Kronenberg, Florian; Fung, Eric T.; Gentry‐Maharaj, Aleksandra; Menon, Usha; Jacobs, Ian

    2014-01-01

    Purpose Ovarian cancer is a devastating disease and biomarkers for its early diagnosis are urgently required. Serum may be a valuable source of biomarkers that may be revealed by proteomic profiling. Herein, complementary serum protein profiling strategies were employed for discovery of biomarkers that could discriminate cases of malignant and benign ovarian cancer. Experimental design Identically collected and processed serum samples from 22 cases of invasive epithelial ovarian cancer, 45 benign ovarian neoplasms, and 64 healthy volunteers were subjected to immunodepletion and protein equalization coupled to 2D‐DIGE/MS and multidimensional fractionation coupled to SELDI‐TOF profiling with MS/MS for protein identification. Selected candidates were verified by ELISA in samples from malignant (n = 70) and benign (n = 89) cases and combined marker panels tested against serum CA125. Results Both profiling platforms were complementary in identifying biomarker candidates, four of which (A1AT, SLPI, APOA4, VDBP) significantly discriminated malignant from benign cases. However, no combination of markers was as good as CA125 for diagnostic accuracy. SLPI was further tested as an early marker using prediagnosis serum samples. While it rose in cases toward diagnosis, it did not discriminate prediagnosis cases from controls. Conclusions and clinical relevance The candidate biomarkers warrant further validation in independent sample sets. PMID:25290619

  19. A Miniaturized Chemical Proteomic Approach for Target Profiling of Clinical Kinase Inhibitors in Tumor Biopsies

    PubMed Central

    Chamrád, Ivo; Rix, Uwe; Stukalov, Alexey; Gridling, Manuela; Parapatics, Katja; Müller, André C.; Altiok, Soner; Colinge, Jacques; Superti-Furga, Giulio; Haura, Eric B.; Bennett, Keiryn L.

    2014-01-01

    While targeted therapy based on the idea of attenuating the activity of a preselected, therapeutically relevant protein has become one of the major trends in modern cancer therapy, no truly specific targeted drug has been developed and most clinical agents have displayed a degree of polypharmacology. Therefore, the specificity of anticancer therapeutics has emerged as a highly important but severely underestimated issue. Chemical proteomics is a powerful technique combining postgenomic drug-affinity chromatography with high-end mass spectrometry analysis and bioinformatic data processing to assemble a target profile of a desired therapeutic molecule. Due to high demands on the starting material, however, chemical proteomic studies have been mostly limited to cancer cell lines. Herein, we report a down-scaling of the technique to enable the analysis of very low abundance samples, as those obtained from needle biopsies. By a systematic investigation of several important parameters in pull-downs with the multikinase inhibitor bosutinib, the standard experimental protocol was optimized to 100 µg protein input. At this level, more than 30 well-known targets were detected per single pull-down replicate with high reproducibility. Moreover, as presented by the comprehensive target profile obtained from miniaturized pull-downs with another clinical drug, dasatinib, the optimized protocol seems to be extendable to other drugs of interest. Sixty distinct human and murine targets were finally identified for bosutinib and dasatinib in chemical proteomic experiments utilizing core needle biopsy samples from xenotransplants derived from patient tumor tissue. Altogether, the developed methodology proves robust and generic and holds many promises for the field of personalized health care. PMID:23901793

  20. A Pro-Atherogenic HDL Profile in Coronary Heart Disease Patients: An iTRAQ Labelling-Based Proteomic Approach

    PubMed Central

    Yan, Li-rong; Wang, Dong-xue; Liu, Hong; Zhang, Xiao-xing; Zhao, Hui; Hua, Lu; Xu, Ping; Li, Yi-shi

    2014-01-01

    Objectives This study aims to compare the protein composition of high-density lipoprotein (HDL) particles in coronary heart disease (CHD) patients and controls by proteomic methods. Background HDL has been reported to exert pro-atherogenic properties in CHD patients. Accumulating evidence indicates that HDL composition, rather than the HDL-C level, determines its functions. The changes in HDL composition involved in the conversion of anti-atherogenic to pro-atherogenic properties in CHD patients are currently unknown. Methods and Results iTRAQ combined with nanoLC-MS/MS was performed to obtain a differential expression profile of the HDL pooled samples of the male age-matched CHD patients and controls (n = 10/group). Of the 196 proteins identified in the examined HDL, 12 were differentially expressed between the CHD patients and the controls, including five up-regulated proteins and seven down-regulated proteins. Using GO analysis, we determined that the up-regulated proteins were mostly involved in inflammatory reactions, displaying a potential pro-atherogenic profile. In contrast, the down-regulated proteins were mostly involved in lipid metabolism processes, displaying anti-atherogenic properties. To confirm the proteomic results, serum amyloid A (SAA) and apoC-I were selected and quantified by ELISA, in the same population as the proteomic analysis, as well as another independent population (n = 120/group). Consistent with the proteomic results, the amount of SAA was significantly increased, and apoC-I was significantly decreased in the HDL particles of CHD patients compared with those of controls (P<0.05). Conclusions Our study shows that the HDL proteome changes to a pro-atherogenic profile in CHD patients, which might compromise the protective effects of HDL. Proteomic analysis of HDL composition may provide more relevant information regarding their functional properties than steady-state HDL-C levels. PMID:24859250

  1. Multimarker proteomic profiling for the prediction of cardiovascular mortality in patients with chronic heart failure.

    PubMed

    Lemesle, Gilles; Maury, Fleur; Beseme, Olivia; Ovart, Lionel; Amouyel, Philippe; Lamblin, Nicolas; de Groote, Pascal; Bauters, Christophe; Pinet, Florence

    2015-01-01

    Risk stratification of patients with systolic chronic heart failure (HF) is critical to better identify those who may benefit from invasive therapeutic strategies such as cardiac transplantation. Proteomics has been used to provide prognostic information in various diseases. Our aim was to investigate the potential value of plasma proteomic profiling for risk stratification in HF. A proteomic profiling using surface enhanced laser desorption ionization - time of flight - mass spectrometry was performed in a case/control discovery population of 198 patients with systolic HF (left ventricular ejection fraction <45%): 99 patients who died from cardiovascular cause within 3 years and 99 patients alive at 3 years. Proteomic scores predicting cardiovascular death were developed using 3 regression methods: support vector machine, sparse partial least square discriminant analysis, and lasso logistic regression. Forty two ion m/z peaks were differentially intense between cases and controls in the discovery population and were used to develop proteomic scores. In the validation population, score levels were higher in patients who subsequently died within 3 years. Similar areas under the curves (0.66 - 0.68) were observed for the 3 methods. After adjustment on confounders, proteomic scores remained significantly associated with cardiovascular mortality. Use of the proteomic scores allowed a significant improvement in discrimination of HF patients as determined by integrated discrimination improvement and net reclassification improvement indexes. In conclusion, proteomic analysis of plasma proteins may help to improve risk prediction in HF patients.

  2. Whole-Proteome Peptide Microarrays for Profiling Autoantibody Repertoires within Multiple Sclerosis and Narcolepsy.

    PubMed

    Zandian, Arash; Forsström, Björn; Häggmark-Månberg, Anna; Schwenk, Jochen M; Uhlén, Mathias; Nilsson, Peter; Ayoglu, Burcu

    2017-02-09

    The underlying molecular mechanisms of autoimmune diseases are poorly understood. To unravel the autoimmune processes across diseases, comprehensive and unbiased analyses of proteins targets recognized by the adaptive immune system are needed. Here we present an approach starting from high-density peptide arrays to characterize autoantibody repertoires and to identify new autoantigens. A set of ten plasma and serum samples from subjects with multiple sclerosis, narcolepsy, and without any disease diagnosis were profiled on a peptide array representing the whole proteome, hosting 2.2 million 12-mer peptides with a six amino acid lateral shift. On the basis of the IgG reactivities found on these whole-proteome peptide microarrays, a set of 23 samples was then studied on a targeted array with 174 000 12-mer peptides of single amino acid lateral shift. Finally, verification of IgG reactivities was conducted with a larger sample set (n = 448) using the bead-based peptide microarrays. The presented workflow employed three different peptide microarray formats to discover and resolve the epitopes of human autoantibodies and revealed two potentially new autoantigens: MAP3K7 in multiple sclerosis and NRXN1 in narcolepsy. The presented strategy provides insights into antibody repertoire reactivity at a peptide level and may accelerate the discovery and validation of autoantigens in human diseases.

  3. Platelets Proteomic Profiles of Acute Ischemic Stroke Patients

    PubMed Central

    Baykal, Ahmet Tarik; Sener, Azize

    2016-01-01

    Platelets play a crucial role in the pathogenesis of stroke and antiplatelet agents exist for its treatment and prevention. Through the use of LC-MS based protein expression profiling, platelets from stroke patients were analyzed and then correlated with the proteomic analyses results in the context of this disease. This study was based on patients who post ischemic stroke were admitted to hospital and had venous blood drawn within 24 hrs of the incidence. Label-free protein expression analyses of the platelets’ tryptic digest was performed in triplicate on a UPLC-ESI-qTOF-MS/MS system and ProteinLynx Global Server (v2.5, Waters) was used for tandem mass data extraction. The peptide sequences were searched against the reviewed homo sapiens database (www.uniprot.org) and the quantitation of protein variation was achieved through Progenesis LC-MS software (V4.0, Nonlinear Dynamics). These Label-free differential proteomics analysis of platelets ensured that 500 proteins were identified and 83 of these proteins were found to be statistically significant. The differentially expressed proteins are involved in various processes such as inflammatory response, cellular movement, immune cell trafficking, cell-to-cell signaling and interaction, hematological system development and function and nucleic acid metabolism. The expressions of myeloperoxidase, arachidonate 12-Lipoxygenase and histidine-rich glycoprotein are involved in cellular metabolic processes, crk-like protein and ras homolog gene family member A involved in cell signaling with vitronectin, thrombospondin 1, Integrin alpha 2b, and integrin beta 3 involved in cell adhesion. Apolipoprotein H, immunoglobulin heavy constant gamma 1 and immunoglobulin heavy constant gamma 3 are involved in structural, apolipoprotein A-I, and alpha-1-microglobulin/bikunin precursor is involved in transport, complement component 3 and clusterin is involved in immunity proteins as has been discussed. Our data provides an insight

  4. 1-Dodecyl-3-methylimidazolium chloride-assisted sample preparation method for efficient integral membrane proteome analysis.

    PubMed

    Zhao, Qun; Fang, Fei; Liang, Yu; Yuan, Huiming; Yang, Kaiguang; Wu, Qi; Liang, Zhen; Zhang, Lihua; Zhang, Yukui

    2014-08-05

    Due to their extremely hydrophobic nature, the analysis of integral membrane proteins (IMPs) is of great challenge. Although various additives have been applied to improve the solubility of IMPs, they still suffer from low solubilization efficiency, incompatibility with trypsin digestion, or interference with MS detection. Herein, the systematic study on the effect of ionic liquid structure on membrane protein solubilization and trypsin biocompatibility was performed, based on which 1-dodecyl-3-methylimidazolium chloride (C12Im-Cl) was selected for the sample preparation of IMPs. Compared with other commonly used additives, such as sodium dodecyl sulfate (SDS), Rapigest, and methanol, C12Im-Cl showed the best performance. In addition, with a strong cation exchange trap column, it could be easily removed after trypsin digestion, which not only was beneficial to avoid protein precipitation during digestion but also had no adverse effect on LC-MS-based separation and detection. Such a C12Im-Cl-assisted sample preparation method was further applied to the membrane proteome analysis of rat brain. Compared with the SDS-assisted method, 1.4 and 3.5 times improvement on the identified IMP and hydrophobic peptide number were achieved (251 vs 178, and 982 vs 279). All these results demonstrated that the C12Im-Cl-assisted sample preparation method is of great promise to promote the large-scale membrane proteome profiling.

  5. S- to N-Palmitoyl Transfer During Proteomic Sample Preparation

    NASA Astrophysics Data System (ADS)

    Ji, Yuhuan; Bachschmid, Markus M.; Costello, Catherine E.; Lin, Cheng

    2016-04-01

    N-palmitoylation has been reported in a number of proteins and suggested to play an important role in protein localization and functions. However, it remains unclear whether N-palmitoylation is a direct enzyme-catalyzed process, or results from intramolecular S- to N-palmitoyl transfer. Here, using the S-palmitoyl peptide standard, GCpalmLGNAK, as the model system, we observed palmitoyl migration from the cysteine residue to either the peptide N-terminus or the lysine side chain during incubation in both neutral and slightly basic buffers commonly used in proteomic sample preparation. Palmitoyl transfer can take place either intra- or inter-molecularly, with the peptide N-terminus being the preferred migration site, presumably because of its lower basicity. The extent of intramolecular palmitoyl migration was low in the system studied, as it required the formation of an entropically unfavored macrocycle intermediate. Intermolecular palmitoyl transfer, however, remained a tangible problem, and may lead to erroneous reporting of in vivo N-palmitoylation. It was found that addition of the MS-compatible detergent RapiGest could significantly inhibit intermolecular palmitoyl transfer, as well as thioester hydrolysis and DTT-induced thioester cleavage. Finally, palmitoyl transfer from the cysteine residue to the peptide N-terminus can also occur in the gas phase, during collision-induced dissociation, and result in false identification of N-palmitoylation. Therefore, one must be careful with both sample preparation and interpretation of tandem mass spectra in the study of N-palmitoylation.

  6. Proteomic Profiling of Nonenzymatically Glycated Proteins in Human Plasma and Erythrocyte Membrane

    SciTech Connect

    Zhang, Qibin; Tang, Ning; Schepmoes, Athena A.; Phillips, Lawrence S.; Smith, Richard D.; Metz, Thomas O.

    2008-05-01

    Non-enzymatic glycation of peptides and proteins by D-glucose has important implications in the pathogenesis of diabetes mellitus, particularly in the development of diabetic complications. In this report, a thorough proteomic profiling of glycated proteins was attempted by using phenylboronate affinity chromatography to enrich glycated proteins and glycated, tryptic peptides from human plasma and erythrocyte membranes. Enriched peptides were subsequently analyzed by liquid chromatography coupled with electron transfer dissociation tandem mass spectrometry, and 76 and 31 proteins were confidently identified as glycated from human plasma and erythrocyte membrane, respectively. It was observed that most of the glycated proteins can be identified in samples from individuals with normal glucose tolerance, although samples from individuals with impaired glucose tolerance and type 2 diabetes mellitus have slightly higher numbers of glycated proteins and more glycation sites identified.

  7. Multiplexed electrokinetic sample fractionation, preconcentration and elution for proteomics.

    PubMed

    Hua, Yujuan; Jemere, Abebaw B; Dragoljic, Jelena; Harrison, D Jed

    2013-07-07

    Both 6 and 8-channel integrated microfluidic sample pretreatment devices capable of performing "in space" sample fractionation, collection, preconcentration and elution of captured analytes via sheath flow assisted electrokinetic pumping are described. Coatings and monolithic polymer beds were developed for the glass devices to provide cationic surface charge and anodal electroosmotic flow for delivery to an electrospray emitter tip. A mixed cationic ([2-(methacryloyloxy)ethyl] trimethylammonium chloride) (META) and hydrophobic butyl methacrylate-based monolithic porous polymer, photopolymerized in the 6- or 8-fractionation channels, was used to capture and preconcentrate samples. A 0.45 wt% META loaded bed generated comparable anodic electroosmotic flow to the cationic polymer PolyE-323 coated channel segments in the device. The balanced electroosmotic flow allowed stable electrokinetic sheath flow to prevent cross contamination of separated protein fractions, while reducing protein/peptide adsorption on the channel walls. Sequential elution of analytes trapped in the SPE beds revealed that the monolithic columns could be efficiently used to provide sheath flow during elution of analytes, as demonstrated for neutral carboxy SNARF (residual signal, 0.08% RSD, n = 40) and charged fluorescein (residual signal, 2.5% n = 40). Elution from monolithic columns showed reproducible performance with peak area reproducibility of ~8% (n = 6 columns) in a single sequential elution and the run-to-run reproducibility was 2.4-6.7% RSD (n = 4) for elution from the same bed. The demonstrated ability of this device design and operation to elute from multiple fractionation beds into a single exit channel for sample analysis by fluorescence or electrospray mass spectrometry is a crucial component of an integrated fractionation and assay system for proteomics.

  8. Human hair shaft proteomic profiling: individual differences, site specificity and cuticle analysis

    PubMed Central

    Laatsch, Chelsea N.; Durbin-Johnson, Blythe P.; Rocke, David M.; Mukwana, Sophie; Newland, Abby B.; Flagler, Michael J.; Davis, Michael G.; Eigenheer, Richard A.; Phinney, Brett S.

    2014-01-01

    Hair from different individuals can be distinguished by physical properties. Although some data exist on other species, examination of the individual molecular differences within the human hair shaft has not been thoroughly investigated. Shotgun proteomic analysis revealed considerable variation in profile among samples from Caucasian, African–American, Kenyan and Korean subjects. Within these ethnic groups, prominent keratin proteins served to distinguish individual profiles. Differences between ethnic groups, less marked, relied to a large extent on levels of keratin associated proteins. In samples from Caucasian subjects, hair shafts from axillary, beard, pubic and scalp regions exhibited distinguishable profiles, with the last being most different from the others. Finally, the profile of isolated hair cuticle cells was distinguished from that of total hair shaft by levels of more than 20 proteins, the majority of which were prominent keratins. The cuticle also exhibited relatively high levels of epidermal transglutaminase (TGM3), accounting for its observed low degree of protein extraction by denaturants. In addition to providing insight into hair structure, present findings may lead to improvements in differentiating hair from various ethnic origins and offer an approach to extending use of hair in crime scene evidence for distinguishing among individuals. PMID:25165623

  9. Human hair shaft proteomic profiling: individual differences, site specificity and cuticle analysis.

    PubMed

    Laatsch, Chelsea N; Durbin-Johnson, Blythe P; Rocke, David M; Mukwana, Sophie; Newland, Abby B; Flagler, Michael J; Davis, Michael G; Eigenheer, Richard A; Phinney, Brett S; Rice, Robert H

    2014-01-01

    Hair from different individuals can be distinguished by physical properties. Although some data exist on other species, examination of the individual molecular differences within the human hair shaft has not been thoroughly investigated. Shotgun proteomic analysis revealed considerable variation in profile among samples from Caucasian, African-American, Kenyan and Korean subjects. Within these ethnic groups, prominent keratin proteins served to distinguish individual profiles. Differences between ethnic groups, less marked, relied to a large extent on levels of keratin associated proteins. In samples from Caucasian subjects, hair shafts from axillary, beard, pubic and scalp regions exhibited distinguishable profiles, with the last being most different from the others. Finally, the profile of isolated hair cuticle cells was distinguished from that of total hair shaft by levels of more than 20 proteins, the majority of which were prominent keratins. The cuticle also exhibited relatively high levels of epidermal transglutaminase (TGM3), accounting for its observed low degree of protein extraction by denaturants. In addition to providing insight into hair structure, present findings may lead to improvements in differentiating hair from various ethnic origins and offer an approach to extending use of hair in crime scene evidence for distinguishing among individuals.

  10. Functional classification of cellular proteome profiles support the identification of drug resistance signatures in melanoma cells.

    PubMed

    Paulitschke, Verena; Haudek-Prinz, Verena; Griss, Johannes; Berger, Walter; Mohr, Thomas; Pehamberger, Hubert; Kunstfeld, Rainer; Gerner, Christopher

    2013-07-05

    Drug resistance is a major obstacle in melanoma treatment. Recognition of specific resistance patterns, the understanding of the patho-physiology of drug resistance, and identification of remaining options for individual melanoma treatment would greatly improve therapeutic success. We performed mass spectrometry-based proteome profiling of A375 melanoma cells and HeLa cells characterized as sensitive to cisplatin in comparison to cisplatin resistant M24met and TMFI melanoma cells. Cells were fractionated into cytoplasm, nuclei and secretome and the proteome profiles classified according to Gene Ontology. The cisplatin resistant cells displayed increased expression of lysosomal as well as Ca²⁺ ion binding and cell adherence proteins. These findings were confirmed using Lysotracker Red staining and cell adhesion assays with a panel of extracellular matrix proteins. To discriminate specific survival proteins, we selected constitutively expressed proteins of resistant M24met cells which were found expressed upon challenging the sensitive A375 cells. Using the CPL/MUW proteome database, the selected lysosomal, cell adherence and survival proteins apparently specifying resistant cells were narrowed down to 47 proteins representing a potential resistance signature. These were tested against our proteomics database comprising more than 200 different cell types/cell states for its predictive power. We provide evidence that this signature enables the automated assignment of resistance features as readout from proteome profiles of any human cell type. Proteome profiling and bioinformatic processing may thus support the understanding of drug resistance mechanism, eventually guiding patient tailored therapy.

  11. Changes of human serum proteome profile during 7-day “dry” immersion

    NASA Astrophysics Data System (ADS)

    Pakharukova, N. A.; Pastushkova, L. Kh.; Larina, I. M.; Grigoriev, A. I.

    2011-05-01

    The aim of this study was to characterize changes of serum proteome profile during 7-day "dry" immersion (DI). The experiment with DI consisted of three series: control group without countermeasures (10 men), with using mechanical stimulation (6 men) and low-frequency myostimulation (5 men) as preventive means. Serum samples were fractionated using ClinProt robot (Bruker Daltonics) on magnetic beads (weak cation exchange magnetic beads—MB WCX) prior to mass-spectral profiling. It was obtained 170 peaks after fractionation of serum samples in each group. On 7th immersion day peak areas of fibrinopeptide A ( m/ z=1206; 1464), angiotensin II ( m/ z=1051), high molecular mass kininogen fragment ( m/ z=2133 Da) and C3-fragment of the complement system ( m/ z=1350 Da) were significantly decreased comparing with pre-experimental values of all experimental series. Peak areas of apolipoprotein C III ( m/ z=9419) and C4a fragment of the complement system ( m/ z=3206 Da) were increased. On 7th day of the recovery peak areas of all changed peaks were not close to pre-experimental values. This fact provided evidence of incomplete recovery of an organism after DI. The depth of the alterations had considerable individual variability. Thereby the detected changes of serum proteome profile in the experiment. They indicated a reorganization of the hormonal, immune systems and lipid metabolism. The use of myostimulation and mechanical stimulation as countermeasures partly compensated adverse effects of 7-day dry immersion on the parameters of coagulation system (fibrinopeptide A) and lipid metabolism (apolipoprotein CIII).

  12. Proteome and transcriptome profiles of a Her2/Neu-driven mouse model of breast cancer.

    PubMed

    Schoenherr, Regine M; Kelly-Spratt, Karen S; Lin, ChenWei; Whiteaker, Jeffrey R; Liu, Tao; Holzman, Ted; Coleman, Ilsa; Feng, Li-Chia; Lorentzen, Travis D; Krasnoselsky, Alexei L; Wang, Pei; Liu, Yan; Gurley, Kay E; Amon, Lynn M; Schepmoes, Athena A; Moore, Ronald J; Camp, David G; Chodosh, Lewis A; Smith, Richard D; Nelson, Peter S; McIntosh, Martin W; Kemp, Christopher J; Paulovich, Amanda G

    2011-04-01

    We generated extensive transcriptional and proteomic profiles from a Her2-driven mouse model of breast cancer that closely recapitulates human breast cancer. This report makes these data publicly available in raw and processed forms, as a resource to the community. Importantly, we previously made biospecimens from this same mouse model freely available through a sample repository, so researchers can obtain samples to test biological hypotheses without the need of breeding animals and collecting biospecimens. Twelve datasets are available, encompassing 841 LC-MS/MS experiments (plasma and tissues) and 255 microarray analyses of multiple tissues (thymus, spleen, liver, blood cells, and breast). Cases and controls were rigorously paired to avoid bias. In total, 18,880 unique peptides were identified (PeptideProphet peptide error rate ≤1%), with 3884 and 1659 non-redundant protein groups identified in plasma and tissue datasets, respectively. Sixty-one of these protein groups overlapped between cancer plasma and cancer tissue. These data are of use for advancing our understanding of cancer biology, for software and quality control tool development, investigations of analytical variation in MS/MS data, and selection of proteotypic peptides for multiple reaction monitoring-MS. The availability of these datasets will contribute positively to clinical proteomics. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Proteome and Transcriptome Profiles of a Her2/Neu-driven Mouse Model of Breast Cancer

    PubMed Central

    Schoenherr, Regine M.; Kelly-Spratt, Karen S.; Lin, ChenWei; Whiteaker, Jeffrey R.; Liu, Tao; Holzman, Ted; Coleman, Ilsa; Feng, Li-Chia; Lorentzen, Travis D.; Krasnoselsky, Alexei L.; Wang, Pei; Liu, Yan; Gurley, Kay E.; Amon, Lynn M.; Schepmoes, Athena A.; Moore, Ronald J.; Camp, David G.; Chodosh, Lewis A.; Smith, Richard D.; Nelson, Peter S.; McIntosh, Martin W.; Kemp, Christopher J.; Paulovich, Amanda G.

    2010-01-01

    Purpose We generated extensive transcriptional and proteomic profiles from a Her2-driven mouse model of breast cancer that closely recapitulates human breast cancer. This report makes these data publicly available in raw and processed forms, as a resource to the community. Importantly, we previously made biospecimens from this same mouse model freely available through a sample repository, so researchers can obtain samples to test biological hypotheses without the need of breeding animals and collecting biospecimens. Experimental design Twelve datasets are available, encompassing 841 LC-MS/MS experiments (plasma and tissues) and 255 microarray analyses of multiple tissues (thymus, spleen, liver, blood cells, and breast). Cases and controls were rigorously paired to avoid bias. Results In total, 18,880 unique peptides were identified (PeptideProphet peptide error rate ≤1%), with 3884 and 1659 non-redundant protein groups identified in plasma and tissue datasets, respectively. Sixty-one of these protein groups overlapped between cancer plasma and cancer tissue. Conclusions and clinical relevance These data are of use for advancing our understanding of cancer biology, for software and quality control tool development, investigations of analytical variation in MS/MS data, and selection of proteotypic peptides for MRM-MS. The availability of these datasets will contribute positively to clinical proteomics. PMID:21448875

  14. Mass spectral analysis of urine proteomic profiles of dairy cows suffering from clinical ketosis.

    PubMed

    Xu, Chuang; Shu, Shi; Xia, Cheng; Wang, Pengxian; Sun, Yuhang; Xu, Chuchu; Li, Changsheng

    2015-01-01

    Ketosis is an important metabolic disorder in dairy cows during the transition period. The urine proteomics of ketosis has not been investigated using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS). The aim is to determine differences between urine proteomic profiles of healthy cows and those with clinical ketosis, and facilitate studies of the underlying physiological and biochemical mechanisms that lead to liver pathology in ketosis. We analyzed the urine samples of 20 cows with clinical ketosis (group 1) and 20 control cows (group 2) using SELDI-TOF-MS. Thirty-nine peptide peaks differed between both groups. Polypeptides corresponding to 26 of these differential peptide peaks were identified using the SWISS-PROT protein database. We found that the peaks of 11 distinct polypeptides from the urine samples of the ketosis group were significantly reduced, compared with those of the control group as based on the Wilcoxon rank sum test. Among these were VGF (non-acronymic) protein, amyloid precursor protein, serum amyloid A (SAA), fibrinogen, C1INH, apolipoprotein C-III, cystatin C, transthyretin, hepcidin, human neutrophil peptides, and osteopontin. These proteins may represent novel biomarkers of the metabolic changes that occur in dairy cows with ketosis. Our results will help to better understand the physiological changes and pathogenesis observed in cows with ketosis. The SELDI-TOF-MS can be used to understand the physiological and biochemical mechanisms of ketosis and identify biomarkers of the disease.

  15. Proteomic profile of Aspergillus flavus in response to water activity.

    PubMed

    Zhang, Feng; Zhong, Hong; Han, Xiaoyun; Guo, Zhenni; Yang, Weiqiang; Liu, Yongfeng; Yang, Kunlong; Zhuang, Zhenhong; Wang, Shihua

    2015-03-01

    Aspergillus flavus, a common contaminant of crops and stored grains, can produce aflatoxins that are harmful to humans and other animals. Water activity (aw) is one of the key factors influencing both fungal growth and mycotoxin production. In this study, we used the isobaric tagging for relative and absolute quantitation (iTRAQ) technique to investigate the effect of aw on the proteomic profile of A. flavus. A total of 3566 proteins were identified, of which 837 were differentially expressed in response to variations in aw. Among these 837 proteins, 403 were over-expressed at 0.99 aw, whereas 434 proteins were over-expressed at 0.93 aw. According to Gene Ontology (GO) analysis, the secretion of extracellular hydrolases increased as aw was raised, suggesting that extracellular hydrolases may play a critical role in induction of aflatoxin biosynthesis. On the basis of Clusters of Orthologous Groups (COG) and Kyoto Encyclopedia of Genes and Genomes (KEGG) categorizations, we identified an exportin protein, KapK, that may down-regulate aflatoxin biosynthesis by changing the location of NirA. Finally, we considered the role of two osmotic stress-related proteins (Sln1 and Glo1) in the Hog1 pathway and investigated the expression patterns of proteins related to aflatoxin biosynthesis. The data uncovered in this study are critical for understanding the effect of water stress on toxin production and for the development of strategies to control toxin contamination of agricultural products. Copyright © 2014 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

  16. Proteomic Profiling of Detergent Resistant Membranes (Lipid Rafts) of Prostasomes.

    PubMed

    Dubois, Louise; Ronquist, Karl K Göran; Ek, Bo; Ronquist, Gunnar; Larsson, Anders

    2015-11-01

    Prostasomes are exosomes derived from prostate epithelial cells through exocytosis by multivesicular bodies. Prostasomes have a bilayered membrane and readily interact with sperm. The membrane lipid composition is unusual with a high contribution of sphingomyelin at the expense of phosphatidylcholine and saturated and monounsaturated fatty acids are dominant. Lipid rafts are liquid-ordered domains that are more tightly packed than the surrounding nonraft phase of the bilayer. Lipid rafts are proposed to be highly dynamic, submicroscopic assemblies that float freely within the liquid disordered membrane bilayer and some proteins preferentially partition into the ordered raft domains. We asked the question whether lipid rafts do exist in prostasomes and, if so, which proteins might be associated with them. Prostasomes of density range 1.13-1.19g/ml were subjected to density gradient ultracentrifugation in sucrose fabricated by phosphate buffered saline (PBS) containing 1% Triton X-100 with capacity for banding at 1.10 g/ml, i.e. the classical density of lipid rafts. Prepared prostasomal lipid rafts (by gradient ultracentrifugation) were analyzed by mass spectrometry. The clearly visible band on top of 1.10g/ml sucrose in the Triton X-100 containing gradient was subjected to liquid chromatography-tandem MS and more than 370 lipid raft associated proteins were identified. Several of them were involved in intraluminal vesicle formation, e.g. tetraspanins, ESCRTs, and Ras-related proteins. This is the first comprehensive liquid chromatography-tandem MS profiling of proteins in lipid rafts derived from exosomes. Data are available via ProteomeXchange with identifier PXD002163.

  17. Proteomics and the Analysis of Proteomic Data: 2013 Overview of Current Protein-Profiling Technologies

    PubMed Central

    Bruce, Can; Stone, Kathryn; Gulcicek, Erol; Williams, Kenneth

    2013-01-01

    Mass spectrometry has become a major tool in the study of proteomes. The analysis of proteolytic peptides and their fragment ions by this technique enables the identification and quantitation of the precursor proteins in a mixture. However, deducing chemical structures and then protein sequences from mass-to-charge ratios is a challenging computational task. Software tools incorporating powerful algorithms and statistical methods improved our ability to process the large quantities of proteomics data. Repositories of spectral data make both data analysis and experimental design more efficient. New approaches in quantitative and statistical proteomics make possible a greater coverage of the proteome, the identification of more post-translational modifications and a greater sensitivity in the quantitation of targeted proteins. PMID:23504934

  18. Proteome Profiling of Paulownia Seedlings Infected with Phytoplasma

    PubMed Central

    Cao, Xibing; Fan, Guoqiang; Dong, Yanpeng; Zhao, Zhenli; Deng, Minjie; Wang, Zhe; Liu, Wenshan

    2017-01-01

    Phytoplasma is an insect-transmitted pathogen that causes witches' broom disease in many plants. Paulownia witches' broom is one of the most destructive diseases threatening Paulownia production. The molecular mechanisms associated with this disease have been investigated by transcriptome sequencing, but changes in protein abundance have not been investigated with isobaric tags for relative and absolute quantitation. Previous results have shown that methyl methane sulfonate (MMS) can help Paulownia seedlings recover from the symptoms of witches' broom and reinstate a healthy morphology. In this study, a transcriptomic-assisted proteomic technique was used to analyze the protein changes in phytoplasma-infected Paulownia tomentosa seedlings, phytoplasma-infected seedlings treated with 20 and 60 mg·L−1 MMS, and healthy seedlings. A total of 2,051 proteins were obtained, 879 of which were found to be differentially abundant in pairwise comparisons between the sample groups. Among the differentially abundant proteins, 43 were related to Paulownia witches' broom disease and many of them were annotated to be involved in photosynthesis, expression of dwarf symptom, energy production, and cell signal pathways. PMID:28344590

  19. Comparative Proteomic Profiling of Pancreatic Ductal Adenocarcinoma Cell Lines

    PubMed Central

    Kim, Yikwon; Han, Dohyun; Min, Hophil; Jin, Jonghwa; Yi, Eugene C.; Kim, Youngsoo

    2014-01-01

    Pancreatic cancer is one of the most fatal cancers and is associated with limited diagnostic and therapeutic modalities. Currently, gemcitabine is the only effective drug and represents the preferred first-line treatment for chemotherapy. However, a high level of intrinsic or acquired resistance of pancreatic cancer to gemcitabine can contribute to the failure of gemcitabine treatment. To investigate the underlying molecular mechanisms for gemcitabine resistance in pancreatic cancer, we performed label-free quantification of protein expression in intrinsic gemcitabine-resistant and - sensitive human pancreatic adenocarcinoma cell lines using our improved proteomic strategy, combined with filter-aided sample preparation, single-shot liquid chromatography-mass spectrometry, enhanced spectral counting, and a statistical method based on a power law global error model. We identified 1931 proteins and quantified 787 differentially expressed proteins in the BxPC3, PANC-1, and HPDE cell lines. Bioinformatics analysis identified 15 epithelial to mesenchymal transition (EMT) markers and 13 EMT-related proteins that were closely associated with drug resistance were differentially expressed. Interestingly, 8 of these proteins were involved in glutathione and cysteine/methionine metabolism. These results suggest that proteins related to the EMT and glutathione metabolism play important roles in the development of intrinsic gemcitabine resistance by pancreatic cancer cell lines. PMID:25518923

  20. Profiling thiol redox proteome using isotope tagging mass spectrometry.

    PubMed

    Parker, Jennifer; Zhu, Ning; Zhu, Mengmeng; Chen, Sixue

    2012-03-24

    Pseudomonas syringae pv. tomato strain DC3000 not only causes bacterial speck disease in Solanum lycopersicum but also on Brassica species, as well as on Arabidopsis thaliana, a genetically tractable host plant(1,2). The accumulation of reactive oxygen species (ROS) in cotyledons inoculated with DC3000 indicates a role of ROS in modulating necrotic cell death during bacterial speck disease of tomato(3). Hydrogen peroxide, a component of ROS, is produced after inoculation of tomato plants with Pseudomonas(3). Hydrogen peroxide can be detected using a histochemical stain 3'-3' diaminobenzidine (DAB)(4). DAB staining reacts with hydrogen peroxide to produce a brown stain on the leaf tissue(4). ROS has a regulatory role of the cellular redox environment, which can change the redox status of certain proteins(5). Cysteine is an important amino acid sensitive to redox changes. Under mild oxidation, reversible oxidation of cysteine sulfhydryl groups serves as redox sensors and signal transducers that regulate a variety of physiological processes(6,7). Tandem mass tag (TMT) reagents enable concurrent identification and multiplexed quantitation of proteins in different samples using tandem mass spectrometry(8,9). The cysteine-reactive TMT (cysTMT) reagents enable selective labeling and relative quantitation of cysteine-containing peptides from up to six biological samples. Each isobaric cysTMT tag has the same nominal parent mass and is composed of a sulfhydryl-reactive group, a MS-neutral spacer arm and an MS/MS reporter(10). After labeling, the samples were subject to protease digestion. The cysteine-labeled peptides were enriched using a resin containing anti-TMT antibody. During MS/MS analysis, a series of reporter ions (i.e., 126-131 Da) emerge in the low mass region, providing information on relative quantitation. The workflow is effective for reducing sample complexity, improving dynamic range and studying cysteine modifications. Here we present redox proteomic

  1. Proteome-Wide Analysis and Diel Proteomic Profiling of the Cyanobacterium Arthrospira platensis PCC 8005

    PubMed Central

    Matallana-Surget, Sabine; Derock, Jérémy; Leroy, Baptiste; Badri, Hanène; Deschoenmaeker, Frédéric; Wattiez, Ruddy

    2014-01-01

    The filamentous cyanobacterium Arthrospira platensis has a long history of use as a food supply and it has been used by the European Space Agency in the MELiSSA project, an artificial microecosystem which supports life during long-term manned space missions. This study assesses progress in the field of cyanobacterial shotgun proteomics and light/dark diurnal cycles by focusing on Arthrospira platensis. Several fractionation workflows including gel-free and gel-based protein/peptide fractionation procedures were used and combined with LC-MS/MS analysis, enabling the overall identification of 1306 proteins, which represents 21% coverage of the theoretical proteome. A total of 30 proteins were found to be significantly differentially regulated under light/dark growth transition. Interestingly, most of the proteins showing differential abundance were related to photosynthesis, the Calvin cycle and translation processes. A novel aspect and major achievement of this work is the successful improvement of the cyanobacterial proteome coverage using a 3D LC-MS/MS approach, based on an immobilized metal affinity chromatography, a suitable tool that enabled us to eliminate the most abundant protein, the allophycocyanin. We also demonstrated that cell growth follows a light/dark cycle in A. platensis. This preliminary proteomic study has highlighted new characteristics of the Arthrospira platensis proteome in terms of diurnal regulation. PMID:24914774

  2. Proteome-wide analysis and diel proteomic profiling of the cyanobacterium Arthrospira platensis PCC 8005.

    PubMed

    Matallana-Surget, Sabine; Derock, Jérémy; Leroy, Baptiste; Badri, Hanène; Deschoenmaeker, Frédéric; Wattiez, Ruddy

    2014-01-01

    The filamentous cyanobacterium Arthrospira platensis has a long history of use as a food supply and it has been used by the European Space Agency in the MELiSSA project, an artificial microecosystem which supports life during long-term manned space missions. This study assesses progress in the field of cyanobacterial shotgun proteomics and light/dark diurnal cycles by focusing on Arthrospira platensis. Several fractionation workflows including gel-free and gel-based protein/peptide fractionation procedures were used and combined with LC-MS/MS analysis, enabling the overall identification of 1306 proteins, which represents 21% coverage of the theoretical proteome. A total of 30 proteins were found to be significantly differentially regulated under light/dark growth transition. Interestingly, most of the proteins showing differential abundance were related to photosynthesis, the Calvin cycle and translation processes. A novel aspect and major achievement of this work is the successful improvement of the cyanobacterial proteome coverage using a 3D LC-MS/MS approach, based on an immobilized metal affinity chromatography, a suitable tool that enabled us to eliminate the most abundant protein, the allophycocyanin. We also demonstrated that cell growth follows a light/dark cycle in A. platensis. This preliminary proteomic study has highlighted new characteristics of the Arthrospira platensis proteome in terms of diurnal regulation.

  3. Shotgun Proteomics of Tomato Fruits: Evaluation, Optimization and Validation of Sample Preparation Methods and Mass Spectrometric Parameters

    PubMed Central

    Kilambi, Himabindu V.; Manda, Kalyani; Sanivarapu, Hemalatha; Maurya, Vineet K.; Sharma, Rameshwar; Sreelakshmi, Yellamaraju

    2016-01-01

    An optimized protocol was developed for shotgun proteomics of tomato fruit, which is a recalcitrant tissue due to a high percentage of sugars and secondary metabolites. A number of protein extraction and fractionation techniques were examined for optimal protein extraction from tomato fruits followed by peptide separation on nanoLCMS. Of all evaluated extraction agents, buffer saturated phenol was the most efficient. In-gel digestion [SDS-PAGE followed by separation on LCMS (GeLCMS)] of phenol-extracted sample yielded a maximal number of proteins. For in-solution digested samples, fractionation by strong anion exchange chromatography (SAX) also gave similar high proteome coverage. For shotgun proteomic profiling, optimization of mass spectrometry parameters such as automatic gain control targets (5E+05 for MS, 1E+04 for MS/MS); ion injection times (500 ms for MS, 100 ms for MS/MS); resolution of 30,000; signal threshold of 500; top N-value of 20 and fragmentation by collision-induced dissociation yielded the highest number of proteins. Validation of the above protocol in two tomato cultivars demonstrated its reproducibility, consistency, and robustness with a CV of < 10%. The protocol facilitated the detection of five-fold higher number of proteins compared to published reports in tomato fruits. The protocol outlined would be useful for high-throughput proteome analysis from tomato fruits and can be applied to other recalcitrant tissues. PMID:27446192

  4. Shotgun Proteomics of Tomato Fruits: Evaluation, Optimization and Validation of Sample Preparation Methods and Mass Spectrometric Parameters.

    PubMed

    Kilambi, Himabindu V; Manda, Kalyani; Sanivarapu, Hemalatha; Maurya, Vineet K; Sharma, Rameshwar; Sreelakshmi, Yellamaraju

    2016-01-01

    An optimized protocol was developed for shotgun proteomics of tomato fruit, which is a recalcitrant tissue due to a high percentage of sugars and secondary metabolites. A number of protein extraction and fractionation techniques were examined for optimal protein extraction from tomato fruits followed by peptide separation on nanoLCMS. Of all evaluated extraction agents, buffer saturated phenol was the most efficient. In-gel digestion [SDS-PAGE followed by separation on LCMS (GeLCMS)] of phenol-extracted sample yielded a maximal number of proteins. For in-solution digested samples, fractionation by strong anion exchange chromatography (SAX) also gave similar high proteome coverage. For shotgun proteomic profiling, optimization of mass spectrometry parameters such as automatic gain control targets (5E+05 for MS, 1E+04 for MS/MS); ion injection times (500 ms for MS, 100 ms for MS/MS); resolution of 30,000; signal threshold of 500; top N-value of 20 and fragmentation by collision-induced dissociation yielded the highest number of proteins. Validation of the above protocol in two tomato cultivars demonstrated its reproducibility, consistency, and robustness with a CV of < 10%. The protocol facilitated the detection of five-fold higher number of proteins compared to published reports in tomato fruits. The protocol outlined would be useful for high-throughput proteome analysis from tomato fruits and can be applied to other recalcitrant tissues.

  5. In-depth proteomic profiling of left ventricular tissues in human end-stage dilated cardiomyopathy

    PubMed Central

    Liu, Shanshan; Xia, Yan; Liu, Xiaohui; Wang, Yi; Chen, Zhangwei; Xie, Juanjuan; Qian, Juying; Shen, Huali; Yang, Pengyuan

    2017-01-01

    Dilated cardiomyopathy (DCM) is caused by reduced left ventricular (LV) myocardial function, which is one of the most common causes of heart failure (HF). We performed iTRAQ-coupled 2D-LC-MS/MS to profile the cardiac proteome of LV tissues from healthy controls and patients with end-stage DCM. We identified 4263 proteins, of which 125 were differentially expressed in DCM tissues compared to LV controls. The majority of these were membrane proteins related to cellular junctions and neuronal metabolism. In addition, these proteins were involved in membrane organization, mitochondrial organization, translation, protein transport, and cell death process. Four key proteins involved in the cell death process were also detected by western blotting, indicated that cell death was activated in DCM tissues. Furthermore, S100A1 and eEF2 were enriched in the “cellular assembly and organization” and “cell cycle” networks, respectively. We verified decreases in these two proteins in end-stage DCM LV samples through multiple reaction monitoring (MRM). These observations demonstrate that our understanding of the mechanisms underlying DCM can be deepened through comparison of the proteomes of normal LV tissues with that from end-stage DCM in humans. PMID:28427148

  6. A Proteomic Study of the HUPO Plasma Proteome Project's Pilot Samples using an Accurate Mass and Time Tag Strategy

    SciTech Connect

    Adkins, Joshua N.; Monroe, Matthew E.; Auberry, Kenneth J.; Shen, Yufeng; Jacobs, Jon M.; Camp, David G.; Vitzthum, Frank; Rodland, Karin D.; Zangar, Richard C.; Smith, Richard D.; Pounds, Joel G.

    2005-08-01

    Characterization of the human blood plasma proteome is critical to the discovery of routinely useful clinical biomarkers. We used an Accurate Mass and Time (AMT) tag strategy with high-resolution mass accuracy capillary liquid chromatography Fourier-Transform Ion Cyclotron Resonance Mass Spectrometry (cLC-FTICR MS) to perform a global proteomic analysis of pilot study samples as part of the HUPO Plasma Proteome Project. HUPO reference serum and citrated plasma samples from African Americans, Asian Americans, and Caucasian Americans were analyzed, in addition to a Pacific Northwest National Laboratory reference serum and plasma. The AMT tag strategy allowed us to leverage two previously published “shotgun” proteomics experiments to perform global analyses on these samples in triplicate in less than 4 days total analysis time. A total of 722 (22% with multiple peptide identifications) International Protein Index (IPI) redundant proteins, or 377 protein families by ProteinProphet, were identified over the 6 individual HUPO serum and plasma samples. The samples yielded a similar number of identified redundant proteins in the plasma samples (average 446 +/-23) as found in the serum samples (average 440+/-20). These proteins were identified by an average of 956+/-35 unique peptides in plasma and 930+/-11 unique peptides in serum. In addition to this high-throughput analysis, the AMT tag approach was used with a Z-score normalization to compare relative protein abundances. This analysis highlighted both known differences in serum and citrated plasma such as fibrinogens, and reproducible differences in peptide abundances from proteins such as soluble activin receptor-like kinase 7b and glycoprotein m6b. The AMT tag strategy not only improved our sample throughput, and provided a basis for estimated quantitation.

  7. Quantitative Proteome Profiling of C. burnetii under Tetracycline Stress Conditions

    PubMed Central

    Vranakis, Iosif; De Bock, Pieter-Jan; Papadioti, Anastasia; Tselentis, Yannis; Gevaert, Kris; Tsiotis, Georgios; Psaroulaki, Anna

    2012-01-01

    The recommended antibiotic regimen against Coxiella burnetii, the etiological agent of Q fever, is based on a semi-synthetic, second-generation tetracycline, doxycycline. Here, we report on the comparison of the proteomes of a C. burnetii reference strain either cultured under control conditions or under tetracycline stress conditions. Using the MS-driven combined fractional diagonal chromatography proteomics technique, out of the 531 proteins identified, 5 and 19 proteins were found significantly up- and down-regulated respectively, under tetracycline stress. Although the predicted cellular functions of these regulated proteins did not point to known tetracycline resistance mechanisms, our data clearly reveal the plasticity of the proteome of C. burnetii to battle tetracycline stress. Finally, we raise several plausible hypotheses that could further lead to more focused experiments on studying tetracycline resistance in C. burnetii and thus reduced treatment failures of Q fever. PMID:22438959

  8. Proteomic profiling reveals insights into Triticeae stigma development and function.

    PubMed

    Nazemof, Nazila; Couroux, Philippe; Rampitsch, Christof; Xing, Tim; Robert, Laurian S

    2014-11-01

    To our knowledge, this study represents the first high-throughput characterization of a stigma proteome in the Triticeae. A total of 2184 triticale mature stigma proteins were identified using three different gel-based approaches combined with mass spectrometry. The great majority of these proteins are described in a Triticeae stigma for the first time. These results revealed many proteins likely to play important roles in stigma development and pollen-stigma interactions, as well as protection against biotic and abiotic stresses. Quantitative comparison of the triticale stigma transcriptome and proteome showed poor correlation, highlighting the importance of having both types of analysis. This work makes a significant contribution towards the elucidation of the Triticeae stigma proteome and provides novel insights into its role in stigma development and function. © The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology.

  9. Protein alterations associated with pancreatic cancer and chronic pancreatitis found in human plasma using global quantitative proteomics profiling

    PubMed Central

    Pan, Sheng; Chen, Ru; Crispin, David A.; May, Damon; Stevens, Tyler; McIntosh, Martin; Bronner, Mary P.; Ziogas, Argyrios; Anton-Culver, Hoda; Brentnall, Teresa A.

    2011-01-01

    Pancreatic cancer is a lethal disease that is difficult to diagnose at early stages when curable treatments are effective. Biomarkers that can improve current pancreatic cancer detection would have great value in improving patient management and survival rate. A large scale quantitative proteomics study was performed to search for the plasma protein alterations associated with pancreatic cancer. The enormous complexity of the plasma proteome and the vast dynamic range of protein concentration therein present major challenges for quantitative global profiling of plasma. To address these challenges, multi-dimensional fractionation at both protein and peptide levels was applied to enhance the depth of proteomics analysis. Employing stringent criteria, more than thirteen hundred proteins total were identified in plasma across 8-orders of magnitude in protein concentration. Differential proteins associated with pancreatic cancer were identified, and their relationship with the proteome of pancreatic tissue and pancreatic juice from our previous studies was discussed. A subgroup of differentially expressed proteins was selected for biomarker testing using an independent cohort of plasma and serum samples from well-diagnosed patients with pancreatic cancer, chronic pancreatitis and non-pancreatic disease controls. Using ELISA methodology, the performance of each of these protein candidates was benchmarked against CA19-9, the current gold standard for a pancreatic cancer blood test. A composite marker of TIMP1 and ICAM1 demonstrate significantly better performance than CA19-9 in distinguishing pancreatic cancer from the non-pancreatic disease controls and chronic pancreatitis controls. In addition, protein AZGP1 was identified as a biomarker candidate for chronic pancreatitis. The discovery and technical challenges associated with plasma-based quantitative proteomics are discussed and may benefit the development of plasma proteomics technology in general. The protein

  10. Sample preparation optimization in fecal metabolic profiling.

    PubMed

    Deda, Olga; Chatziioannou, Anastasia Chrysovalantou; Fasoula, Stella; Palachanis, Dimitris; Raikos, Νicolaos; Theodoridis, Georgios A; Gika, Helen G

    2017-03-15

    Metabolomic analysis of feces can provide useful insight on the metabolic status, the health/disease state of the human/animal and the symbiosis with the gut microbiome. As a result, recently there is increased interest on the application of holistic analysis of feces for biomarker discovery. For metabolomics applications, the sample preparation process used prior to the analysis of fecal samples is of high importance, as it greatly affects the obtained metabolic profile, especially since feces, as matrix are diversifying in their physicochemical characteristics and molecular content. However there is still little information in the literature and lack of a universal approach on sample treatment for fecal metabolic profiling. The scope of the present work was to study the conditions for sample preparation of rat feces with the ultimate goal of the acquisition of comprehensive metabolic profiles either untargeted by NMR spectroscopy and GC-MS or targeted by HILIC-MS/MS. A fecal sample pooled from male and female Wistar rats was extracted under various conditions by modifying the pH value, the nature of the organic solvent and the sample weight to solvent volume ratio. It was found that the 1/2 (wf/vs) ratio provided the highest number of metabolites under neutral and basic conditions in both untargeted profiling techniques. Concerning LC-MS profiles, neutral acetonitrile and propanol provided higher signals and wide metabolite coverage, though extraction efficiency is metabolite dependent. Copyright © 2016 Elsevier B.V. All rights reserved.

  11. Comparative bioinformatics analyses and profiling of lysosome-related organelle proteomes

    NASA Astrophysics Data System (ADS)

    Hu, Zhang-Zhi; Valencia, Julio C.; Huang, Hongzhan; Chi, An; Shabanowitz, Jeffrey; Hearing, Vincent J.; Appella, Ettore; Wu, Cathy

    2007-01-01

    Complete and accurate profiling of cellular organelle proteomes, while challenging, is important for the understanding of detailed cellular processes at the organelle level. Mass spectrometry technologies coupled with bioinformatics analysis provide an effective approach for protein identification and functional interpretation of organelle proteomes. In this study, we have compiled human organelle reference datasets from large-scale proteomic studies and protein databases for seven lysosome-related organelles (LROs), as well as the endoplasmic reticulum and mitochondria, for comparative organelle proteome analysis. Heterogeneous sources of human organelle proteins and rodent homologs are mapped to human UniProtKB protein entries based on ID and/or peptide mappings, followed by functional annotation and categorization using the iProXpress proteomic expression analysis system. Cataloging organelle proteomes allows close examination of both shared and unique proteins among various LROs and reveals their functional relevance. The proteomic comparisons show that LROs are a closely related family of organelles. The shared proteins indicate the dynamic and hybrid nature of LROs, while the unique transmembrane proteins may represent additional candidate marker proteins for LROs. This comparative analysis, therefore, provides a basis for hypothesis formulation and experimental validation of organelle proteins and their functional roles.

  12. Proteomic analysis of minute amount of colonic biopsies by enteroscopy sampling

    SciTech Connect

    Liu, Xing; Xu, Yanli; Meng, Qian; Zheng, Qingqing; Wu, Jianhong; Wang, Chen; Jia, Weiping; Figeys, Daniel; Chang, Ying; Zhou, Hu

    2016-08-05

    Colorectal cancer (CRC) is one of the most common types of malignant tumor worldwide. Currently, although many researchers have been devoting themselves in CRC studies, the process of locating biomarkers for CRC early diagnosis and prognostic is still very slow. Using a centrifugal proteomic reactor-based proteomic analysis of minute amount of colonic biopsies by enteroscopy sampling, 2620 protein groups were quantified between cancer mucosa and adjacent normal colorectal mucosa. Of which, 403 protein groups were differentially expressed with statistic significance between cancer and normal tissues, including 195 up-regulated and 208 down-regulated proteins in cancer tissues. Three proteins (SOD3, PRELP and NGAL) were selected for further Western blot validation. And the resulting Western blot experimental results were consistent with the quantitative proteomic data. SOD3 and PRELP are down-regulated in CRC mucosa comparing to adjacent normal tissue, while NGAL is up-regulated in CRC mucosa. In conclusion, the centrifugal proteomic reactor-based label-free quantitative proteomic approach provides a highly sensitive and powerful tool for analyzing minute protein sample from tiny colorectal biopsies, which may facilitate CRC biomarkers discovery for diagnoses and prognoses. -- Highlights: •Minute amount of colonic biopsies by endoscopy is suitable for proteomic analysis. •Centrifugal proteomic reactor can be used for processing tiny clinic biopsy sample. •SOD3 and PRELP are down-regulated in CRC, while NGAL is up-regulated in CRC.

  13. Proteome-Wide Profiling of Targets of Cysteine reactive Small Molecules by Using Ethynyl Benziodoxolone Reagents.

    PubMed

    Abegg, Daniel; Frei, Reto; Cerato, Luca; Prasad Hari, Durga; Wang, Chao; Waser, Jerome; Adibekian, Alexander

    2015-09-07

    In this study, we present a highly efficient method for proteomic profiling of cysteine residues in complex proteomes and in living cells. Our method is based on alkynylation of cysteines in complex proteomes using a "clickable" alkynyl benziodoxolone bearing an azide group. This reaction proceeds fast, under mild physiological conditions, and with a very high degree of chemoselectivity. The formed azide-capped alkynyl-cysteine adducts are readily detectable by LC-MS/MS, and can be further functionalized with TAMRA or biotin alkyne via CuAAC. We demonstrate the utility of alkynyl benziodoxolones for chemical proteomics applications by identifying the proteomic targets of curcumin, a diarylheptanoid natural product that was and still is part of multiple human clinical trials as anticancer agent. Our results demonstrate that curcumin covalently modifies several key players of cellular signaling and metabolism, most notably the enzyme casein kinase I gamma. We anticipate that this new method for cysteine profiling will find broad application in chemical proteomics and drug discovery.

  14. Fractionation profiling: a fast and versatile approach for mapping vesicle proteomes and protein–protein interactions

    PubMed Central

    Borner, Georg H. H.; Hein, Marco Y.; Hirst, Jennifer; Edgar, James R.; Mann, Matthias; Robinson, Margaret S.

    2014-01-01

    We developed “fractionation profiling,” a method for rapid proteomic analysis of membrane vesicles and protein particles. The approach combines quantitative proteomics with subcellular fractionation to generate signature protein abundance distribution profiles. Functionally associated groups of proteins are revealed through cluster analysis. To validate the method, we first profiled >3500 proteins from HeLa cells and identified known clathrin-coated vesicle proteins with >90% accuracy. We then profiled >2400 proteins from Drosophila S2 cells, and we report the first comprehensive insect clathrin-coated vesicle proteome. Of importance, the cluster analysis extends to all profiled proteins and thus identifies a diverse range of known and novel cytosolic and membrane-associated protein complexes. We show that it also allows the detailed compositional characterization of complexes, including the delineation of subcomplexes and subunit stoichiometry. Our predictions are presented in an interactive database. Fractionation profiling is a universal method for defining the clathrin-coated vesicle proteome and may be adapted for the analysis of other types of vesicles and particles. In addition, it provides a versatile tool for the rapid generation of large-scale protein interaction maps. PMID:25165137

  15. Subnanogram proteomics: impact of LC column selection, MS instrumentation and data analysis strategy on proteome coverage for trace samples

    DOE PAGES

    Zhu, Ying; Zhao, Rui; Piehowski, Paul D.; ...

    2017-09-01

    One of the greatest challenges for mass spectrometry (MS)-based proteomics is the limited ability to analyze small samples. Here in this study, we investigate the relative contributions of liquid chromatography (LC), MS instrumentation and data analysis methods with the aim of improving proteome coverage for sample sizes ranging from 0.5 ng to 50 ng. We show that the LC separations utilizing 30-μm-i.d. columns increase signal intensity by >3-fold relative to those using 75-μm-i.d. columns, leading to 32% increase in peptide identifications. The Orbitrap Fusion Lumos MS significantly boosted both sensitivity and sequencing speed relative to earlier generation Orbitraps (e.g., LTQ-Orbitrap),more » leading to a ~3-fold increase in peptide identifications and 1.7-fold increase in identified protein groups for 2 ng tryptic digests of the bacterium S. oneidensis. The Match Between Runs algorithm of open-source MaxQuant software further increased proteome coverage by ~95% for 0.5 ng samples and by ~42% for 2 ng samples. Using the best combination of the above variables, we were able to identify >3,000 proteins from 10 ng tryptic digests from both HeLa and THP-1 mammalian cell lines. We also identified >950 proteins from subnanogram archaeal/bacterial cocultures. Finally, the present ultrasensitive LC-MS platform achieves a level of proteome coverage not previously realized for ultra-small sample loadings, and is expected to facilitate the analysis of subnanogram samples, including single mammalian cells.« less

  16. Extracellular Proteome Profiling of Bacillus pumilus SCU11 Producing Alkaline Protease for Dehairing.

    PubMed

    Wang, Chao; Yu, Shiqiang; Song, Ting; He, Tingting; Shao, Huanhuan; Wang, Haiyan

    2016-11-28

    Bacillus pumilus is one of the most characterized microorganisms that are used for high-level production of select industrial enzymes. A novel B. pumilus SCU11 strain possessing high alkaline protease activity was obtained in our previous work. The culture supernatant of this strain showed efficient dehairing capability with minimal collagen damage, indicating promising potential applications in the leather industry. In this study, the strain's extracellular proteome was identified by LC-MS/MS-based shotgun proteomic analysis, and their related secretory pathways were characterized by BLAST searches. A total of 513 proteins, including 100 actual secreted and 413 intracellular proteins, were detected in the extracellular proteome. The functions of these secreted proteins were elucidated and four complete secretory systems (Sec, Tat, Com, and ABC transporter) were proposed for B. pumilus. These data provide B. pumilus a comprehensive extracellular proteome profile, which is a valuable theoretical and applicative basis for future genetic modifications and development of industrial enzymes.

  17. Chemical proteomic probes for profiling cytochrome P450 activities and drug interactions in vivo

    PubMed Central

    Wright, Aaron T.; Cravatt, Benjamin F.

    2007-01-01

    The cytochrome P450 (P450) superfamily metabolizes many endogenous signaling molecules and drugs. P450 enzymes are regulated by post-translational mechanisms in vivo, which hinders their functional characterization by conventional genomic or proteomic methods. Here, we describe a chemical proteomic strategy to profile P450 activities directly in living systems. Derivatization of a mechanism-based inhibitor with a “clickable” handle provided an activity-based probe that labels multiple P450s both in proteomic extracts and in vivo. This probe was used to record alterations in liver P450 activities triggered by chemical agents, including inducers of P450 expression and direct P450 inhibitors. The chemical proteomic strategy described herein thus offers a versatile method to monitor P450 activities and small molecule interactions in any biological system and, through doing so, should facilitate the functional characterization of this large and diverse enzyme class. PMID:17884636

  18. The proteome of Hypobaric Induced Hypoxic Lung: Insights from Temporal Proteomic Profiling for Biomarker Discovery

    PubMed Central

    Ahmad, Yasmin; Sharma, Narendra K.; Ahmad, Mohammad Faiz; Sharma, Manish; Garg, Iti; Srivastava, Mousami; Bhargava, Kalpana

    2015-01-01

    Exposure to high altitude induces physiological responses due to hypoxia. Lungs being at the first level to face the alterations in oxygen levels are critical to counter and balance these changes. Studies have been done analysing pulmonary proteome alterations in response to exposure to hypobaric hypoxia. However, such studies have reported the alterations at specific time points and do not reflect the gradual proteomic changes. These studies also identify the various biochemical pathways and responses induced after immediate exposure and the resolution of these effects in challenge to hypobaric hypoxia. In the present study, using 2-DE/MS approach, we attempt to resolve these shortcomings by analysing the proteome alterations in lungs in response to different durations of exposure to hypobaric hypoxia. Our study thus highlights the gradual and dynamic changes in pulmonary proteome following hypobaric hypoxia. For the first time, we also report the possible consideration of SULT1A1, as a biomarker for the diagnosis of high altitude pulmonary edema (HAPE). Higher SULT1A1 levels were observed in rats as well as in humans exposed to high altitude, when compared to sea-level controls. This study can thus form the basis for identifying biomarkers for diagnostic and prognostic purposes in responses to hypobaric hypoxia. PMID:26022216

  19. Bead Based Proteome Enrichment Enhances Features of the Protein Elution Plate (PEP) for Functional Proteomic Profiling

    PubMed Central

    Wang, Xing; Davies, Michael; Roy, Swapan; Kuruc, Matthew

    2015-01-01

    A novel functional proteomics technology called PEP(Protein Elution Plate) was developed to separate complex proteomes from natural sources and analyze protein functions systematically. The technology takes advantage of the powerful resolution of two-dimensional gel electrophoresis (2-D Gels). The modification of electrophoretic conditions in combination with a high-resolution protein elution plate supports the recovery of functionally active proteins. As 2DE(2-Dimensional Electrophoresis) resolution can be limited by protein load, we investigated the use of bead based enrichment technologies, called AlbuVoid™ and KinaSorb™ to determine their effect on the proteomic features which can be generated from the PEP platform. Using a variety of substrates and enzyme activity assays, we report on the benefits of combining bead based enrichment to improve the signal report and the features generated for Hexokinase, Protein Kinase, Protease, and Alkaline Phosphatase activities. As a result, the PEP technology allows systematic analysis of large enzyme families and can build a comprehensive picture of protein function from a complex proteome, providing biological insights that could otherwise not be observed if only protein abundances were analyzed. PMID:28248280

  20. Proteomic Profiling of Bladders from Mice Exposed with Sodium Arsenite

    EPA Science Inventory

    Arsenic, an environmental contaminant, has been linked with cancer of the bladder in humans. To study the mode of action of arsenic, female CH3 mice were exposed to 85 ppm sodium arsenite in their drinking water for 30 days. Following the exposure a comparative proteomic analysis...

  1. Proteome Profiling of Wheat Shoots from Different Cultivars

    PubMed Central

    Vu, Lam Dai; Verstraeten, Inge; Stes, Elisabeth; Van Bel, Michiel; Coppens, Frederik; Gevaert, Kris; De Smet, Ive

    2017-01-01

    Wheat is a cereal grain and one of the world’s major food crops. Recent advances in wheat genome sequencing are by now facilitating its genomic and proteomic analyses. However, little is known about possible differences in total protein levels of hexaploid versus tetraploid wheat cultivars, and also knowledge of phosphorylated wheat proteins is still limited. Here, we performed a detailed analysis of the proteome of seedling leaves from two hexaploid wheat cultivars (Triticum aestivum L. Pavon 76 and USU-Apogee) and one tetraploid wheat (T. turgidum ssp. durum cv. Senatore Cappelli). Our shotgun proteomics data revealed that, whereas we observed some significant differences, overall a high similarity between hexaploid and tetraploid varieties with respect to protein abundance was observed. In addition, already at the seedling stage, a small set of proteins was differential between the small (USU-Apogee) and larger hexaploid wheat cultivars (Pavon 76), which could potentially act as growth predictors. Finally, the phosphosites identified in this study can be retrieved from the in-house developed plant PTM-Viewer (bioinformatics.psb.ugent.be/webtools/ptm_viewer/), making this the first searchable repository for phosphorylated wheat proteins. This paves the way for further in depth, quantitative (phospho)proteome-wide differential analyses upon a specific trigger or environmental change. PMID:28348574

  2. Proteomic technology for biomarker profiling in cancer: an update*

    PubMed Central

    Alaoui-Jamali, Moulay A.; Xu, Ying-jie

    2006-01-01

    The progress in the understanding of cancer progression and early detection has been slow and frustrating due to the complex multifactorial nature and heterogeneity of the cancer syndrome. To date, no effective treatment is available for advanced cancers, which remain a major cause of morbidity and mortality. Clearly, there is urgent need to unravel novel biomarkers for early detection. Most of the functional information of the cancer-associated genes resides in the proteome. The later is an exceptionally complex biological system involving several proteins that function through posttranslational modifications and dynamic intermolecular collisions with partners. These protein complexes can be regulated by signals emanating from cancer cells, their surrounding tissue microenvironment, and/or from the host. Some proteins are secreted and/or cleaved into the extracellular milieu and may represent valuable serum biomarkers for diagnosis purpose. It is estimated that the cancer proteome may include over 1.5 million proteins as a result of posttranslational processing and modifications. Such complexity clearly highlights the need for ultra-high resolution proteomic technology for robust quantitative protein measurements and data acquisition. This review is to update the current research efforts in high-resolution proteomic technology for discovery and monitoring cancer biomarkers. PMID:16625706

  3. Proteomic Profiling of Bladders from Mice Exposed with Sodium Arsenite

    EPA Science Inventory

    Arsenic, an environmental contaminant, has been linked with cancer of the bladder in humans. To study the mode of action of arsenic, female CH3 mice were exposed to 85 ppm sodium arsenite in their drinking water for 30 days. Following the exposure a comparative proteomic analysis...

  4. Selecting Sample Preparation Workflows for Mass Spectrometry-Based Proteomic and Phosphoproteomic Analysis of Patient Samples with Acute Myeloid Leukemia

    PubMed Central

    Hernandez-Valladares, Maria; Aasebø, Elise; Selheim, Frode; Berven, Frode S.; Bruserud, Øystein

    2016-01-01

    Global mass spectrometry (MS)-based proteomic and phosphoproteomic studies of acute myeloid leukemia (AML) biomarkers represent a powerful strategy to identify and confirm proteins and their phosphorylated modifications that could be applied in diagnosis and prognosis, as a support for individual treatment regimens and selection of patients for bone marrow transplant. MS-based studies require optimal and reproducible workflows that allow a satisfactory coverage of the proteome and its modifications. Preparation of samples for global MS analysis is a crucial step and it usually requires method testing, tuning and optimization. Different proteomic workflows that have been used to prepare AML patient samples for global MS analysis usually include a standard protein in-solution digestion procedure with a urea-based lysis buffer. The enrichment of phosphopeptides from AML patient samples has previously been carried out either with immobilized metal affinity chromatography (IMAC) or metal oxide affinity chromatography (MOAC). We have recently tested several methods of sample preparation for MS analysis of the AML proteome and phosphoproteome and introduced filter-aided sample preparation (FASP) as a superior methodology for the sensitive and reproducible generation of peptides from patient samples. FASP-prepared peptides can be further fractionated or IMAC-enriched for proteome or phosphoproteome analyses. Herein, we will review both in-solution and FASP-based sample preparation workflows and encourage the use of the latter for the highest protein and phosphorylation coverage and reproducibility. PMID:28248234

  5. Selecting Sample Preparation Workflows for Mass Spectrometry-Based Proteomic and Phosphoproteomic Analysis of Patient Samples with Acute Myeloid Leukemia.

    PubMed

    Hernandez-Valladares, Maria; Aasebø, Elise; Selheim, Frode; Berven, Frode S; Bruserud, Øystein

    2016-08-22

    Global mass spectrometry (MS)-based proteomic and phosphoproteomic studies of acute myeloid leukemia (AML) biomarkers represent a powerful strategy to identify and confirm proteins and their phosphorylated modifications that could be applied in diagnosis and prognosis, as a support for individual treatment regimens and selection of patients for bone marrow transplant. MS-based studies require optimal and reproducible workflows that allow a satisfactory coverage of the proteome and its modifications. Preparation of samples for global MS analysis is a crucial step and it usually requires method testing, tuning and optimization. Different proteomic workflows that have been used to prepare AML patient samples for global MS analysis usually include a standard protein in-solution digestion procedure with a urea-based lysis buffer. The enrichment of phosphopeptides from AML patient samples has previously been carried out either with immobilized metal affinity chromatography (IMAC) or metal oxide affinity chromatography (MOAC). We have recently tested several methods of sample preparation for MS analysis of the AML proteome and phosphoproteome and introduced filter-aided sample preparation (FASP) as a superior methodology for the sensitive and reproducible generation of peptides from patient samples. FASP-prepared peptides can be further fractionated or IMAC-enriched for proteome or phosphoproteome analyses. Herein, we will review both in-solution and FASP-based sample preparation workflows and encourage the use of the latter for the highest protein and phosphorylation coverage and reproducibility.

  6. Proteome Profile and Quantitative Proteomic Analysis of Buffalo (Bubalusbubalis) Follicular Fluid during Follicle Development.

    PubMed

    Fu, Qiang; Huang, Yulin; Wang, Zhiqiang; Chen, Fumei; Huang, Delun; Lu, Yangqing; Liang, Xianwei; Zhang, Ming

    2016-04-29

    Follicular fluid (FF) accumulates in the antrum of the ovarian follicle and provides the microenvironment for oocyte development. FF plays an important role in follicle growth and oocyte maturation. The FF provides a unique window to investigate the processes occurring during buffalo follicular development. The observed low quality of buffalo oocytes may arise from the poor follicular microenvironment. Investigating proteins found in buffalo FF (BFF) should provide insight into follicular development processes and provide further understanding of intra-follicular maturation and oocytes quality. Here, a proteomic-based approach was used to analyze the proteome of BFF. SDS-PAGE separation combined with mass spectrometry was used to generate the proteomic dataset. In total, 363 proteins were identified and classified by Gene Ontology terms. The proteins were assigned to 153 pathways, including signaling pathways. To evaluate difference in proteins expressed between BFF with different follicle size (small, <4 mm; and large, >8 mm), a quantitative proteomic analysis based on multi-dimensional liquid chromatography pre-fractionation tandem Orbitrap mass spectrometry identification was performed. Eleven differentially expressed proteins (six downregulated and five upregulated in large BFF) were identified and assigned to a variety of functional processes, including serine protease inhibition, oxidation protection and the complement cascade system. Three differentially expressed proteins, Vimentin, Peroxiredoxin-1 and SERPIND1, were verified by Western blotting, consistent with the quantitative proteomics results. Our datasets offers new information about proteins present in BFF and should facilitate the development of new biomarkers. These differentially expressed proteins illuminate the size-dependent protein changes in follicle microenvironment.

  7. Proteome and Transcriptome Profiles of a Her2/Neu-driven Mouse Model of Breast Cancer

    SciTech Connect

    Schoenherr, Regine M.; Kelly-Spratt, Karen S.; Lin, Chen Wei; Whiteaker, Jeffrey R.; Liu, Tao; Holzman, Ted; Coleman, Ilsa; Feng, Li-Chia; Lorentzen, Travis D.; Krasnoselsky, Alexei L.; Wang, Pei; Liu, Yan; Gurley, Kay E.; Amon, Lynn M.; Schepmoes, Athena A.; Moore, Ronald J.; Camp, David G.; Chodosh, Lewis A.; Smith, Richard D.; Nelson, Peter S.; McIntosh, Martin; Kemp, Christopher; Paulovich, Amanda G.

    2011-04-01

    In recent years, mouse models have proven to be invaluable in expanding our understanding of cancer biology. We have amassed a tremendous amount of proteomics and transcriptomics data profiling blood and tissues from a Her2-driven mouse model of breast cancer that closely recapitulates the pathology and natural history of human breast cancer. The purpose of this report is to make all of these data publicly available in raw and processed forms, as a resource to the community. Importantly, high quality biospecimens from this same mouse model are freely available through a sample repository that we established, so researchers can readily obtain samples to test biological hypotheses without the need of breeding animals and collecting biospecimens. Specifically, six proteomics and six transcriptomics datasets are available, with the former encompassing 841 liquid chromatography-tandem mass spectrometry (LC-MS/MS) experiments of both plasma and tissue samples, and the latter including 255 individual microarray analyses of five different tissue types (thymus, spleen, liver, blood cells, and breast ± laser capture microdissection). A total of 18,880 unique peptides were identified with a PeptideProphet error rate ≤1%, with 3884 non-redundant protein groups identified in five plasma datasets, and 1659 non-redundant protein groups in a tissue dataset (4977 non-redundant protein groups in total). We anticipate that these data will be of use to the community for software tool development, investigations of analytical variation in MS/MS data, development of quality control tools (multiple technical replicates are provided for a subset of the data), empirical selection of proteotypic peptides for multiple reaction monitoring mass spectrometry, and for advancing our understanding of cancer biology.

  8. Analysis and comparison of proteomic profiles of tear fluid from human, cow, sheep, and camel eyes.

    PubMed

    Shamsi, Farrukh A; Chen, Ziyan; Liang, Jingwen; Li, Kaijun; Al-Rajhi, Ali A; Chaudhry, Imtiaz A; Li, Mingtao; Wu, Kaili

    2011-11-25

    To investigate the tear proteome profiles of human, cow, sheep, and camel comparatively and to explore the difference of tear protein profiles among different species. Tears were collected from both eyes of 25 clinically healthy volunteers, 50 cows, 25 sheep, and 50 camels. Pooled tear protein samples were separated by SDS-PAGE and two-dimensional electrophoresis. Protein spots of differential expression were excised and subjected to in-gel digestion and identification by matrix assisted laser desorption/ionization-time-of-flight/time-of-flight mass spectrum analysis. Because of the incomplete genomic data of cow, sheep, and camel, a combined strategy of de novo sequencing and BLAST (Best Local Alignment Search Tool) homology searching was also used for protein identification. The differentially expressed proteins were validated by Western blot analysis. On comparison with human tears (182 ± 6 spots), 223 ± 8, 217 ± 11, and 241 ± 3 well-resolved protein spots were detected in triphenylmethane dye-stained gels of cow, sheep, and camel tears, respectively. Similar high-abundant proteins (lactoferrin, lysozyme, etc.) were found in all tear fluids. Tear lipocalins have been identified in cow and sheep tears. BLAST searching revealed a 21-kDa protein, identical with human vitelline membrane outer layer protein 1 (VMO1) homolog, in camel tears. The Western blot confirmed that VMO1 homolog was present in both camel and sheep tears but not in human and cow tears. The comparative proteomic analyses of tears from healthy humans, cows, sheep, and camels were first reported. Differential protein expression existed in the tear among species, offering useful information for further study on tear proteins and the related ocular diseases.

  9. Time-Resolved Proteomics Extends Ribosome Profiling-Based Measurements of Protein Synthesis Dynamics.

    PubMed

    Liu, Tzu-Yu; Huang, Hector H; Wheeler, Diamond; Xu, Yichen; Wells, James A; Song, Yun S; Wiita, Arun P

    2017-06-28

    Ribosome profiling is a widespread tool for studying translational dynamics in human cells. Its central assumption is that ribosome footprint density on a transcript quantitatively reflects protein synthesis. Here, we test this assumption using pulsed-SILAC (pSILAC) high-accuracy targeted proteomics. We focus on multiple myeloma cells exposed to bortezomib, a first-line chemotherapy and proteasome inhibitor. In the absence of drug effects, we found that direct measurement of protein synthesis by pSILAC correlated well with indirect measurement of synthesis from ribosome footprint density. This correlation, however, broke down under bortezomib-induced stress. By developing a statistical model integrating longitudinal proteomic and mRNA-sequencing measurements, we found that proteomics could directly detect global alterations in translational rate caused by bortezomib; these changes are not detectable by ribosomal profiling alone. Further, by incorporating pSILAC data into a gene expression model, we predict cell-stress specific proteome remodeling events. These results demonstrate that pSILAC provides an important complement to ribosome profiling in measuring proteome dynamics. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  10. Restoring Aperture Profile At Sample Plane

    SciTech Connect

    Jackson, J L; Hackel, R P; Lungershausen, A W

    2003-08-03

    Off-line conditioning of full-size optics for the National Ignition Facility required a beam delivery system to allow conditioning lasers to rapidly raster scan samples while achieving several technical goals. The main purpose of the optical system designed was to reconstruct at the sample plane the flat beam profile found at the laser aperture with significant reductions in beam wander to improve scan times. Another design goal was the ability to vary the beam size at the sample to scan at different fluences while utilizing all of the laser power and minimizing processing time. An optical solution was developed using commercial off-the-shelf lenses. The system incorporates a six meter relay telescope and two sets of focusing optics. The spacing of the focusing optics is changed to allow the fluence on the sample to vary from 2 to 14 Joules per square centimeter in discrete steps. More importantly, these optics use the special properties of image relaying to image the aperture plane onto the sample to form a pupil relay with a beam profile corresponding almost exactly to the flat profile found at the aperture. A flat beam profile speeds scanning by providing a uniform intensity across a larger area on the sample. The relayed pupil plane is more stable with regards to jitter and beam wander. Image relaying also reduces other perturbations from diffraction, scatter, and focus conditions. Image relaying, laser conditioning, and the optical system designed to accomplish the stated goals are discussed.

  11. Stromal proteome expression profile and muscle-invasive bladder cancer research

    PubMed Central

    2012-01-01

    Background To globally characterize the cancer stroma expression profile of muscle-invasive transitional cell carcinoma and to discuss the cancer biology as well as biomarker discovery from stroma. Laser capture micro dissection was used to harvest purified muscle-invasive bladder cancer stromal cells and normal urothelial stromal cells from 4 paired samples. Two-dimensional liquid chromatography tandem mass spectrometry was used to identify the proteome expression profile. The differential proteins were further analyzed using bioinformatics tools and compared with the published literature. Results We identified 868/872 commonly expressed proteins and 978 differential proteins from 4 paired cancer and normal stromal samples using laser capture micro dissection coupled with two-dimensional liquid chromatography tandem mass spectrometry. 487/491 proteins uniquely expressed in cancer/normal stroma. Differential proteins were compared with the entire list of the international protein index (IPI), and there were 42/42 gene ontology (GO) terms exhibited as enriched and 8/5 exhibited as depleted in cellular Component, respectively. Significantly altered pathways between cancer/normal stroma mainly include metabolic pathways, ribosome, focal adhesion, etc. Finally, descriptive statistics show that the stromal proteins with extremes of PI and MW have the same probability to be a biomarker. Conclusions Based on our results, stromal cells are essential component of the cancer, biomarker discovery and network based multi target therapy should consider neoplastic cells itself and corresponding stroma as whole one. PMID:22920603

  12. Mass Spectrometry–based Proteomic Profiling of Lung Cancer

    PubMed Central

    Ocak, Sebahat; Chaurand, Pierre; Massion, Pierre P.

    2009-01-01

    In an effort to further our understanding of lung cancer biology and to identify new candidate biomarkers to be used in the management of lung cancer, we need to probe these tissues and biological fluids with tools that address the biology of lung cancer directly at the protein level. Proteins are responsible of the function and phenotype of cells. Cancer cells express proteins that distinguish them from normal cells. Proteomics is defined as the study of the proteome, the complete set of proteins produced by a species, using the technologies of large-scale protein separation and identification. As a result, new technologies are being developed to allow the rapid and systematic analysis of thousands of proteins. The analytical advantages of mass spectrometry (MS), including sensitivity and high-throughput, promise to make it a mainstay of novel biomarker discovery to differentiate cancer from normal cells and to predict individuals likely to develop or recur with lung cancer. In this review, we summarize the progress made in clinical proteomics as it applies to the management of lung cancer. We will focus our discussion on how MS approaches may advance the areas of early detection, response to therapy, and prognostic evaluation. PMID:19349484

  13. Proteome profiling of human epithelial ovarian cancer cell line TOV-112D.

    PubMed

    Gagné, Jean-Philippe; Gagné, Pierre; Hunter, Joanna M; Bonicalzi, Marie-Eve; Lemay, Jean-François; Kelly, Isabelle; Le Page, Cécile; Provencher, Diane; Mes-Masson, Anne-Marie; Droit, Amaud; Bourgais, David; Poirier, Guy G

    2005-07-01

    A proteome profiling of the epithelial ovarian cancer cell line TOV-112D was initiated as a protein expression reference in the study of ovarian cancer. Two complementary proteomic approaches were used in order to maximise protein identification: two-dimensional gel electrophoresis (2DE) protein separation coupled to matrix assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) and one-dimensional gel electrophoresis (1DE) coupled to liquid-chromatography tandem mass spectrometry (LC MS/MS). One hundred and seventy-two proteins have been identified among 288 spots selected on two-dimensional gels and a total of 579 proteins were identified with the 1DE LC MS/MS approach. This proteome profiling covers a wide range of protein expression and identifies several proteins known for their oncogenic properties. Bioinformatics tools were used to mine databases in order to determine whether the identified proteins have previously been implicated in pathways associated with carcinogenesis or cell proliferation. Indeed, several of the proteins have been reported to be specific ovarian cancer markers while others are common to many tumorigenic tissues or proliferating cells. The diversity of proteins found and their association with known oncogenic pathways validate this proteomic approach. The proteome 2D map of the TOV-112D cell line will provide a valuable resource in studies on differential protein expression of human ovarian carcinomas while the 1DE LC MS/MS approach gives a picture of the actual protein profile of the TOV-112D cell line. This work represents one of the most complete ovarian protein expression analysis reports to date and the first comparative study of gene expression profiling and proteomic patterns in ovarian cancer.

  14. [Direct proteomic profiling of human urine and blood serum in an experiment with 5-day dry immersion].

    PubMed

    2012-01-01

    Changes in proteome of urine and blood serum obtained from 14 healthy humans (age 21-29 yrs) medically certified for an experiment with dry immersion were analyzed. Urine and serum samples were pre-fractionated and enriched with magnetic particles MB-WCX and MB-HIC, respectively, on robot ClinProt (Bruker Daltonics) for direct mass-spectrometry profiling by MALDI-TOF. As a result, 143 protein peaks on the average were identified in urine samples. It was shown that a high variation coefficient in 23.7% of protein peaks, i.e. double technical, points to the most plastic fraction of the urine proteome. In blood serum, 175 peaks were identified in a sample on the average. Comparison of baseline and immersion mass-spectra of the blood proteome revealed significant differences. Increased peak areas of several protein fragments--C3 and C4 fragments of complement system, high-molecular kininogen and fibrinogen--can be ascribed to human body adaptation to the experimental conditions.

  15. Comparative proteomic and transcriptomic profile of Staphylococcus epidermidis biofilms grown in glucose-enriched medium.

    PubMed

    Carvalhais, Virginia; França, Angela; Pier, Gerald B; Vilanova, Manuel; Cerca, Nuno; Vitorino, Rui

    2015-01-01

    Staphylococcus epidermidis is an important nosocomial agent among carriers of indwelling medical devices, due to its strong ability to form biofilms on inert surfaces. Contrary to some advances made in the transcriptomic field, proteome characterization of S. epidermidis biofilms is less developed. To highlight the relation between transcripts and proteins of S. epidermidis biofilms, we analyzed the proteomic profile obtained by two mechanical lysis methods (sonication and bead beating), associated with two distinct detergent extraction buffers, namely SDS and CHAPS. Based on gel electrophoresis-LC-MS/MS, we identified a total of 453 proteins. While lysis with glass beads provided greater amounts of protein, CHAPS extraction buffer allowed identification of a higher number of proteins compared to SDS. Our data shows the impact of different protein isolation methods in the characterization of the S. epidermidis biofilm proteome. Furthermore, the correlation between proteomic and transcriptomic profiles was evaluated. The results confirmed that proteomic and transcriptomic data should be analyzed simultaneously in order to have a comprehensive understanding of a specific microbiological condition. Copyright © 2014 Elsevier B.V. All rights reserved.

  16. Investigating the Correspondence Between Transcriptomic and Proteomic Expression Profiles Using Coupled Cluster Models.

    SciTech Connect

    Rogers, Simon; Girolami, Mark; Kolch, Walter; Waters, Katrina M.; Liu, Tao; Thrall, Brian D.; Wiley, H. S.

    2008-12-01

    Modern transcriptomics and proteomics enable us to survey the expression of RNAs and proteins at large scales. While these data are usually generated and analysed separately, there is an increasing interest in comparing and co-analysing transcriptome and proteome expression data. A major open question is whether transcriptome and proteome expression is linked and how it is coordinated. Results: Here we have developed a probabilistic clustering model that permits analysis of the links between transcriptomic and proteomic profiles in a sensible and flexible manner. Our coupled mixture model defines a prior probability distribution over the component to which a protein profile should be assigned conditioned on which component the associated mRNA profile belongs to. By providing probabilistic assignments this approach sits between the two extremes of concatenating the data on the assumption that mRNA and protein clusters would have a one-to-one relationship, and independent clustering where the mRNA profile provides no information on the protein profile and vice-versa. We apply this approach to a large dataset of quantitative transcriptomic and proteomic expression data obtained from a human breast epithelial cell line (HMEC) stimulated by epidermal growth factor (EGF) over a series of timepoints corresponding to one cell cycle. The results reveal a complex relationship between transcriptome and proteome with most mRNA clusters linked to at least two protein clusters, and vice versa. A more detailed analysis incorporating information on gene function from the gene ontology database shows that a high correlation of mRNA and protein expression is limited to the components of some molecular machines, such as the ribosome, cell adhesion complexes and the TCP-1 chaperonin involved in protein folding. Conclusions: The dynamic regulation of the transcriptome and proteome in mammalian cells in response to an acute mitogenic stimulus appears largely independent with very little

  17. An individual urinary proteome analysis in normal human beings to define the minimal sample number to represent the normal urinary proteome

    PubMed Central

    2012-01-01

    Background The urinary proteome has been widely used for biomarker discovery. A urinary proteome database from normal humans can provide a background for discovery proteomics and candidate proteins/peptides for targeted proteomics. Therefore, it is necessary to define the minimum number of individuals required for sampling to represent the normal urinary proteome. Methods In this study, inter-individual and inter-gender variations of urinary proteome were taken into consideration to achieve a representative database. An individual analysis was performed on overnight urine samples from 20 normal volunteers (10 males and 10 females) by 1DLC/MS/MS. To obtain a representative result of each sample, a replicate 1DLCMS/MS analysis was performed. The minimal sample number was estimated by statistical analysis. Results For qualitative analysis, less than 5% of new proteins/peptides were identified in a male/female normal group by adding a new sample when the sample number exceeded nine. In addition, in a normal group, the percentage of newly identified proteins/peptides was less than 5% upon adding a new sample when the sample number reached 10. Furthermore, a statistical analysis indicated that urinary proteomes from normal males and females showed different patterns. For quantitative analysis, the variation of protein abundance was defined by spectrum count and western blotting methods. And then the minimal sample number for quantitative proteomic analysis was identified. Conclusions For qualitative analysis, when considering the inter-individual and inter-gender variations, the minimum sample number is 10 and requires a balanced number of males and females in order to obtain a representative normal human urinary proteome. For quantitative analysis, the minimal sample number is much greater than that for qualitative analysis and depends on the experimental methods used for quantification. PMID:23170922

  18. Extending the limits of quantitative proteome profiling with data-independent acquisition and application to acetaminophen-treated three-dimensional liver microtissues.

    PubMed

    Bruderer, Roland; Bernhardt, Oliver M; Gandhi, Tejas; Miladinović, Saša M; Cheng, Lin-Yang; Messner, Simon; Ehrenberger, Tobias; Zanotelli, Vito; Butscheid, Yulia; Escher, Claudia; Vitek, Olga; Rinner, Oliver; Reiter, Lukas

    2015-05-01

    The data-independent acquisition (DIA) approach has recently been introduced as a novel mass spectrometric method that promises to combine the high content aspect of shotgun proteomics with the reproducibility and precision of selected reaction monitoring. Here, we evaluate, whether SWATH-MS type DIA effectively translates into a better protein profiling as compared with the established shotgun proteomics. We implemented a novel DIA method on the widely used Orbitrap platform and used retention-time-normalized (iRT) spectral libraries for targeted data extraction using Spectronaut. We call this combination hyper reaction monitoring (HRM). Using a controlled sample set, we show that HRM outperformed shotgun proteomics both in the number of consistently identified peptides across multiple measurements and quantification of differentially abundant proteins. The reproducibility of HRM in peptide detection was above 98%, resulting in quasi complete data sets compared with 49% of shotgun proteomics. Utilizing HRM, we profiled acetaminophen (APAP)(1)-treated three-dimensional human liver microtissues. An early onset of relevant proteome changes was revealed at subtoxic doses of APAP. Further, we detected and quantified for the first time human NAPQI-protein adducts that might be relevant for the toxicity of APAP. The adducts were identified on four mitochondrial oxidative stress related proteins (GATM, PARK7, PRDX6, and VDAC2) and two other proteins (ANXA2 and FTCD). Our findings imply that DIA should be the preferred method for quantitative protein profiling.

  19. A proteogenomics approach integrating proteomics and ribosome profiling increases the efficiency of protein identification and enables the discovery of alternative translation start sites.

    PubMed

    Koch, Alexander; Gawron, Daria; Steyaert, Sandra; Ndah, Elvis; Crappé, Jeroen; De Keulenaer, Sarah; De Meester, Ellen; Ma, Ming; Shen, Ben; Gevaert, Kris; Van Criekinge, Wim; Van Damme, Petra; Menschaert, Gerben

    2014-12-01

    Next-generation transcriptome sequencing is increasingly integrated with MS to enhance MS-based protein and peptide identification. Recently, a breakthrough in transcriptome analysis was achieved with the development of ribosome profiling (ribo-seq). This technology is based on the deep sequencing of ribosome-protected mRNA fragments, thereby enabling the direct observation of in vivo protein synthesis at the transcript level. In order to explore the impact of a ribo-seq-derived protein sequence search space on MS/MS spectrum identification, we performed a comprehensive proteome study on a human cancer cell line, using both shotgun and N-terminal proteomics, next to ribosome profiling, which was used to delineate (alternative) translational reading frames. By including protein-level evidence of sample-specific genetic variation and alternative translation, this strategy improved the identification score of 69 proteins and identified 22 new proteins in the shotgun experiment. Furthermore, we discovered 18 new alternative translation start sites in the N-terminal proteomics data and observed a correlation between the quantitative measures of ribo-seq and shotgun proteomics with a Pearson correlation coefficient ranging from 0.483 to 0.664. Overall, this study demonstrated the benefits of ribosome profiling for MS-based protein and peptide identification and we believe this approach could develop into a common practice for next-generation proteomics.

  20. Extending the Limits of Quantitative Proteome Profiling with Data-Independent Acquisition and Application to Acetaminophen-Treated Three-Dimensional Liver Microtissues*

    PubMed Central

    Bruderer, Roland; Bernhardt, Oliver M.; Gandhi, Tejas; Miladinović, Saša M.; Cheng, Lin-Yang; Messner, Simon; Ehrenberger, Tobias; Zanotelli, Vito; Butscheid, Yulia; Escher, Claudia; Vitek, Olga; Rinner, Oliver; Reiter, Lukas

    2015-01-01

    The data-independent acquisition (DIA) approach has recently been introduced as a novel mass spectrometric method that promises to combine the high content aspect of shotgun proteomics with the reproducibility and precision of selected reaction monitoring. Here, we evaluate, whether SWATH-MS type DIA effectively translates into a better protein profiling as compared with the established shotgun proteomics. We implemented a novel DIA method on the widely used Orbitrap platform and used retention-time-normalized (iRT) spectral libraries for targeted data extraction using Spectronaut. We call this combination hyper reaction monitoring (HRM). Using a controlled sample set, we show that HRM outperformed shotgun proteomics both in the number of consistently identified peptides across multiple measurements and quantification of differentially abundant proteins. The reproducibility of HRM in peptide detection was above 98%, resulting in quasi complete data sets compared with 49% of shotgun proteomics. Utilizing HRM, we profiled acetaminophen (APAP)1-treated three-dimensional human liver microtissues. An early onset of relevant proteome changes was revealed at subtoxic doses of APAP. Further, we detected and quantified for the first time human NAPQI-protein adducts that might be relevant for the toxicity of APAP. The adducts were identified on four mitochondrial oxidative stress related proteins (GATM, PARK7, PRDX6, and VDAC2) and two other proteins (ANXA2 and FTCD). Our findings imply that DIA should be the preferred method for quantitative protein profiling. PMID:25724911

  1. Identification of a metabolizing enzyme in human kidney by proteomic correlation profiling.

    PubMed

    Sakurai, Hidetaka; Kubota, Kazuishi; Inaba, Shin-Ichi; Takanaka, Kaoru; Shinagawa, Akira

    2013-08-01

    Molecular identification of endogenous enzymes and biologically active substances from complex biological sources remains a challenging task, and although traditional biochemical purification is sometimes regarded as outdated, it remains one of the most powerful methodologies for this purpose. While biochemical purification usually requires large amounts of starting material and many separation steps, we developed an advanced method named "proteomic correlation profiling" in our previous study. In proteomic correlation profiling, we first fractionated biological material by column chromatography, and then calculated each protein's correlation coefficient between the enzyme activity profile and protein abundance profile determined by proteomics technology toward fractions. Thereafter, we could choose possible candidates for the enzyme among proteins with a high correlation value by domain predictions using informatics tools. Ultimately, this streamlined procedure requires fewer purification steps and reduces starting materials dramatically due to low required purity compared with conventional approaches. To demonstrate the generality of this approach, we have now applied an improved workflow of proteomic correlation profiling to a drug metabolizing enzyme and successfully identified alkaline phosphatase, tissue-nonspecific isozyme (ALPL) as a phosphatase of CS-0777 phosphate (CS-0777-P), a selective sphingosine 1-phosphate receptor 1 modulator with potential benefits in the treatment of autoimmune diseases including multiple sclerosis, from human kidney extract. We identified ALPL as a candidate protein only by the 200-fold purification and only from 1 g of human kidney. The identification of ALPL as CS-0777-P phosphatase was strongly supported by a recombinant protein, and contribution of the enzyme in human kidney extract was validated by immunodepletion and a specific inhibitor. This approach can be applied to any kind of enzyme class and biologically active

  2. Redox Proteomics in Human Biofluids: Sample Preparation, Separation and Immunochemical Tagging for Analysis of Protein Oxidation.

    PubMed

    Di Domenico, Fabio; Perluigi, Marzia; Butterfield, D Allan

    2016-01-01

    Proteomics offers the simultaneous detection of a large number of proteins in a single experiment and can provide important information regarding crucial aspects of specific proteins, particularly post-translational modifications (PTMs). Investigations of oxidative PTMs are currently performed using focused redox proteomics techniques, which rely on gel electrophoresis separations of intact proteins with the final detection of oxidative PTMs being performed by mass spectrometry (MS) analysis. The application of this technique to human biofluids is being subject of increasing investigation and is expected to provide new insights on the oxidative status of the peripheral proteome in neurological diseases such as Alzheimer's disease, towards purposes of early diagnosis and prognosis. This chapter describes all the experimental steps to perform redox proteomics analysis of cerebrospinal fluid and plasma/serum samples.

  3. Simple Sodium Dodecyl Sulfate-Assisted Sample Preparation Method for LC-MS-based Proteomic Applications

    SciTech Connect

    Zhou, Jianying; Dann, Geoffrey P.; Shi, Tujin; Wang, Lu; Gao, Xiaoli; Su, Dian; Nicora, Carrie D.; Shukla, Anil K.; Moore, Ronald J.; Liu, Tao; Camp, David G.; Smith, Richard D.; Qian, Weijun

    2012-03-10

    Sodium dodecyl sulfate (SDS) is one of the most popular laboratory reagents used for highly efficient biological sample extraction; however, SDS presents a significant challenge to LC-MS-based proteomic analyses due to its severe interference with reversed-phase LC separations and electrospray ionization interfaces. This study reports a simple SDS-assisted proteomic sample preparation method facilitated by a novel peptide-level SDS removal protocol. After SDS-assisted protein extraction and digestion, SDS was effectively (>99.9%) removed from peptides through ion substitution-mediated DS- precipitation with potassium chloride (KCl) followed by {approx}10 min centrifugation. Excellent peptide recovery (>95%) was observed for less than 20 {mu}g of peptides. Further experiments demonstrated the compatibility of this protocol with LC-MS/MS analyses. The resulting proteome coverage from this SDS-assisted protocol was comparable to or better than those obtained from other standard proteomic preparation methods in both mammalian tissues and bacterial samples. These results suggest that this SDS-assisted protocol is a practical, simple, and broadly applicable proteomic sample processing method, which can be particularly useful when dealing with samples difficult to solubilize by other methods.

  4. Proteomics in globe artichoke: protein extraction and sample complexity reduction by PEG fractionation.

    PubMed

    Acquadro, Alberto; Falvo, Sara; Mila, Silvia; Giuliano Albo, Alessandra; Comino, Cinzia; Moglia, Andrea; Lanteri, Sergio

    2009-05-01

    Here, we report the first leaf proteome analysis for globe artichoke. Three protein extraction protocols were tested and a reproducible Mg/NP-40-based method was established. Ribulose-1,5-biphosphate carboxylase-oxygenase (RuBisCO) is a highly abundant leaf protein, and its presence masks co-localizing, less abundant proteins. To remove RuBisCO from the sample, and thereby improve spot resolution, a PEG fractionation approach was elaborated. 2-DE profiles of various PEG fractions showed that the fractionation procedure was successful in excluding most of the RuBisCO, allowing for the detection of many low-abundance proteins. Western blot analysis was able to confirm the reduction in RuBisCO content achieved by PEG fractionation. In all, 841 distinct protein spots were detected, and 40 of these, selected from the RuBisCO region of the 2-DE profile, were successfully identified by MS. A number of homologues of these proteins also co-localize with RuBisCO in Arabidopsis thaliana.

  5. Serum proteome profiling detects myelodysplastic syndromes and identifies CXC chemokine ligands 4 and 7 as markers for advanced disease

    PubMed Central

    Aivado, Manuel; Spentzos, Dimitrios; Germing, Ulrich; Alterovitz, Gil; Meng, Xiao-Ying; Grall, Franck; Giagounidis, Aristoteles A. N.; Klement, Giannoula; Steidl, Ulrich; Otu, Hasan H.; Czibere, Akos; Prall, Wolf C.; Iking-Konert, Christof; Shayne, Michelle; Ramoni, Marco F.; Gattermann, Norbert; Haas, Rainer; Mitsiades, Constantine S.; Fung, Eric T.; Libermann, Towia A.

    2007-01-01

    Myelodysplastic syndromes (MDS) are among the most frequent hematologic malignancies. Patients have a short survival and often progress to acute myeloid leukemia. The diagnosis of MDS can be difficult; there is a paucity of molecular markers, and the pathophysiology is largely unknown. Therefore, we conducted a multicenter study investigating whether serum proteome profiling may serve as a noninvasive platform to discover novel molecular markers for MDS. We generated serum proteome profiles from 218 individuals by MS and identified a profile that distinguishes MDS from non-MDS cytopenias in a learning sample set. This profile was validated by testing its ability to predict MDS in a first independent validation set and a second, prospectively collected, independent validation set run 5 months apart. Accuracy was 80.5% in the first and 79.0% in the second validation set. Peptide mass fingerprinting and quadrupole TOF MS identified two differential proteins: CXC chemokine ligands 4 (CXCL4) and 7 (CXCL7), both of which had significantly decreased serum levels in MDS, as confirmed with independent antibody assays. Western blot analyses of platelet lysates for these two platelet-derived molecules revealed a lack of CXCL4 and CXCL7 in MDS. Subtype analyses revealed that these two proteins have decreased serum levels in advanced MDS, suggesting the possibility of a concerted disturbance of transcription or translation of these chemokines in advanced MDS. PMID:17220270

  6. PiB-PET Imaging-Based Serum Proteome Profiles Predict Mild Cognitive Impairment and Alzheimer's Disease.

    PubMed

    Kang, Seokjo; Jeong, Hyobin; Baek, Je-Hyun; Lee, Seung-Jin; Han, Sun-Ho; Cho, Hyun Jin; Kim, Hee; Hong, Hyun Seok; Kim, Young Ho; Yi, Eugene C; Seo, Sang Won; Na, Duk L; Hwang, Daehee; Mook-Jung, Inhee

    2016-07-06

    Development of a simple, non-invasive early diagnosis platform of Alzheimer's disease (AD) using blood is urgently required. Recently, PiB-PET imaging has been shown to be powerful to quantify amyloid-β plaque loads leading to pathophysiological alterations in AD brains. Thus, there has been a need for serum biomarkers reflecting PiB-PET imaging data as an early diagnosis platform of AD. Here, using LC-MS/MS analysis coupled with isobaric tagging, we performed comprehensive proteome profiling of serum samples from cognitively normal controls, mild cognitive impairment (MCI), and AD patients, who were selected using PiB-PET imaging. Comparative analysis of the proteomes revealed 79 and 72 differentially expressed proteins in MCI and AD, respectively, compared to controls. Integrated analysis of these proteins with genomic and proteomic data of AD brain tissues, together with network analysis, identified three biomarker candidates representing the altered proteolysis-related process in MCI or AD: proprotein convertase subtilisin/kexin type 9 (PCSK9), coagulation factor XIII, A1 polypeptide (F13A1), and dermcidin (DCD). In independent serum samples of MCI and AD, we confirmed the elevation of the candidates using western blotting and ELISA. Our results suggest that these biomarker candidates can serve as a potential non-invasive early diagnosis platform reflecting PiB-PET imaging for MCI and AD.

  7. Quantitative Proteomic Profiling of Early and Late Responses to Salicylic Acid in Cucumber Leaves

    PubMed Central

    Li, Liang; Shang, Qing-Mao

    2016-01-01

    Salicylic acid (SA) is an important phytohormone that plays vital regulatory roles in plant growth, development, and stress responses. However, studies on the molecular mechanism of SA, especially during the early SA responses, are lagging behind. In this study, we initiated a comprehensive isobaric tag for relative and absolute quantitation (iTRAQ)-based proteomic analysis to explore the early and late SA-responsive proteins in leaves of cucumber (Cucumis sativus L.) seedlings. Upon SA application through the roots, endogenous SA accumulated in cucumber leaves. By assaying the changes in marker gene expression and photosynthetic rate, we collected samples at 12 h and 72 h post treatment (hpt) to profile the early and late SA responsiveness, respectively. The iTRAQ assay followed by tandem mass spectrometry revealed 135 differentially expressed proteins (DEPs) at 12 hpt and 301 DEPs at 72 hpt. The functional categories for these SA-responsive proteins included in a variety of biochemical processes, including photosynthesis, redox homeostasis, carbohydrate and energy metabolism, lipid metabolism, transport, protein folding and modification, proteolysis, cell wall organization, and the secondary phenylpropanoid pathway. Conclusively, based on the abundant changes of these DEPs, together with their putative functions, we proposed a possible SA-responsive protein network. It appears that SA could elicit reactive oxygen species (ROS) production via enhancing the photosynthetic electron transferring, and then confer some growth-promoting and stress-priming effects on cells during the late phase, including enhanced photosynthesis and ROS scavenging, altered carbon metabolic flux for the biosynthesis of amino acids and nucleotides, and cell wall reorganization. Overall, the present iTRAQ assay provides higher proteome coverage and deepened our understanding of the molecular basis of SA-responses. PMID:27551830

  8. Proteomic profiling of the infective trophozoite stage of Acanthamoeba polyphaga.

    PubMed

    Caumo, Karin Silva; Monteiro, Karina Mariante; Ott, Thiely Rodrigues; Maschio, Vinicius José; Wagner, Glauber; Ferreira, Henrique Bunselmeyer; Rott, Marilise Brittes

    2014-12-01

    Acanthamoeba polyphaga is a free-living protozoan pathogen, whose infective trophozoite form is capable of causing a blinding keratitis and fatal granulomatous encephalitis in humans. The damage caused by A. polyphaga trophozoites in human corneal or brain infections is the result of several different pathogenic mechanisms that have not yet been elucidated at the molecular level. We performed a comprehensive analysis of the proteins expressed by A. polyphaga trophozoites, based on complementary 2-DE MS/MS and gel-free LC-MS/MS approaches. Overall, 202 non-redundant proteins were identified. An A. polyphaga proteomic map in the pH range 3-10 was produced, with protein identification for 184 of 370 resolved spots, corresponding to 142 proteins. Additionally, 94 proteins were identified by gel-free LC-MS/MS. Functional classification revealed several proteins with potential importance for pathogen survival and infection of mammalian hosts, including surface proteins and proteins related to defense mechanisms. Our study provided the first comprehensive proteomic survey of the trophozoite infective stage of an Acanthamoeba species, and established foundations for prospective, comparative and functional studies of proteins involved in mechanisms of survival, development, and pathogenicity in A. polyphaga and other pathogenic amoebae.

  9. Transcriptional and proteomic profiling of flatfish (Solea senegalensis) spermatogenesis.

    PubMed

    Forné, Ignasi; Castellana, Bárbara; Marín-Juez, Rubén; Cerdà, Joan; Abián, Joaquín; Planas, Josep V

    2011-06-01

    The Senegalese sole (Solea senegalensis) is a marine flatfish of high economic value and a target species for aquaculture. The efforts to reproduce this species in captivity have been hampered by the fact that farmed males (F1) often show lower sperm production and fertilization capacity than wild-type males (F0). Our knowledge on spermatogenesis is however limited to a few studies. In a previous work, we identified by 2-D DIGE several potential protein markers in testis for the poor reproductive performance of F1 males. Therefore, the objectives of the present study were, first, to investigate changes in genes and proteins expressed in the testis throughout spermatogenesis in F0 males by using a combination of transcriptomic and proteomic approaches and, second, to further compare the testis proteome between late spermatogenic stages of F0 and F1 fish to identify potential indicators of hampered reproductive performance in F1 fish. We identified approximately 400 genes and 49 proteins that are differentially expressed during the progression of spermatogenesis and that participate in processes such as transcriptional activation, the ubiquitin-proteasome system, sperm maturation and motility or cytoskeletal remodeling. Interestingly, a number of these proteins differed in abundance between F0 and F1 fish, pointing toward alterations in cytoskeleton, sperm motility, the ubiquitin-proteasome system and the redox state during spermiogenesis as possible causes for the decreased fertility of F1 fish.

  10. Radiation-related changes in serum proteome profiles detected by mass spectrometry in blood of patients treated with radiotherapy due to larynx cancer.

    PubMed

    Widłak, Piotr; Pietrowska, Monika; Wojtkiewicz, Katarzyna; Rutkowski, Tomasz; Wygoda, Andrzej; Marczak, Lukasz; Marczyk, Michał; Polańska, Joanna; Walaszczyk, Anna; Domińczyk, Iwona; Składowski, Krzysztof; Stobiecki, Maciej; Polański, Andrzej

    2011-01-01

    The study aimed to detect features of human serum proteome that were associated with exposure to ionizing radiation. The analyzed group consisted of 46 patients treated with radical radiotherapy for larynx cancer; patients were irradiated with total doses in a range from 51 to 72 Gy. Three consecutive blood samples were collected from each patient: before the start, 2 weeks after the start, and 4-6 weeks after the end of radiotherapy. The low-molecular-weight fraction of the serum proteome (2,000-13,000 Da) was analyzed by the MALDI-ToF mass spectrometry. Proteome profiles of serum samples collected before the start of radiotherapy and during the early stage of the treatment were similar. In marked contrast, mass profiles of serum samples collected several weeks after the end of the treatment revealed clear changes. We found that 41 out of 312 registered peptide ions changed their abundance significantly when serum samples collected after the final irradiation were compared with samples collected at the two earlier time points. We also found that abundances of certain serum peptides were associated with total doses of radiation received by patients. The results of this pilot study indicate that features of serum proteome analyzed by mass spectrometry have potential applicability as a retrospective marker of exposure to ionizing radiation.

  11. Methods for Investigation of Targeted Kinase Inhibitor Therapy using Chemical Proteomics and Phosphorylation Profiling

    PubMed Central

    Fang, Bin; Haura, Eric B.; Smalley, Keiran S.; Eschrich, Steven A.; Koomen, John M.

    2010-01-01

    Phosphorylation acts as a molecular switch for many regulatory events in signaling pathways that drive cell division, proliferation, and apoptosis. Because of the critical nature of these protein post-translational modifications in cancer, drug development programs often focus on inhibitors for kinases and phosphatases, which control protein phosphorylation. Numerous kinase inhibitors have entered clinical use, but prediction of their efficacy and a molecular basis for patient response remain uncertain. Chemical proteomics, the combination of drug affinity chromatography with mass spectrometry, identifies potential target proteins that bind to the drugs. Phosphorylation profiling can complement chemical proteomics by cataloging modifications in the target kinases and their downstream substrates using phosphopeptide enrichment and quantitative mass spectrometry. These experiments shed light on the mechanism of disease development and illuminate candidate biomarkers to guide personalized therapeutic strategies. In this review, commonly applied technologies and workflows are discussed to illustrate the role of proteomics in examining tumor biology and therapeutic intervention using kinase inhibitors. PMID:20361944

  12. Functionalized magnetic nanoparticles for sample preparation in proteomics and peptidomics analysis.

    PubMed

    Li, Yan; Zhang, Xiangmin; Deng, Chunhui

    2013-11-07

    Sample preparation is a fundamental step in the proteomics and peptidomics workflow. Due to their good biocompatibility, superparamagnetic property, and high binding capacity, magnetic nanoparticles (MNPs) functionalized with different active moieties have been widely applied in recent years in various sample preparation procedures in proteomics and peptidomics analysis. The magnetic cores of the MNPs facilitate elegant handling using only magnetic devices and their small diameters are advantageous for increasing the sensitivity when using subsequent mass spectrometry (MS) analysis or gel electrophoresis. This review mainly focuses on overviewing present advances in the preparation and application of functionalized magnetic nanoparticles for sample preparation in proteomics and peptidomics analysis, including protein digestion, enrichment of low-abundance peptides/proteins and specific enrichment of peptides/proteins with post-translational modifications, such as phosphorylation and glycosylation.

  13. Profiling the Aspergillus fumigatus proteome in response to caspofungin.

    PubMed

    Cagas, Steven E; Jain, Mohit Raja; Li, Hong; Perlin, David S

    2011-01-01

    The proteomic response of Aspergillus fumigatus to caspofungin was evaluated by gel-free isobaric tagging for relative and absolute quantitation (iTRAQ) as a means to determine potential biomarkers of drug action. A cell fractionation approach yielding 4 subcellular compartment fractions was used to enhance the resolution of proteins for proteomic analysis. Using iTRAQ, a total of 471 unique proteins were identified in soluble and cell wall/plasma membrane fractions at 24 and 48 h of growth in rich media in a wild-type drug-susceptible strain. A total of 122 proteins showed at least a 2-fold change in relative abundance following exposure to caspofungin (CSF) at just below the minimum effective concentration (0.12 μg/ml). The largest changes were seen in the mitochondrial hypoxia response domain protein (AFUA_1G12250), the level of which decreased >16-fold in the secreted fraction, and ChiA1, the level of which decreased 12.1-fold in the cell wall/plasma membrane fraction. The level of the major allergen and cytotoxin AspF1 was also shown to decrease by 12.1-fold upon the addition of drug. A subsequent iTRAQ analysis of an echinocandin-resistant strain (fks1-S678P) was used to validate proteins specific to drug action. A total of 103 proteins in the 2 fractions tested by iTRAQ were differentially expressed in the wild-type susceptible strain but not significantly changed in the resistant strain. Of these potential biomarkers, 11 had levels that changed at least 12-fold. Microarray analysis of the susceptible strain was performed to evaluate the correlation between proteomics and genomics, with a total of 117 genes found to be changing at least 2-fold. Of these, a total of 22 proteins with significant changes identified by iTRAQ also showed significant gene expression level changes by microarray. Overall, these data have the potential to identify biomarkers that assess the relative efficacy of echinocandin drug therapy.

  14. Quantitative Proteome Profiling of Street Rabies Virus-Infected Mouse Hippocampal Synaptosomes.

    PubMed

    Sun, Xiaoning; Shi, Ning; Li, Ying; Dong, Chunyan; Zhang, Maolin; Guan, Zhenhong; Duan, Ming

    2016-09-01

    It is well established now that neuronal dysfunction rather than structural damage may be responsible for the development of rabies. In order to explore the underlying mechanisms in rabies virus (RABV) and synaptic dysfunctions, a quantitative proteome profiling was carried out on synaptosome samples from mice hippocampus. Synaptosome samples from mice hippocampus were isolated and confirmed by Western blot and transmission electron microscopy. Synaptosome protein content changes were quantitatively detected by Nano-LC-MS/MS. Protein functions were classified by the Gene Ontology (GO) and KEGG pathway. PSICQUIC was used to create a network. MCODE algorithm was applied to obtain subnetworks. Of these protein changes, 45 were upregulated and 14 were downregulated following RABV infection relative to non-infected (mock) synaptosomes. 28 proteins were unique to mock treatment and 12 were unique to RABV treatment. Proteins related to metabolism and synaptic vesicle showed the most changes in expression levels. Furthermore, protein-protein interaction (PPI) networks revealed that several key biological processes related to synaptic functions potentially were modulated by RABV, including energy metabolism, cytoskeleton organization, and synaptic transmission. These data will be useful for better understanding of neuronal dysfunction of rabies and provide the foundation for future research.

  15. Proteomic Profiling of Bifidobacterium bifidum S17 Cultivated Under In Vitro Conditions

    PubMed Central

    Wei, Xiao; Wang, Simiao; Zhao, Xiangna; Wang, Xuesong; Li, Huan; Lin, Weishi; Lu, Jing; Zhurina, Daria; Li, Boxing; Riedel, Christian U.; Sun, Yansong; Yuan, Jing

    2016-01-01

    Bifidobacteria are frequently used in probiotic food and dairy products. Bifidobacterium bifidum S17 is a promising probiotic candidate strain that displays strong adhesion to intestinal epithelial cells and elicits potent anti-inflammatory capacity both in vitro and in murine models of colitis. The recently sequenced genome of B. bifidum S17 has a size of about 2.2 Mb and encodes 1,782 predicted protein-coding genes. In the present study, a comprehensive proteomic profiling was carried out to identify and characterize proteins expressed by B. bifidum S17. A total of 1148 proteins entries were identified by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), representing 64.4% of the predicted proteome. 719 proteins could be assigned to functional categories according to cluster of orthologous groups of proteins (COGs). The COG distribution of the detected proteins highly correlates with that of the complete predicted proteome suggesting a good coverage and representation of the genomic content of B. bifidum S17 by the proteome. COGs that were highly present in the proteome of B. bifidum S17 were Translation, Amino Acid Transport and Metabolism, and Carbohydrate Transport and Metabolism. Complete sets of enzymes for both the bifidus shunt and the Embden-Meyerh of pathway were identified. Further bioinformatic analysis yielded 28 proteins with a predicted extracellular localization including 14 proteins with an LPxTG-motif for cell wall anchoring and two proteins (elongation factor Tu and enolase) with a potential moonlighting function in adhesion. Amongst the predicted extracellular proteins were five of six pilin proteins encoded in the B. bifidum S17 genome as well as several other proteins with a potential role in interaction with host structures. The presented results are the first compilation of a proteomic reference profile for a B. bifidum strain and will facilitate analysis of the molecular mechanisms of physiology, host-interactions and

  16. Proteomics profiling of ethylene-induced tomato flower pedicel abscission.

    PubMed

    Zhang, Xiao-lin; Qi, Ming-fang; Xu, Tao; Lu, Xiu-jun; Li, Tian-lai

    2015-05-21

    The control of abscission is an important agricultural concern because of its substantial effect on crop yield and quality. Changes in gene expression are correlated with the ethylene-mediated execution of abscission. However, only few large-scale proteomic studies focused on tomato pedicel abscission. Isobaric tag for relative and absolute quantification labeling was used to examine the protein and phosphoprotein changes in the tomato pedicel AZ (AZ) treated with ethylene or 1-methylcyclopropene. Among the 1429 quantified proteins, 383 unique peptides corresponding to 166 proteins showed higher than 1.5-fold change in abundance. A total of 450 phosphopeptides were detected, among which 85 phosphopeptides corresponding to 73 phosphoproteins were significantly regulated (>1.5-fold abundance change) in response to ethylene. Protein and phosphoprotein sets showed 26 similar proteins. Six phosphorylation motifs were extracted from the 138 phosphorylation sites. By analyzing translational and modification levels, we found that the modification level was not due to the translational changes. Comparison between the protein and phosphoprotein functions revealed that the proteins acted mainly in the metabolic process and showed catalytic activity, whereas most of the phosphoproteins showed signaling and transporting activities. Data revealed the unique features of the AZ phosphoproteomics, thereby suggesting the involvement of a complex network of kinase-substrate and phosphatase-substrate interactions in response to ethylene. Some phosphorylation sites from calcium-dependent protein kinase (CDPK5(S523)), CDPK5(S527), and SRL3(S329) were also found to perform protective functions for AZ and to be helpful in ethylene signal transduction. Organ abscission has both positive and negative roles. Abscission is conducive for the fall of ripe fruits and the release and dispersion of seeds, but abscission has been a major limiting factor for crop productivity. Hence, more details

  17. Application of proteomics in the discovery of candidate protein biomarkers in a Diabetes Autoantibody Standardization Program (DASP) sample subset

    PubMed Central

    Metz, Thomas O.; Qian, Wei-Jun; Jacobs, Jon M.; Gritsenko, Marina A.; Moore, Ronald J.; Polpitiya, Ashoka D.; Monroe, Matthew E.; Camp, David G.; Mueller, Patricia W.; Smith, Richard D.

    2009-01-01

    Novel biomarkers of type 1 diabetes must be identified and validated in initial, exploratory studies before they can be assessed in proficiency evaluations. Currently, untargeted “-omics” approaches are under-utilized in profiling studies of clinical samples. This report describes the evaluation of capillary liquid chromatography (LC) coupled with mass spectrometry (MS) in a pilot proteomic analysis of human plasma and serum from a subset of control and type 1 diabetic individuals enrolled in the Diabetes Autoantibody Standardization Program with the goal of identifying candidate biomarkers of type 1 diabetes. Initial high-resolution capillary LC-MS/MS experiments were performed to augment an existing plasma peptide database, while subsequent LC-FTICR studies identified quantitative differences in the abundance of plasma proteins. Analysis of LC-FTICR proteomic data identified five candidate protein biomarkers of type 1 diabetes. Alpha-2-glycoprotein 1 (zinc), corticosteroid-binding globulin, and lumican were 2-fold up-regulated in type 1 diabetic samples relative to control samples, whereas clusterin and serotransferrin were 2-fold up-regulated in control samples relative to type 1 diabetic samples. Observed perturbations in the levels of all five proteins are consistent with the metabolic aberrations found in type 1 diabetes. While the discovery of these candidate protein biomarkers of type 1 diabetes is encouraging, follow up studies are required for validation in a larger population of individuals and for determination of laboratory-defined sensitivity and specificity values using blinded samples. PMID:18092746

  18. Unravelling the proteomic profile of rice meiocytes during early meiosis

    PubMed Central

    Collado-Romero, Melania; Alós, Enriqueta; Prieto, Pilar

    2014-01-01

    Transfer of genetic traits from wild or related species into cultivated rice is nowadays an important aim in rice breeding. Breeders use genetic crosses to introduce desirable genes from exotic germplasms into cultivated rice varieties. However, in many hybrids there is only a low level of pairing (if existing) and recombination at early meiosis between cultivated rice and wild relative chromosomes. With the objective of getting deeper into the knowledge of the proteins involved in early meiosis, when chromosomes associate correctly in pairs and recombine, the proteome of isolated rice meiocytes has been characterized by nLC-MS/MS at every stage of early meiosis (prophase I). Up to 1316 different proteins have been identified in rice isolated meiocytes in early meiosis, being 422 exclusively identified in early prophase I (leptotene, zygotene, or pachytene). The classification of proteins in functional groups showed that 167 were related to chromatin structure and remodeling, nucleic acid binding, cell-cycle regulation, and cytoskeleton. Moreover, the putative roles of 16 proteins which have not been previously associated to meiosis or were not identified in rice before, are also discussed namely: seven proteins involved in chromosome structure and remodeling, five regulatory proteins [such as SKP1 (OSK), a putative CDK2 like effector], a protein with RNA recognition motifs, a neddylation-related protein, and two microtubule-related proteins. Revealing the proteins involved in early meiotic processes could provide a valuable tool kit to manipulate chromosome associations during meiosis in rice breeding programs. The data have been deposited to the ProteomeXchange with the PXD001058 identifier. PMID:25104955

  19. Optimization of Proteomic Sample Preparation Procedures for Comprehensive Protein Characterization of Pathogenic Systems

    PubMed Central

    Mottaz-Brewer, Heather M.; Norbeck, Angela D.; Adkins, Joshua N.; Manes, Nathan P.; Ansong, Charles; Shi, Liang; Rikihisa, Yasuko; Kikuchi, Takane; Wong, Scott W.; Estep, Ryan D.; Heffron, Fred; Pasa-Tolic, Ljiljana; Smith, Richard D.

    2008-01-01

    Mass spectrometry-based proteomics is a powerful analytical tool for investigating pathogens and their interactions within a host. The sensitivity of such analyses provides broad proteome characterization, but the sample-handling procedures must first be optimized to ensure compatibility with the technique and to maximize the dynamic range of detection. The decision-making process for determining optimal growth conditions, preparation methods, sample analysis methods, and data analysis techniques in our laboratory is discussed herein with consideration of the balance in sensitivity, specificity, and biomass losses during analysis of host-pathogen systems. PMID:19183792

  20. Riboflavin deficiency induces a significant change in proteomic profiles in HepG2 cells

    PubMed Central

    Xin, Zhonghao; Pu, Lingling; Gao, Weina; Wang, Yawen; Wei, Jingyu; Shi, Tala; Yao, Zhanxin; Guo, Changjiang

    2017-01-01

    Riboflavin deficiency is widespread in many regions over the world, especially in underdeveloped countries. In this study, we investigated the effects of riboflavin deficiency on protein expression profiles in HepG2 cells in order to provide molecular information for the abnormalities induced by riboflavin deficiency. HepG2 cells were cultured in media containing different concentrations of riboflavin. Changes of cell viability and apoptosis were assessed. A comparative proteomic analysis was performed using a label-free shotgun method with LC–MS/MS to investigate the global changes of proteomic profiles in response to riboflavin deficiency. Immunoblotting test was used to validate the results of proteomic approach. The cell viability and apoptosis tests showed that riboflavin was vital in maintaining the cytoactivity of HepG2 cells. The label-free proteomic analysis revealed that a total of 37 proteins showing differential expression (±2 fold, p < 0.05) were identified after riboflavin deficiency. Bioinformatics analysis indicated that the riboflavin deficiency caused an up-regulation of Parkinson’s disease pathway, steroid catabolism, endoplasmic reticulum stress and apoptotic process, while the fatty acid metabolism, tricarboxylic citrate cycle, oxidative phosphorylation and iron metabolism were down-regulated. These findings provide a molecular basis for the elucidation of the effects caused by riboflavin deficiency. PMID:28367977

  1. Riboflavin deficiency induces a significant change in proteomic profiles in HepG2 cells.

    PubMed

    Xin, Zhonghao; Pu, Lingling; Gao, Weina; Wang, Yawen; Wei, Jingyu; Shi, Tala; Yao, Zhanxin; Guo, Changjiang

    2017-04-03

    Riboflavin deficiency is widespread in many regions over the world, especially in underdeveloped countries. In this study, we investigated the effects of riboflavin deficiency on protein expression profiles in HepG2 cells in order to provide molecular information for the abnormalities induced by riboflavin deficiency. HepG2 cells were cultured in media containing different concentrations of riboflavin. Changes of cell viability and apoptosis were assessed. A comparative proteomic analysis was performed using a label-free shotgun method with LC-MS/MS to investigate the global changes of proteomic profiles in response to riboflavin deficiency. Immunoblotting test was used to validate the results of proteomic approach. The cell viability and apoptosis tests showed that riboflavin was vital in maintaining the cytoactivity of HepG2 cells. The label-free proteomic analysis revealed that a total of 37 proteins showing differential expression (±2 fold, p < 0.05) were identified after riboflavin deficiency. Bioinformatics analysis indicated that the riboflavin deficiency caused an up-regulation of Parkinson's disease pathway, steroid catabolism, endoplasmic reticulum stress and apoptotic process, while the fatty acid metabolism, tricarboxylic citrate cycle, oxidative phosphorylation and iron metabolism were down-regulated. These findings provide a molecular basis for the elucidation of the effects caused by riboflavin deficiency.

  2. Protease specificity profiling by tandem mass spectrometry using proteome-derived peptide libraries.

    PubMed

    Schilling, Oliver; auf dem Keller, Ulrich; Overall, Christopher M

    2011-01-01

    Protease specificity profiling using proteome-derived, database-searchable peptide libraries is a novel approach to define the active site specificity of proteolytic enzymes we call PICS (Proteomic Identification of protease Cleavage Sites). Proteome-derived peptide libraries are generated by trypsin, GluC, or chymotrypsin digestion of biologically relevant proteomes, such as cytosolic lysates, to generate three separate libraries that each differ from the others in their C-terminal amino acid residues according to the protease specificity. Primary amines of all peptides are then chemically protected so that after incubation with a test protease, the neo-N-termini of the prime-side cleavage products with exposed α-amines can be specifically biotinylated, enriched, and identified by liquid chromatography-tandem mass spectrometry. The corresponding nonprime-side sequences are derived bioinformatically. Suited for all protease classes except carboxyproteases and those aminoproteases and dipeptidases requiring a free α-amine for cleavage, PICS simultaneously profiles the specificity of prime and nonprime positions and directly determines scissile peptide bonds of up to hundreds of cleavage site sequences in a single experiment. This wealth of sequence specificity information also allows for the investigation of subsite cooperativity. Herein we describe a simplified procedure to produce PICS peptide libraries, the methods to perform a PICS assay, and a new method of data analysis.

  3. Filter-Aided Sample Preparation (FASP) for Improved Proteome Analysis of Recombinant Chinese Hamster Ovary Cells.

    PubMed

    Coleman, Orla; Henry, Michael; Clynes, Martin; Meleady, Paula

    2017-01-01

    Chinese hamster ovary (CHO) cells are the most commonly used mammalian host cell line for biopharmaceutical production because of their ability to correctly fold and posttranslationally modify recombinant proteins that are compatible with human use. Proteomics, along with other 'omic platforms, are being used to understand the biology of CHO cells with the ultimate aim of enhancing CHO cell factories for more efficient production of biopharmaceuticals. In this chapter, we will describe an efficient protocol called Filter Aided Sample Preparation (FASP) for the extraction of proteins from CHO cells for proteomic studies. FASP uses a common ultrafiltration device whereby the membrane pores are small enough to allow contaminating detergents to pass through, while proteins are too large and are retained and concentrated in the filter unit. This method of sample preparation and protein digestion is universally applicable and can be easily employed in any proteomics facilities as standard everyday laboratory reagents and equipment are used.

  4. Improved proteomic discovery by sample pre-fractionation using dual-column ion-exchange high performance liquid chromatography.

    PubMed

    Havugimana, Pierre C; Wong, Peter; Emili, Andrew

    2007-02-15

    Clinically relevant biomarkers are urgently needed for improving patient diagnosis, risk stratification, prognosis and therapeutic treatments. There is a particularly compelling motivation for identifying protein-based indicators of early-stage disease for more effective interventions. Despite recent progress, the proteomic discovery process remains a daunting challenge due to the sheer heterogeneity and skewed protein abundances in biofluids. Even the most advanced mass spectrometry systems exhibit limiting overall dynamic ranges and sensitivities relative to the needs of modern biomedical applications. To this end, we report the development of a robust, rapid, and reproducible high performance ion-exchange liquid chromatography pre-fractionation method that allows for improved proteomic detection coverage of complex biological specimens using basic tandem mass spectrometry screening procedures. This form of sample simplification prior to global proteomic profiling, which we refer to collectively as 'fractionomics', increases the number and diversity of proteins that can be confidently identified in tissue and cell lysates as compared to the straight analysis of unfractionated crude extracts.

  5. Comparative Proteomic Analysis of Whole-Gut Lavage Fluid and Pancreatic Juice Reveals a Less Invasive Method of Sampling Pancreatic Secretions.

    PubMed

    Rocker, Jana M; Tan, Marcus C; Thompson, Lee W; Contreras, Carlo M; DiPalma, Jack A; Pannell, Lewis K

    2016-05-26

    There are currently no reliable, non-invasive screening tests for pancreatic ductal adenocarcinoma. The fluid secreted from the pancreatic ductal system ("pancreatic juice") has been well-studied as a potential source of cancer biomarkers. However, it is invasive to collect. We recently observed that the proteomic profile of intestinal effluent from the bowel in response to administration of an oral bowel preparation solution (also known as whole-gut lavage fluid, WGLF) contains large amounts of pancreas-derived proteins. We therefore hypothesized that the proteomic profile is similar to that of pancreatic juice. In this study, we compared the proteomic profiles of 77 patients undergoing routine colonoscopy with the profiles of 19 samples of pure pancreatic juice collected during surgery. WGLF was collected from patients undergoing routine colonoscopy, and pancreatic juice was collected from patients undergoing pancreatic surgery. Protein was isolated from both samples using an optimized method and analyzed by LC-MS/MS. Identified proteins were compared between samples and groups to determine similarity of the two fluids. We then compared our results with literature reports of pancreatic juice-based studies to determine similarity. We found 104 proteins in our pancreatic juice samples, of which 90% were also found in our WGLF samples. The majority (67%) of the total proteins found in the WGLF were common to pancreatic juice, with intestine-specific proteins making up a smaller proportion. WGLF and pancreatic juice appear to have similar proteomic profiles. This supports the notion that WGLF is a non-invasive, surrogate bio-fluid for pancreatic juice. Further studies are required to further elucidate its role in the diagnosis of pancreatic cancer.

  6. An Integrated Platform for Isolation, Processing, and Mass Spectrometry-based Proteomic Profiling of Rare Cells in Whole Blood*

    PubMed Central

    Li, Siyang; Plouffe, Brian D.; Belov, Arseniy M.; Ray, Somak; Wang, Xianzhe; Murthy, Shashi K.; Karger, Barry L.; Ivanov, Alexander R.

    2015-01-01

    Isolation and molecular characterization of rare cells (e.g. circulating tumor and stem cells) within biological fluids and tissues has significant potential in clinical diagnostics and personalized medicine. The present work describes an integrated platform of sample procurement, preparation, and analysis for deep proteomic profiling of rare cells in blood. Microfluidic magnetophoretic isolation of target cells spiked into 1 ml of blood at the level of 1000–2000 cells/ml, followed by focused acoustics-assisted sample preparation has been coupled with one-dimensional PLOT-LC-MS methodology. The resulting zeptomole detection sensitivity enabled identification of ∼4000 proteins with injection of the equivalent of only 100–200 cells per analysis. The characterization of rare cells in limited volumes of physiological fluids is shown by the isolation and quantitative proteomic profiling of first MCF-7 cells spiked into whole blood as a model system and then two CD133+ endothelial progenitor and hematopoietic cells in whole blood from volunteers. PMID:25755294

  7. Proteomic profiling of 16 cereal grains and the application of targeted proteomics to detect wheat contamination.

    PubMed

    Colgrave, Michelle L; Goswami, Hareshwar; Byrne, Keren; Blundell, Malcolm; Howitt, Crispin A; Tanner, Gregory J

    2015-06-05

    Global proteomic analysis utilizing SDS-PAGE, Western blotting and LC-MS/MS of total protein and gluten-enriched extracts derived from 16 economically important cereals was undertaken, providing a foundation for the development of MS-based quantitative methodologies that would enable the detection of wheat contamination in foods. The number of proteins identified in each grain correlated with the number of entries in publicly available databases, highlighting the importance of continued advances in genome sequencing to facilitate accurate protein identification. Subsequently, candidate wheat-specific peptide markers were evaluated by multiple-reaction monitoring MS. The selected markers were unique to wheat, yet present in a wide range of wheat varieties that represent up to 80% of the bread wheat genome. The final analytical method was rapid (15 min) and robust (CV < 10%), showed linearity (R(2) > 0.98) spanning over 3 orders of magnitude, and was highly selective and sensitive with detection down to 15 mg/kg in intentionally contaminated soy flour. Furthermore, application of this technology revealed wheat contamination in commercially sourced flours, including rye, millet, oats, sorghum, buckwheat and three varieties of soy.

  8. Proteomic profiling of the cancer microenvironment by antibody arrays.

    PubMed

    Knezevic, V; Leethanakul, C; Bichsel, V E; Worth, J M; Prabhu, V V; Gutkind, J S; Liotta, L A; Munson, P J; Petricoin, E F; Krizman, D B

    2001-10-01

    Critical changes in protein expression that enable tumors to initiate and progress originate in the local tissue microenvironment, and there are increasing indications that these microenvironmental alterations in protein expression play critical roles in shaping and directing this process. As a model to better understand how patterns of protein expression shape the tissue microenvironment, we analyzed protein expression in tissue derived from squamous cell carcinoma of the oral cavity through an antibody microarray approach for high-throughput proteomic analysis. Utilizing laser capture microdissection to procure total protein from specific microscopic cellular populations, we demonstrate that quantitative, and potentially qualitative, differences in expression patterns of multiple proteins within epithelial cells reproducibly correlate with oral cavity tumor progression. Furthermore, differential expression of multiple proteins was also found in stromal cells surrounding and adjacent to regions of diseased epithelium that directly correlated with tumor progression of the epithelium. Most of the proteins identified in both cell types are involved in signal transduction pathways, thus we hypothesize that extensive molecular communication involving complex cellular signaling between epithelium and stroma play a key role in driving oral cavity cancer progression.

  9. Transcriptomic and proteomic profiling of maize embryos exposed to camptothecin

    PubMed Central

    2011-01-01

    Background Camptothecin is a plant alkaloid that specifically binds topoisomerase I, inhibiting its activity and inducing double stranded breaks in DNA, activating the cell responses to DNA damage and, in response to severe treatments, triggering cell death. Results Comparative transcriptomic and proteomic analyses of maize embryos that had been exposed to camptothecin were conducted. Under the conditions used in this study, camptothecin did not induce extensive degradation in the genomic DNA but induced the transcription of genes involved in DNA repair and repressed genes involved in cell division. Camptothecin also affected the accumulation of several proteins involved in the stress response and induced the activity of certain calcium-dependent nucleases. We also detected changes in the expression and accumulation of different genes and proteins involved in post-translational regulatory processes. Conclusions This study identified several genes and proteins that participate in DNA damage responses in plants. Some of them may be involved in general responses to stress, but others are candidate genes for specific involvement in DNA repair. Our results open a number of new avenues for researching and improving plant resistance to DNA injury. PMID:21595924

  10. Proteomic Profiling of Mouse Liver following Acute Toxoplasma gondii Infection.

    PubMed

    He, Jun-Jun; Ma, Jun; Elsheikha, Hany M; Song, Hui-Qun; Zhou, Dong-Hui; Zhu, Xing-Quan

    2016-01-01

    Toxoplasma gondii remains a global public health problem. However, its pathophysiology is still not-completely understood particularly the impact of infection on host liver metabolism. We performed iTRAQ-based proteomic analysis to evaluate early liver protein responses in BALB/c mice following infection with T. gondii PYS strain (genotype ToxoDB#9) infection. Our data revealed modification of protein expression in key metabolic pathways, as indicated by the upregulation of immune response and downregulation of mitochondrial respiratory chain, and the metabolism of fatty acids, lipids and xenobiotics. T. gondii seems to hijack host PPAR signaling pathway to downregulate the metabolism of fatty acids, lipids and energy in the liver. The metabolism of over 400 substances was affected by the downregulation of genes involved in xenobiotic metabolism. The top 10 transcription factors used by upregulated genes were Stat2, Stat1, Irf2, Irf1, Sp2, Egr1, Stat3, Klf4, Elf1 and Gabpa, while the top 10 transcription factors of downregulated genes were Hnf4A, Ewsr1, Fli1, Hnf4g, Nr2f1, Pparg, Rxra, Hnf1A, Foxa1 and Foxo1. These findings indicate global reprogramming of the metabolism of the mouse liver after acute T. gondii infection. Functional characterization of the altered proteins may enhance understanding of the host responses to T. gondii infection and lead to the identification of new therapeutic targets.

  11. Proteomic Profiling of Mouse Liver following Acute Toxoplasma gondii Infection

    PubMed Central

    He, Jun-Jun; Ma, Jun; Elsheikha, Hany M.; Song, Hui-Qun; Zhou, Dong-Hui; Zhu, Xing-Quan

    2016-01-01

    Toxoplasma gondii remains a global public health problem. However, its pathophysiology is still not-completely understood particularly the impact of infection on host liver metabolism. We performed iTRAQ-based proteomic analysis to evaluate early liver protein responses in BALB/c mice following infection with T. gondii PYS strain (genotype ToxoDB#9) infection. Our data revealed modification of protein expression in key metabolic pathways, as indicated by the upregulation of immune response and downregulation of mitochondrial respiratory chain, and the metabolism of fatty acids, lipids and xenobiotics. T. gondii seems to hijack host PPAR signaling pathway to downregulate the metabolism of fatty acids, lipids and energy in the liver. The metabolism of over 400 substances was affected by the downregulation of genes involved in xenobiotic metabolism. The top 10 transcription factors used by upregulated genes were Stat2, Stat1, Irf2, Irf1, Sp2, Egr1, Stat3, Klf4, Elf1 and Gabpa, while the top 10 transcription factors of downregulated genes were Hnf4A, Ewsr1, Fli1, Hnf4g, Nr2f1, Pparg, Rxra, Hnf1A, Foxa1 and Foxo1. These findings indicate global reprogramming of the metabolism of the mouse liver after acute T. gondii infection. Functional characterization of the altered proteins may enhance understanding of the host responses to T. gondii infection and lead to the identification of new therapeutic targets. PMID:27003162

  12. Identification and proteomic profiling of exosomes in human urine.

    PubMed

    Pisitkun, Trairak; Shen, Rong-Fong; Knepper, Mark A

    2004-09-07

    Urine provides an alternative to blood plasma as a potential source of disease biomarkers. One urinary biomarker already exploited in clinical studies is aquaporin-2. However, it remains a mystery how aquaporin-2 (an integral membrane protein) and other apical transporters are delivered to the urine. Here we address the hypothesis that these proteins reach the urine through the secretion of exosomes [membrane vesicles that originate as internal vesicles of multivesicular bodies (MVBs)]. Low-density urinary membrane vesicles from normal human subjects were isolated by differential centrifugation. ImmunoGold electron microscopy using antibodies directed to cytoplasmic or anticytoplasmic epitopes revealed that the vesicles are oriented "cytoplasmic-side inward," consistent with the unique orientation of exosomes. The vesicles were small (<100 nm), consistent with studies of MVBs and exosomes from other tissues. Proteomic analysis of urinary vesicles through nanospray liquid chromatography-tandem mass spectrometry identified numerous protein components of MVBs and of the endosomal pathway in general. Full liquid chromatography-tandem MS analysis revealed 295 proteins, including multiple protein products of genes already known to be responsible for renal and systemic diseases, including autosomal dominant polycystic kidney disease, Gitelman syndrome, Bartter syndrome, autosomal recessive syndrome of osteopetrosis with renal tubular acidosis, and familial renal hypomagnesemia. The results indicate that exosome isolation may provide an efficient first step in biomarker discovery in urine.

  13. Exploration of Serum Proteomic Profiling and Diagnostic Model That Differentiate Crohn's Disease and Intestinal Tuberculosis

    PubMed Central

    Zhang, Fenming; Xu, Chengfu; Ning, Longgui; Hu, Fengling; Shan, Guodong; Chen, Hongtan; Yang, Ming; Chen, Wenguo; Yu, Jiekai; Xu, Guoqiang

    2016-01-01

    Aim To explore the diagnostic models of Crohn’s disease (CD), Intestinal tuberculosis (ITB) and the differential diagnostic model between CD and ITB by analyzing serum proteome profiles. Methods Serum proteome profiles from 30 CD patients, 21 ITB patients and 30 healthy controls (HCs) were analyzed by using weak cationic magnetic beads combined with MALDI-TOF-MS technique to detect the differentially expressed proteins of serum samples. Three groups were made and compared accordingly: group of CD patients and HCs, group of ITB patients and HCs, group of CD patients and ITB patients. Wilcoxon rank sum test was used to screen the ten most differentiated protein peaks (P < 0.05). Genetic algorithm combining with support vector machine (SVM) was utilized to establish the optimal diagnostic models for CD, ITB and the optimal differential diagnostic model between CD and ITB. The predictive effects of these models were evaluated by Leave one out (LOO) cross validation method. Results There were 236 protein peaks differently expressed between group of CD patients and HCs, 305 protein peaks differently expressed between group of ITB patients and HCs, 332 protein peaks differently expressed between group of CD patients and ITB patients. Ten most differentially expressed peaks were screened out between three groups respectively (P < 0.05) to establish diagnostic models and differential diagnostic model. A diagnostic model comprising of four protein peaks (M/Z 4964, 3029, 2833, 2900) can well distinguish CD patients and HCs, with a specificity and sensitivity of 96.7% and 96.7% respectively. A diagnostic model comprising four protein peaks (M/Z 3030, 2105, 2545, 4210) can well distinguish ITB patients and HCs, with a specificity and sensitivity of 93.3% and 95.2% respectively. A differential diagnostic model comprising three potential biomarkers protein peaks (M/Z 4267, 4223, 1541) can well distinguish CD patients and ITB patients, with a specificity and sensitivity of 76

  14. Towards the profiling of the Arabidopsis thaliana plasma membrane transportome by targeted proteomics.

    PubMed

    Monneuse, Jean-Marc; Sugano, Madeleine; Becue, Thierry; Santoni, Véronique; Hem, Sonia; Rossignol, Michel

    2011-05-01

    Plant membranes bear a variety of transporters belonging to multigene families that are affected by environmental and nutritional conditions. In addition, they often display high-sequence identity, making difficult in-depth investigation by current shot-gun strategies. In this study, we set up a targeted proteomics approach aimed at identifying and quantifying within single experiments the five major proton pumps of the autoinhibited H(+) ATPases (AHA) family, the 13 plasma membrane intrinsic proteins (PIP) water channels (PIPs), and ten members of ammonium transporters (AMTs) and nitrate transporter (NRT) families. Proteotypic peptides were selected and isotopically labeled heavy versions were used for technical optimization and for quantification of the corresponding light version in biological samples. This approach allowed to quantify simultaneously nine PIPs in leaf membranes and 13 PIPs together with three autoinhibited H(+) ATPases, two ammonium transporters, and two NRTs in root membranes. Similarly, it was used to investigate the effect of a salt stress on the expression of these latter 20 transporters in roots. These novel isoform-specific data were compared with published transcriptome information and revealed a close correlation between PIP isoforms and transcripts levels. The obtained resource is reusable and can be expanded to other transporter families for large-scale profiling of membrane transporters. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. Proteomic Analysis of the Protein Expression Profile in the Mature Nigella sativa (Black Seed).

    PubMed

    Alanazi, Ibrahim O; Benabdelkamel, Hicham; Alfadda, Assim A; AlYahya, Sami A; Alghamdi, Waleed M; Aljohi, Hasan A; Almalik, Abdulaziz; Masood, Afshan

    2016-08-01

    Nigella sativa (N. sativa) seed has been used as an important nutritional flavoring agent and in traditional medicine for treating many illnesses since ancient times. Understanding the proteomic component of the seed may lead to enhance the understanding of its structural and biological functional complexity. In this study, we have analyzed its proteome profile based on gel-based proteome mapping technique that includes one-dimensional gel electrophoresis followed by liquid chromatography and tandem mass spectrometry strategy. We have not come across any such studies that have been performed in N. sativa seeds up to date. A total of 277 proteins were identified, and their functional, metabolic, and location-wise annotations were carried out using the UniProt database. The majority of proteins identified in the proteome dataset based on their function were those involved in enzyme catalytic activity, nucleotide binding, and protein binding while the major cellular processes included regulation of biological process followed by regulation of secondary biological process, cell organization and biogenesis, protein metabolism, and transport. The identified proteome was localized mainly to the nucleus then to the cytoplasm, plasma membrane, mitochondria, plastid, and others. A majority of the proteins were involved in biochemical pathways involving carbohydrate metabolism, amino acid and shikimate pathway, lipid metabolism, nucleotide, cell organization and biogenesis, transport, and defense processes. The identified proteins in the dataset help to improve our understanding of the pathways involved in N. sativa seed metabolism and its biochemical features and detail out useful information that may help to utilize these proteins. This study could thus pave a way for future further high-throughput studies using a more targeted proteomic approach.

  16. Proteomic profiling of plasma in Huntington's disease reveals neuroinflammatory activation and biomarker candidates.

    PubMed

    Dalrymple, Annette; Wild, Edward J; Joubert, Richard; Sathasivam, Kirupa; Björkqvist, Maria; Petersén, Asa; Jackson, Graham S; Isaacs, Jeremy D; Kristiansen, Mark; Bates, Gillian P; Leavitt, Blair R; Keir, Geoff; Ward, Malcolm; Tabrizi, Sarah J

    2007-07-01

    Huntington's disease (HD) causes widespread CNS changes and systemic abnormalities including endocrine and immune dysfunction. HD biomarkers are needed to power clinical trials of potential treatments. We used multiplatform proteomic profiling to reveal plasma changes with HD progression. Proteins of interest were evaluated using immunoblotting and ELISA in plasma from 2 populations, CSF and R6/2 mice. The identified proteins demonstrate neuroinflammation in HD and warrant further investigation as possible biomarkers.

  17. Multilineage potential and proteomic profiling of human dental stem cells derived from a single donor

    SciTech Connect

    Patil, Rajreddy; Kumar, B. Mohana; Lee, Won-Jae; Jeon, Ryoung-Hoon; Jang, Si-Jung; Lee, Yeon-Mi; Park, Bong-Wook; Byun, June-Ho; Ahn, Chun-Seob; Kim, Jae-Won; Rho, Gyu-Jin

    2014-01-01

    Dental tissues provide an alternative autologous source of mesenchymal stem cells (MSCs) for regenerative medicine. In this study, we isolated human dental MSCs of follicle, pulp and papilla tissue from a single donor tooth after impacted third molar extraction by excluding the individual differences. We then compared the morphology, proliferation rate, expression of MSC-specific and pluripotency markers, and in vitro differentiation ability into osteoblasts, adipocytes, chondrocytes and functional hepatocyte-like cells (HLCs). Finally, we analyzed the protein expression profiles of undifferentiated dental MSCs using 2DE coupled with MALDI-TOF-MS. Three types of dental MSCs largely shared similar morphology, proliferation potential, expression of surface markers and pluripotent transcription factors, and differentiation ability into osteoblasts, adipocytes, and chondrocytes. Upon hepatogenic induction, all MSCs were transdifferentiated into functional HLCs, and acquired hepatocyte functions by showing their ability for glycogen storage and urea production. Based on the proteome profiling results, we identified nineteen proteins either found commonly or differentially expressed among the three types of dental MSCs. In conclusion, three kinds of dental MSCs from a single donor tooth possessed largely similar cellular properties and multilineage potential. Further, these dental MSCs had similar proteomic profiles, suggesting their interchangeable applications for basic research and call therapy. - Highlights: • Isolated and characterized three types of human dental MSCs from a single donor. • MSCs of dental follicle, pulp and papilla had largely similar biological properties. • All MSCs were capable of transdifferentiating into functional hepatocyte-like cells. • 2DE proteomics with MALDI-TOF/MS identified 19 proteins in three types of MSCs. • Similar proteomic profiles suggest interchangeable applications of dental MSCs.

  18. Label-Free Proteome Profiling of Carbapenem-Resistant Klebsiella pneumoniae LC-MS/MS

    DTIC Science & Technology

    2016-12-12

    Title Carbapenem- resistant Klebsiella pneumoniae LC-MS/MS Description A comprehensive analysis of proteome changes in CRKP cells in response to...the sub-MIC doses of antibiotics was performed by using label-free quantitative mass spectrometry. Sample Processing Protocol The pellets were

  19. A DATABASE FOR TRACKING TOXICOGENOMIC SAMPLES AND PROCEDURES WITH GENOMIC, PROTEOMIC AND METABONOMIC COMPONENTS

    EPA Science Inventory

    A Database for Tracking Toxicogenomic Samples and Procedures with Genomic, Proteomic and Metabonomic Components
    Wenjun Bao1, Jennifer Fostel2, Michael D. Waters2, B. Alex Merrick2, Drew Ekman3, Mitchell Kostich4, Judith Schmid1, David Dix1
    Office of Research and Developmen...

  20. A DATABASE FOR TRACKING TOXICOGENOMIC SAMPLES AND PROCEDURES WITH GENOMIC, PROTEOMIC AND METABONOMIC COMPONENTS

    EPA Science Inventory

    A Database for Tracking Toxicogenomic Samples and Procedures with Genomic, Proteomic and Metabonomic Components
    Wenjun Bao1, Jennifer Fostel2, Michael D. Waters2, B. Alex Merrick2, Drew Ekman3, Mitchell Kostich4, Judith Schmid1, David Dix1
    Office of Research and Developmen...

  1. [Search for potential gastric cancer biomarkers using low molecular weight blood plasma proteome profiling by mass spectrometry].

    PubMed

    Shevchenko, V E; Arnotskaia, N E; Ogorodnikova, E V; Davydov, M M; Ibraev, M A; Turkin, I N; Davydov, M I

    2014-01-01

    Gastric cancer, one of the most widespread malignant tumors, still lacks reliable serum/plasma biomarkers of its early detection. In this study we have developed, unified, and tested a new methodology for search of gastric cancer biomarkers based on profiling of low molecular weight proteome (LMWP) (1-17 kDa). This approach included three main components: sample pre-fractionation, matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS), data analysis by a bioinformatics software package. Applicability and perspectives of the developed approach for detection of potential gastric cancer markers during LMWP analysis have been demonstrated using 69 plasma samples from patients with gastric cancer (stages I-IV) and 238 control samples. The study revealed peptides/polypeptides, which may be potentially used for detection of this pathology.

  2. Proteomic profiling of metalloprotease activities with cocktails of active-site probes

    PubMed Central

    Sieber, Stephan A; Niessen, Sherry; Hoover, Heather S; Cravatt, Benjamin F

    2006-01-01

    Metalloproteases are a large, diverse class of enzymes involved in many physiological and disease processes. Metalloproteases are regulated by post-translational mechanisms that diminish the effectiveness of conventional genomic and proteomic methods for their functional characterization. Chemical probes directed at active sites offer a potential way to measure metalloprotease activities in biological systems; however, large variations in structure limit the scope of any single small-molecule probe aimed at profiling this enzyme class. Here, we address this problem by creating a library of metalloprotease-directed probes that show complementary target selectivity. These probes were applied as a ‘cocktail’ to proteomes and their labeling profiles were analyzed collectively using an advanced liquid chromatography-mass spectrometry platform. More than 20 metalloproteases were identified, including members from nearly all of the major branches of this enzyme class. These findings suggest that chemical proteomic methods can serve as a universal strategy to profile the activity of the metalloprotease superfamily in complex biological systems. PMID:16565715

  3. Functional profiling of the Tritrichomonas foetus transcriptome and proteome.

    PubMed

    Huang, Kuo-Yang; Shin, Jyh-Wei; Huang, Po-Jung; Ku, Fu-Man; Lin, Wei-Chen; Lin, Rose; Hsu, Wei-Min; Tang, Petrus

    2013-01-01

    Tritrichomonas foetus is a potent veterinary pathogen, causing bovine and feline trichomoniasis. The principal clinical manifestation of infection in cattle is inflammation of the genital tract and infertility. In feline, the parasite causes large-bowel disease resulting in chronic diarrhea. In contrast to other well-studied protozoan, genetic data regarding the molecular characterization and expression in T. foetus is far less understood. In this study, the first large-scale T. foetus expressed sequence tag (TfEST) project was conducted on 5064 randomly selected EST clones from a non-normalized unidirectional Tf30924 cDNA library. Assembling of 5064 single-pass sequences from the 5' end resulted in 713 contigs and 1961 singlets. BLAST search revealed that 53.52% of the unigenes showed significant similarity to known sequences or protein motifs/domains. Functional classifications indicated that most of the unigenes are involved in translation, ribosomal structure and ribosome biogenesis. The average GC content of the T. foetus transcriptome is 40.93%. Intriguingly, only 31.29% of the unigenes contain the classical AAUAAA polyadenylation signal sequence at the 3'-UTR region. Furthermore, a panel of potential chemotherapeutic targets was also identified for the first time in T. foetus. The protein expression levels were verified by using two-dimensional electrophoresis and matrix-assisted laser desorption ionization time-of-flight mass spectrometry. A total of 68 highly abundant protein spots were successfully identified in the reference 2-DE map based on our T. foetus-specific protein database. The EST dataset and the reference 2-DE map established in the present study will provide a foundation for future whole genome sequencing project and comparative transcriptomic/proteomic analyses to provide potential drug targets against T. foetus infection.

  4. Proteomic analysis of propiconazole responses in mouse liver: comparison of genomic and proteomic profiles

    EPA Science Inventory

    We have performed for the first time a comprehensive profiling of changes in protein expression of soluble proteins in livers from mice treated with the mouse liver tumorigen, propiconazole, to uncover the pathways and networks altered by this fungicide. Utilizing twodimensional...

  5. Proteomic Analysis of Propiconazole Responses in Mouse Liver-Comparison of Genomic and Proteomic Profiles

    EPA Science Inventory

    We have performed for the first time a comprehensive profiling of changes in protein expression of soluble proteins in livers from mice treated with the mouse liver tumorigen, propiconazole, to uncover the pathways and networks altered by this commonly used fungicide. Utilizing t...

  6. Proteomic analysis of propiconazole responses in mouse liver: comparison of genomic and proteomic profiles

    EPA Science Inventory

    We have performed for the first time a comprehensive profiling of changes in protein expression of soluble proteins in livers from mice treated with the mouse liver tumorigen, propiconazole, to uncover the pathways and networks altered by this fungicide. Utilizing twodimensional...

  7. Proteomic Analysis of Propiconazole Responses in Mouse Liver-Comparison of Genomic and Proteomic Profiles

    EPA Science Inventory

    We have performed for the first time a comprehensive profiling of changes in protein expression of soluble proteins in livers from mice treated with the mouse liver tumorigen, propiconazole, to uncover the pathways and networks altered by this commonly used fungicide. Utilizing t...

  8. Sources of Technical Variability in Quantitative LC-MS Proteomics: Human Brain Tissue Sample Analysis.

    SciTech Connect

    Piehowski, Paul D.; Petyuk, Vladislav A.; Orton, Daniel J.; Xie, Fang; Moore, Ronald J.; Ramirez Restrepo, Manuel; Engel, Anzhelika; Lieberman, Andrew P.; Albin, Roger L.; Camp, David G.; Smith, Richard D.; Myers, Amanda J.

    2013-05-03

    To design a robust quantitative proteomics study, an understanding of both the inherent heterogeneity of the biological samples being studied as well as the technical variability of the proteomics methods and platform is needed. Additionally, accurately identifying the technical steps associated with the largest variability would provide valuable information for the improvement and design of future processing pipelines. We present an experimental strategy that allows for a detailed examination of the variability of the quantitative LC-MS proteomics measurements. By replicating analyses at different stages of processing, various technical components can be estimated and their individual contribution to technical variability can be dissected. This design can be easily adapted to other quantitative proteomics pipelines. Herein, we applied this methodology to our label-free workflow for the processing of human brain tissue. For this application, the pipeline was divided into four critical components: Tissue dissection and homogenization (extraction), protein denaturation followed by trypsin digestion and SPE clean-up (digestion), short-term run-to-run instrumental response fluctuation (instrumental variance), and long-term drift of the quantitative response of the LC-MS/MS platform over the 2 week period of continuous analysis (instrumental stability). From this analysis, we found the following contributions to variability: extraction (72%) >> instrumental variance (16%) > instrumental stability (8.4%) > digestion (3.1%). Furthermore, the stability of the platform and its’ suitability for discovery proteomics studies is demonstrated.

  9. Methodologies and perspectives of proteomics applied to filamentous fungi: from sample preparation to secretome analysis.

    PubMed

    Bianco, Linda; Perrotta, Gaetano

    2015-03-12

    Filamentous fungi possess the extraordinary ability to digest complex biomasses and mineralize numerous xenobiotics, as consequence of their aptitude to sensing the environment and regulating their intra and extra cellular proteins, producing drastic changes in proteome and secretome composition. Recent advancement in proteomic technologies offers an exciting opportunity to reveal the fluctuations of fungal proteins and enzymes, responsible for their metabolic adaptation to a large variety of environmental conditions. Here, an overview of the most commonly used proteomic strategies will be provided; this paper will range from sample preparation to gel-free and gel-based proteomics, discussing pros and cons of each mentioned state-of-the-art technique. The main focus will be kept on filamentous fungi. Due to the biotechnological relevance of lignocellulose degrading fungi, special attention will be finally given to their extracellular proteome, or secretome. Secreted proteins and enzymes will be discussed in relation to their involvement in bio-based processes, such as biomass deconstruction and mycoremediation.

  10. Sources of technical variability in quantitative LC-MS proteomics: human brain tissue sample analysis.

    PubMed

    Piehowski, Paul D; Petyuk, Vladislav A; Orton, Daniel J; Xie, Fang; Moore, Ronald J; Ramirez-Restrepo, Manuel; Engel, Anzhelika; Lieberman, Andrew P; Albin, Roger L; Camp, David G; Smith, Richard D; Myers, Amanda J

    2013-05-03

    To design a robust quantitative proteomics study, an understanding of both the inherent heterogeneity of the biological samples being studied as well as the technical variability of the proteomics methods and platform is needed. Additionally, accurately identifying the technical steps associated with the largest variability would provide valuable information for the improvement and design of future processing pipelines. We present an experimental strategy that allows for a detailed examination of the variability of the quantitative LC-MS proteomics measurements. By replicating analyses at different stages of processing, various technical components can be estimated and their individual contribution to technical variability can be dissected. This design can be easily adapted to other quantitative proteomics pipelines. Herein, we applied this methodology to our label-free workflow for the processing of human brain tissue. For this application, the pipeline was divided into four critical components: Tissue dissection and homogenization (extraction), protein denaturation followed by trypsin digestion and SPE cleanup (digestion), short-term run-to-run instrumental response fluctuation (instrumental variance), and long-term drift of the quantitative response of the LC-MS/MS platform over the 2 week period of continuous analysis (instrumental stability). From this analysis, we found the following contributions to variability: extraction (72%) > instrumental variance (16%) > instrumental stability (8.4%) > digestion (3.1%). Furthermore, the stability of the platform and its suitability for discovery proteomics studies is demonstrated.

  11. Methodologies and Perspectives of Proteomics Applied to Filamentous Fungi: From Sample Preparation to Secretome Analysis

    PubMed Central

    Bianco, Linda; Perrotta, Gaetano

    2015-01-01

    Filamentous fungi possess the extraordinary ability to digest complex biomasses and mineralize numerous xenobiotics, as consequence of their aptitude to sensing the environment and regulating their intra and extra cellular proteins, producing drastic changes in proteome and secretome composition. Recent advancement in proteomic technologies offers an exciting opportunity to reveal the fluctuations of fungal proteins and enzymes, responsible for their metabolic adaptation to a large variety of environmental conditions. Here, an overview of the most commonly used proteomic strategies will be provided; this paper will range from sample preparation to gel-free and gel-based proteomics, discussing pros and cons of each mentioned state-of-the-art technique. The main focus will be kept on filamentous fungi. Due to the biotechnological relevance of lignocellulose degrading fungi, special attention will be finally given to their extracellular proteome, or secretome. Secreted proteins and enzymes will be discussed in relation to their involvement in bio-based processes, such as biomass deconstruction and mycoremediation. PMID:25775160

  12. Spatial differences in an integral membrane proteome detected in laser capture microdissected samples.

    PubMed

    Wang, Zhen; Han, Jun; Schey, Kevin L

    2008-07-01

    The combination of laser capture microdissection and mass spectrometry represents a powerful technology for studying spatially resolved proteomes. Moreover, the compositions of integral membrane proteomes have rarely been studied in a spatially resolved manner. In this study, ocular lens tissue was carefully dissected by laser capture microdissection and conditions for membrane protein enrichment, trypsin digestion, and mass spectrometry analysis were optimized. Proteomic analysis allowed the identification of 170 proteins, 136 of which were identified with more than one peptide match. Spatial differences in protein expression were observed between cortical and nuclear samples. In addition, the spatial distribution of post-translational modifications to lens membrane proteins, such as the lens major intrinsic protein AQP0, were investigated and regional differences were measured for AQP0 C-terminal phosphorylation and truncation.

  13. Proteomic profiling of gill GSTs in Mytilus galloprovincialis from the North of Portugal and Galicia evidences variations at protein isoform level with a possible relation with water quality.

    PubMed

    Azevedo, Catarina C; Guzmán-Guillén, Remédios; Martins, José C; Osório, Hugo; Vasconcelos, Vitor; da Fonseca, Rute R; Campos, Alexandre

    2015-09-01

    Glutathione transferases (GSTs) are key for xenobiotic detoxification at the molecular level across phyla. These enzymes are therefore likely to be part of the defence mechanisms used by marine organisms, such as mussels, that thrive in highly polluted environments. Taking this hypothesis into account, we used proteomics to characterize the profile of GSTs from the gills of marine mussel Mytilus galloprovincialis in order to discriminate natural mussel populations exposed to different levels of pollution. Samples were collected between Cabo Home (Spain) and Matosinhos (Portugal) covering a north-south transect of approximately 122 Km of the Atlantic Ocean along the Western Coast of the Iberian Peninsula. GSTs from mussel gills were extracted and purified by affinity chromatography with glutathione as the binding substrate to the solid medium. We studied the abundance of GST isoforms by two-dimensional gel electrophoresis and matrix assisted laser desorption/ionization-time of flight mass spectrometry and assessed total activity. Eleven putative individual GSTs from classes Mu, Pi and Sigma were identified by proteomics. Few variations were observed in total GST activity of post-mitochondrial samples between sampling sites, with animals from Matosinhos (polluted site) showing highest GST activity and Cabo Home (clean site) showing lowest. This contrasts with the increased number of differences in the individual GST isoforms. Each mussel population showed unique GST proteomic profiles. Based on the results we conclude that proteomics surpasses the conventional GST enzymatic activity method to discriminate natural mussel populations and has potential application in environmental monitoring. It is reasonable to suggest that the GST proteomic profiles observed may reflect differences in contamination levels.

  14. Proteomic Profiling in Multiple Sclerosis Clinical Courses Reveals Potential Biomarkers of Neurodegeneration

    PubMed Central

    Liguori, Maria; Qualtieri, Antonio; Tortorella, Carla; Direnzo, Vita; Bagalà, Angelo; Mastrapasqua, Mariangela; Spadafora, Patrizia; Trojano, Maria

    2014-01-01

    The aim of our project was to perform an exploratory analysis of the cerebrospinal fluid (CSF) proteomic profiles of Multiple Sclerosis (MS) patients, collected in different phases of their clinical course, in order to investigate the existence of peculiar profiles characterizing the different MS phenotypes. The study was carried out on 24 Clinically Isolated Syndrome (CIS), 16 Relapsing Remitting (RR) MS, 11 Progressive (Pr) MS patients. The CSF samples were analysed using the Matrix Assisted Laser Desorption Ionisation Time Of Flight (MALDI-TOF) mass spectrometer in linear mode geometry and in delayed extraction mode (m/z range: 1000–25000 Da). Peak lists were imported for normalization and statistical analysis. CSF data were correlated with demographic, clinical and MRI parameters. The evaluation of MALDI-TOF spectra revealed 348 peak signals with relative intensity ≥1% in the study range. The peak intensity of the signals corresponding to Secretogranin II and Protein 7B2 were significantly upregulated in RRMS patients compared to PrMS (p<0.05), whereas the signals of Fibrinogen and Fibrinopeptide A were significantly downregulated in CIS compared to PrMS patients (p<0.04). Additionally, the intensity of the Tymosin β4 peak was the only signal to be significantly discriminated between the CIS and RRMS patients (p = 0.013). Although with caution due to the relatively small size of the study populations, and considering that not all the findings remained significant after adjustment for multiple comparisons, in our opinion this mass spectrometry evaluation confirms that this technique may provide useful and important information to improve our understanding of the complex pathogenesis of MS. PMID:25098164

  15. Proteomic profiling in multiple sclerosis clinical courses reveals potential biomarkers of neurodegeneration.

    PubMed

    Liguori, Maria; Qualtieri, Antonio; Tortorella, Carla; Direnzo, Vita; Bagalà, Angelo; Mastrapasqua, Mariangela; Spadafora, Patrizia; Trojano, Maria

    2014-01-01

    The aim of our project was to perform an exploratory analysis of the cerebrospinal fluid (CSF) proteomic profiles of Multiple Sclerosis (MS) patients, collected in different phases of their clinical course, in order to investigate the existence of peculiar profiles characterizing the different MS phenotypes. The study was carried out on 24 Clinically Isolated Syndrome (CIS), 16 Relapsing Remitting (RR) MS, 11 Progressive (Pr) MS patients. The CSF samples were analysed using the Matrix Assisted Laser Desorption Ionisation Time Of Flight (MALDI-TOF) mass spectrometer in linear mode geometry and in delayed extraction mode (m/z range: 1000-25000 Da). Peak lists were imported for normalization and statistical analysis. CSF data were correlated with demographic, clinical and MRI parameters. The evaluation of MALDI-TOF spectra revealed 348 peak signals with relative intensity ≥ 1% in the study range. The peak intensity of the signals corresponding to Secretogranin II and Protein 7B2 were significantly upregulated in RRMS patients compared to PrMS (p<0.05), whereas the signals of Fibrinogen and Fibrinopeptide A were significantly downregulated in CIS compared to PrMS patients (p<0.04). Additionally, the intensity of the Tymosin β4 peak was the only signal to be significantly discriminated between the CIS and RRMS patients (p = 0.013). Although with caution due to the relatively small size of the study populations, and considering that not all the findings remained significant after adjustment for multiple comparisons, in our opinion this mass spectrometry evaluation confirms that this technique may provide useful and important information to improve our understanding of the complex pathogenesis of MS.

  16. Proteome-wide reactivity profiling identifies diverse carbamate chemotypes tuned for serine hydrolase inhibition.

    PubMed

    Chang, Jae Won; Cognetta, Armand B; Niphakis, Micah J; Cravatt, Benjamin F

    2013-07-19

    Serine hydrolases are one of the largest and most diverse enzyme classes in Nature. Inhibitors of serine hydrolases are used to treat many diseases, including obesity, diabetes, cognitive dementia, and bacterial and viral infections. Nonetheless, the majority of the 200+ serine hydrolases in mammals still lack selective inhibitors for their functional characterization. We and others have shown that activated carbamates, through covalent reaction with the conserved serine nucleophile of serine hydrolases, can serve as useful inhibitors for members of this enzyme family. The extent to which carbamates, however, cross-react with other protein classes remains mostly unexplored. Here, we address this problem by investigating the proteome-wide reactivity of a diverse set of activated carbamates in vitro and in vivo, using a combination of competitive and click chemistry (CC)-activity-based protein profiling (ABPP). We identify multiple classes of carbamates, including O-aryl, O-hexafluoroisopropyl (HFIP), and O-N-hydroxysuccinimidyl (NHS) carbamates that react selectively with serine hydrolases across entire mouse tissue proteomes in vivo. We exploit the proteome-wide specificity of HFIP carbamates to create in situ imaging probes for the endocannabinoid hydrolases monoacylglycerol lipase (MAGL) and α-β hydrolase-6 (ABHD6). These findings, taken together, designate the carbamate as a privileged reactive group for serine hydrolases that can accommodate diverse structural modifications to produce inhibitors that display exceptional potency and selectivity across the mammalian proteome.

  17. Proteome-wide reactivity profiling identifies diverse carbamate chemotypes tuned for serine hydrolase inhibition

    PubMed Central

    Chang, Jae Won; Cognetta, Armand B.; Niphakis, Micah J.; Cravatt, Benjamin F.

    2013-01-01

    Serine hydrolases are one of the largest and most diverse enzyme classes in Nature. Inhibitors of serine hydrolases are used to treat many diseases, including obesity, diabetes, cognitive dementia, and bacterial and viral infections. Nonetheless, the majority of the 200+ serine hydrolases in mammals still lack selective inhibitors for their functional characterization. We and others have shown that activated carbamates, through covalent reaction with the conserved serine nucleophile of serine hydrolases, can serve as useful inhibitors for members of this enzyme family. The extent to which carbamates, however, cross-react with other protein classes remains mostly unexplored. Here, we address this problem by investigating the proteome-wide reactivity of a diverse set of activated carbamates in vitro and in vivo using a combination of competitive and click chemistry (CC)-activity-based protein profiling (ABPP). We identify multiple classes of carbamates, including O-aryl, O-hexafluoroisopropyl (HFIP), and O-N-hydroxysuccinimidyl (NHS) carbamates that react selectively with serine hydrolases across entire mouse tissue proteomes in vivo. We exploit the proteome-wide specificity of HFIP carbamates to create in situ imaging probes for the endocannabinoid hydrolases monoacylglycerol lipase (MAGL) and alpha-beta hydrolase-6 (ABHD6). These findings, taken together, designate the carbamate as a privileged reactive group for serine hydrolases that can accommodate diverse structural modifications to produce inhibitors that display exceptional potency and selectivity across the mammalian proteome. PMID:23701408

  18. Distinctive proteomic profiles among different regions of human carotid plaques in men and women

    PubMed Central

    Liang, Wenzhao; Ward, Liam J.; Karlsson, Helen; Ljunggren, Stefan A.; Li, Wei; Lindahl, Mats; Yuan, Xi-Ming

    2016-01-01

    The heterogeneity of atherosclerotic tissue has limited comprehension in proteomic and metabolomic analyses. To elucidate the functional implications, and differences between genders, of atherosclerotic lesion formation we investigated protein profiles from different regions of human carotid atherosclerotic arteries; internal control, fatty streak, plaque shoulder, plaque centre, and fibrous cap. Proteomic analysis was performed using 2-DE with MALDI-TOF, with validation using nLC-MS/MS. Protein mapping of 2-DE identified 52 unique proteins, including 15 previously unmapped proteins, of which 41 proteins were confirmed by nLC-MS/MS analysis. Expression levels of 18 proteins were significantly altered in plaque regions compared to the internal control region. Nine proteins showed site-specific alterations, irrespective of gender, with clear associations to extracellular matrix remodelling. Five proteins display gender-specific alterations with 2-DE, with two alterations validated by nLC-MS/MS. Gender differences in ferritin light chain and transthyretin were validated using both techniques. Validation of immunohistochemistry confirmed significantly higher levels of ferritin in plaques from male patients. Proteomic analysis of different plaque regions has reduced the effects of plaque heterogeneity, and significant differences in protein expression are determined in specific regions and between genders. These proteomes have functional implications in plaque progression and are of importance in understanding gender differences in atherosclerosis. PMID:27198765

  19. Proteomic profiling of salivary gland after nonviral gene transfer mediated by conventional plasmids and minicircles

    PubMed Central

    Geguchadze, Ramaz; Wang, Zhimin; Zourelias, Lee; Perez-Riveros, Paola; Edwards, Paul C; Machen, Laurie; Passineau, Michael J

    2014-01-01

    In this study, we compared gene transfer efficiency and host response to ultrasound-assisted, nonviral gene transfer with a conventional plasmid and a minicircle vector in the submandibular salivary glands of mice. Initially, we looked at gene transfer efficiency with equimolar amounts of the plasmid and minicircle vectors, corroborating an earlier report showing that minicircle is more efficient in the context of a physical method of gene transfer. We then sought to characterize the physiological response of the salivary gland to exogenous gene transfer using global proteomic profiling. Somewhat surprisingly, we found that sonoporation alone, without a gene transfer vector present, had virtually no effect on the salivary gland proteome. However, when a plasmid vector was used, we observed profound perturbations of the salivary gland proteome that compared in magnitude to that seen in a previous report after high doses of adeno-associated virus. Finally, we found that gene transfer with a minicircle induces only minor proteomic alterations that were similar to sonoporation alone. Using mass spectrometry, we assigned protein IDs to 218 gel spots that differed between plasmid and minicircle. Bioinformatic analysis of these proteins demonstrated convergence on 68 known protein interaction pathways, most notably those associated with innate immunity, cellular stress, and morphogenesis. PMID:25414909

  20. Whole proteomes as internal standards in quantitative proteomics.

    PubMed

    Ong, Shao-En

    2010-07-30

    As mass-spectrometry-based quantitative proteomics approaches become increasingly powerful, researchers are taking advantage of well established methodologies and improving instrumentation to pioneer new protein expression profiling methods. For example, pooling several proteomes labeled using the stable isotope labeling by amino acids in cell culture (SILAC) method yields a whole-proteome stable isotope-labeled internal standard that can be mixed with a tissue-derived proteome for quantification. By increasing quantitative accuracy in the analysis of tissue proteomes, such methods should improve integration of protein expression profiling data with transcriptomic data and enhance downstream bioinformatic analyses. An accurate and scalable quantitative method to analyze tumor proteomes at the depth of several thousand proteins provides a powerful tool for global protein quantification of tissue samples and promises to redefine our understanding of tumor biology.

  1. Global and comparative proteomic profiling of overwintering and developing mountain pine beetle, Dendroctonus ponderosae (Coleoptera: Curculionidae), larvae.

    PubMed

    Bonnett, Tiffany R; Robert, Jeanne A; Pitt, Caitlin; Fraser, Jordie D; Keeling, Christopher I; Bohlmann, Jörg; Huber, Dezene P W

    2012-12-01

    Mountain pine beetles, Dendroctonus ponderosae Hopkins (Coleoptera: Curculionidae), are native to western North America, but have recently begun to expand their range across the Canadian Rocky Mountains. The requirement for larvae to withstand extremely cold winter temperatures and potentially toxic host secondary metabolites in the midst of their ongoing development makes this a critical period of their lives. We have uncovered global protein profiles for overwintering mountain pine beetle larvae. We have also quantitatively compared the proteomes for overwintering larvae sampled during autumn cooling and spring warming using iTRAQ methods. We identified 1507 unique proteins across all samples. In total, 33 proteins exhibited differential expression (FDR < 0.05) when compared between larvae before and after a cold snap in the autumn; and 473 proteins exhibited differential expression in the spring when measured before and after a steady incline in mean daily temperature. Eighteen proteins showed significant changes in both autumn and spring samples. These first proteomic data for mountain pine beetle larvae show evidence of the involvement of trehalose, 2-deoxyglucose, and antioxidant enzymes in overwintering physiology; confirm and expand upon previous work implicating glycerol in cold tolerance in this insect; and provide new, detailed information on developmental processes in beetles. These results and associated data will be an invaluable resource for future targeted research on cold tolerance mechanisms in the mountain pine beetle and developmental biology in coleopterans. Copyright © 2012 Elsevier Ltd. All rights reserved.

  2. Genetic differences in the serum proteome of horses, donkeys and mules are detectable by protein profiling.

    PubMed

    Henze, Andrea; Aumer, Franziska; Grabner, Arthur; Raila, Jens; Schweigert, Florian J

    2011-10-01

    Although horses and donkeys belong to the same genus, their genetic characteristics probably result in specific proteomes and post-translational modifications (PTM) of proteins. Since PTM can alter protein properties, specific PTM may contribute to species-specific characteristics. Therefore, the aim of the present study was to analyse differences in serum protein profiles of horses and donkeys as well as mules, which combine the genetic backgrounds of both species. Additionally, changes in PTM of the protein transthyretin (TTR) were analysed. Serum protein profiles of each species (five animals per species) were determined using strong anion exchanger ProteinChips® (Bio-Rad, Munich, Germany) in combination with surface-enhanced laser desorption ionisation-time of flight MS. The PTM of TTR were analysed subsequently by immunoprecipitation in combination with matrix-assisted laser desorption ionisation-time of flight MS. Protein profiling revealed species-specific differences in the proteome, with some protein peaks present in all three species as well as protein peaks that were unique for donkeys and mules, horses and mules or for horses alone. The molecular weight of TTR of horses and donkeys differed by 30 Da, and both species revealed several modified forms of TTR besides the native form. The mass spectra of mules represented a merging of TTR spectra of horses and donkeys. In summary, the present study indicated that there are substantial differences in the proteome of horses and donkeys. Additionally, the results probably indicate that the proteome of mules reveal a higher similarity to donkeys than to horses.

  3. HUPO Plasma Proteome Project specimen collection and handling: towards the standardization of parameters for plasma proteome samples.

    PubMed

    Rai, Alex J; Gelfand, Craig A; Haywood, Bruce C; Warunek, David J; Yi, Jizu; Schuchard, Mark D; Mehigh, Richard J; Cockrill, Steven L; Scott, Graham B I; Tammen, Harald; Schulz-Knappe, Peter; Speicher, David W; Vitzthum, Frank; Haab, Brian B; Siest, Gerard; Chan, Daniel W

    2005-08-01

    There is a substantial list of pre-analytical variables that can alter the analysis of blood-derived samples. We have undertaken studies on some of these issues including choice of sample type, stability during storage, use of protease inhibitors, and clinical standardization. As there is a wide range of sample variables and a broad spectrum of analytical techniques in the HUPO PPP effort, it is not possible to define a single list of pre-analytical standards for samples or their processing. We present here a compendium of observations, drawing on actual results and sound clinical theories and practices. Based on our data, we find that (1) platelet-depleted plasma is preferable to serum for certain peptidomic studies; (2) samples should be aliquoted and stored preferably in liquid nitrogen; (3) the addition of protease inhibitors is recommended, but should be incorporated early and used judiciously, as some form non specific protein adducts and others interfere with peptide studies. Further, (4) the diligent tracking of pre-analytical variables and (5) the use of reference materials for quality control and quality assurance, are recommended. These findings help provide guidance on sample handling issues, with the overall suggestion being to be conscious of all possible pre-analytical variables as a prerequisite of any proteomic study.

  4. Elucidation of Xenobiotic Metabolism Pathways in Human Skin and Human Skin Models by Proteomic Profiling

    PubMed Central

    van Eijl, Sven; Zhu, Zheying; Cupitt, John; Gierula, Magdalena; Götz, Christine; Fritsche, Ellen; Edwards, Robert J.

    2012-01-01

    Background Human skin has the capacity to metabolise foreign chemicals (xenobiotics), but knowledge of the various enzymes involved is incomplete. A broad-based unbiased proteomics approach was used to describe the profile of xenobiotic metabolising enzymes present in human skin and hence indicate principal routes of metabolism of xenobiotic compounds. Several in vitro models of human skin have been developed for the purpose of safety assessment of chemicals. The suitability of these epidermal models for studies involving biotransformation was assessed by comparing their profiles of xenobiotic metabolising enzymes with those of human skin. Methodology/Principal Findings Label-free proteomic analysis of whole human skin (10 donors) was applied and analysed using custom-built PROTSIFT software. The results showed the presence of enzymes with a capacity for the metabolism of alcohols through dehydrogenation, aldehydes through dehydrogenation and oxidation, amines through oxidation, carbonyls through reduction, epoxides and carboxylesters through hydrolysis and, of many compounds, by conjugation to glutathione. Whereas protein levels of these enzymes in skin were mostly just 4–10 fold lower than those in liver and sufficient to support metabolism, the levels of cytochrome P450 enzymes were at least 300-fold lower indicating they play no significant role. Four epidermal models of human skin had profiles very similar to one another and these overlapped substantially with that of whole skin. Conclusions/Significance The proteomics profiling approach was successful in producing a comprehensive analysis of the biotransformation characteristics of whole human skin and various in vitro skin models. The results show that skin contains a range of defined enzymes capable of metabolising different classes of chemicals. The degree of similarity of the profiles of the in vitro models indicates their suitability for epidermal toxicity testing. Overall, these results provide a

  5. Proteomic Profiling of Exosomes Leads to the Identification of Novel Biomarkers for Prostate Cancer

    SciTech Connect

    Duijvesz, Diederick; Burnum-Johnson, Kristin E.; Gritsenko, Marina A.; Hoogland, Marije; Vredenbregt-van den Berg, Mirella S.; Willemsen, Rob; Luider, Theo N.; Pasa-Tolic, Ljiljana; Jenster, Guido

    2013-12-31

    Introduction: Current markers for prostate cancer, such as PSA lack specificity. Therefore, novel biomarkers are needed. Unfortunately, biomarker discovery from body fluids is often hampered by the high abundance of many proteins unrelated to disease. An attractive alternative biomarker discovery approach is the isolation of small vesicles (exosomes, ~100 nm). They contain proteins that are specific to the tissue from which they are derived and therefore can be considered as treasure chests for disease-specific marker discovery. Profiling prostate cancer-derived exosomes could reveal new markers for this malignancy. Materials and Methods: Exosomes were isolated from 2 immortalized primary prostate epithelial cells (PNT2C2 and RWPE-1) and 2 PCa cell lines (PC346C and VCaP) by ultracentrifugation. Proteomic analyses utilized a nanoLC coupled with an LTQ-Orbitrap operated in tandem MS (MS/MS) mode, followed by the Accurate Mass and Time (AMT) tag approach. Exosomal proteins were validated by Western blotting. A Tissue Micro Array, containing 481 different PCa samples (radical prostatectomy), was used to correlate candidate markers with several clinical-pathological parameters such as PSA, Gleason score, biochemical recurrence, and (PCa-related) death. Results: Proteomic characterization resulted in the identification of 263 proteins by at least 2 peptides. Specifically analysis of exosomes from PNT2C2, RWPE-1, PC346C, and VCaP identified 248, 233, 169, and 216 proteins, respectively. Statistical analyses revealed 52 proteins differently expressed between PCa and control cells, 9 of which were more abundant in PCa. Validation by Western blotting confirmed a higher abundance of FASN, XPO1 and PDCD6IP (ALIX) in PCa exosomes. The Tissue Micro 4 Array showed strong correlation of higher Gleason scores and local recurrence with increased cytoplasmic XPO1 (P<0.001). Conclusions: Differentially abundant proteins of cell line-derived exosomes make a clear subdivision between

  6. O-GlcNAc profiling: from proteins to proteomes

    PubMed Central

    2014-01-01

    O-linked β-D-N-acetylglucosamine (O-GlcNAc) modification (O-GlcNAcylation) onto serine and threonine residues of proteins is an important post-translational modification (PTM), which is involved in many crucial biological processes including transcription, translation, proteasomal degradation, and signal transduction. Aberrant protein O-GlcNAcylation is directly linked to the pathological progression of chronic diseases including diabetes, cancer, and neurodegenerative disorders. Identification, site mapping, and quantification of O-GlcNAc proteins are a prerequisite to decipher their functions. In this review, we mainly focus on technological developments regarding O-GlcNAc protein profiling. Specifically, on one hand, we show how these techniques are being used for the comprehensive characterization of certain targeted proteins in which biologists are most interested. On the other hand, we present several newly developed approaches for O-GlcNAcomic profiling as well as how they provide us with a systems perspective to crosstalk amongst different PTMs and complicated biological events. Promising technical trends are also highlighted to evoke more efforts by diverse laboratories, which would further expand our understanding of the physiological and pathological roles of protein O-GlcNAcylation in chronic diseases. PMID:24593906

  7. Comparative proteomic profiles of Pinus monticola needles during early compatible and incompatible interactions with Cronartium ribicola.

    PubMed

    Zamany, Arezoo; Liu, Jun-Jun; Ekramoddoullah, Abul K M

    2012-12-01

    The proteomic profiles of primary needles from Cr2-resistant and cr2-susceptible Pinus monticola seedlings were analysed post Cronartium ribicola inoculation by 2-DE. One hundred-and-five protein spots exhibiting significant differential expression were identified using LC-MS/MS. Functional classification showed that the most numerous proteins are involved in defence signalling, oxidative burst, metabolic pathways, and other physiological processes. Our results revealed that differential expression of proteins in response to C. ribicola inoculation was genotype- and infection-stage dependent. Responsive proteins in resistant seedlings with incompatible white pine blister rust (WPBR) interaction included such well-characterized proteins as heat shock proteins (HSPs), reactive oxygen species (ROS) scavenging enzymes, and intermediate factors functioning in the signal transduction pathways triggered by well-known plant R genes, as well as new candidates in plant defence like sugar epimerase, GTP-binding proteins, and chloroplastic ribonucleoproteins. Fewer proteins were regulated in susceptible seedlings; most of them were in common with resistant seedlings and related to photosynthesis among others. Quantitative RT-PCR analysis confirmed HSP- and ROS-related genes played an important role in host defence in response to C. ribicola infection. To the best of our knowledge, this is the first comparative proteomics study on WPBR interactions at the early stages of host defence, which provides a reference proteomic profile for other five-needle pines as well as resistance candidates for further understanding of host resistance in the WPBR pathosystem.

  8. Dynamic proteomic profiling of a unicellular cyanobacterium Cyanothece ATCC51142 across light-dark diurnal cycles

    SciTech Connect

    Aryal, Uma K.; Stockel, Jana; Krovvidi, Ravi K.; Gritsenko, Marina A.; Monroe, Matthew E.; Moore, Ronald J.; Koppenaal, David W.; Smith, Richard D.; Pakrasi, Himadri B.; Jacobs, Jon M.

    2011-12-01

    Unicellular cyanobacteria of the genus Cyanothece are recognized for their ability to execute nitrogen (N2)-fixation in the dark and photosynthesis in the light. Systems-wide dynamic proteomic profiling with mass spectrometry (MS) analysis reveals fundamental insights into the control and regulation of these functions. To expand upon the current knowledge of protein expression patterns in Cyanothece ATCC51142, we performed quantitative proteomic analysis using partial ("unsaturated") metabolic labeling and high mass accuracy LC-MS analysis. This dynamic proteomic profiling identified 721 actively synthesized proteins with significant temporal changes in expression throughout the light-dark cycles, of which 425 proteins matched with previously characterized cycling transcripts. The remaining 296 proteins contained a cluster of proteins uniquely involved in DNA replication and repair, protein degradation, tRNA synthesis and modification, transport and binding, and regulatory functions. Analysis of protein functions revealed that the expression of nitrogenase in the dark is mediated by higher respiration and glycogen metabolism. We have also shown that Cyanothece ATCC51142 utilizes alternative pathways for carbon (C) and nitrogen (N) acquisition, particularly, aspartic acid and glutamate as substrates of C and N, respectively. Utilization of phosphoketolase (PHK) pathway for the conversion of xylulose-5P to pyruvate and acetyl-P likely constitutes an alternative strategy to compensate higher ATP and NADPH demand. In conclusion, this study provides a deeper insight into how Cyanothece ATCC51142 modulates cellular functions to accommodate photosynthesis and N2-fixation within the single cell.

  9. Proteomic profile of saliva and plasma from women with impalpable breast lesions

    PubMed Central

    Delmonico, Lucas; Bravo, Maryah; Silvestre, Rafaele Tavares; Ornellas, Maria Helena Faria; De Azevedo, Carolina Maria; Alves, Gilda

    2016-01-01

    The present study evaluated the proteomic profile of saliva and plasma from women with impalpable breast lesions using nano-liquid chromatography-quadrupole-time-of-flight (nLC-Q-TOF) technology. Plasma and saliva from patients with fibroadenoma (n=10), infiltrating ductal carcinoma (n=10) and healthy control groups (n=8) were assessed by combinations of inter/intra-group analyses, revealing significant quantitative and qualitative differences. The major differentially-expressed proteins in the saliva of patients compared with the controls were α2-macroglobulin and ceruloplasmin, but the proteins that met the minimum fold-change and P-value cut-offs were leukocyte elastase inhibitor and α-enolase, and deleted in malignant brain tumors 1. Concerning plasma, α-2-macroglobulin and ceruplasmin were upregulated, while other proteins such as haptoglobin, hemopexin and vitamin D-binding protein were downregulated compared with the control. The changes in immune, molecular transport and signaling pathways were the most representative in the proteomic profile of the saliva and plasma. This is the first study to describe the proteome of saliva and plasma from the same women with impalpable breast lesions. PMID:27602154

  10. Spatially-Resolved Proteomics: Rapid Quantitative Analysis of Laser Capture Microdissected Alveolar Tissue Samples.

    PubMed

    Clair, Geremy; Piehowski, Paul D; Nicola, Teodora; Kitzmiller, Joseph A; Huang, Eric L; Zink, Erika M; Sontag, Ryan L; Orton, Daniel J; Moore, Ronald J; Carson, James P; Smith, Richard D; Whitsett, Jeffrey A; Corley, Richard A; Ambalavanan, Namasivayam; Ansong, Charles

    2016-12-22

    Laser capture microdissection (LCM)-enabled region-specific tissue analyses are critical to better understand complex multicellular processes. However, current proteomics workflows entail several manual sample preparation steps and are challenged by the microscopic mass-limited samples generated by LCM, impacting measurement robustness, quantification and throughput. Here, we coupled LCM with a proteomics workflow that provides fully automated analysis of proteomes from microdissected tissues. Benchmarking against the current state-of-the-art in ultrasensitive global proteomics (FASP workflow), our approach demonstrated significant improvements in quantification (~2-fold lower variance) and throughput (>5 times faster). Using our approach we for the first time characterized, to a depth of >3,400 proteins, the ontogeny of protein changes during normal lung development in microdissected alveolar tissue containing only 4,000 cells. Our analysis revealed seven defined modules of coordinated transcription factor-signaling molecule expression patterns, suggesting a complex network of temporal regulatory control directs normal lung development with epigenetic regulation fine-tuning pre-natal developmental processes.

  11. Spatially-Resolved Proteomics: Rapid Quantitative Analysis of Laser Capture Microdissected Alveolar Tissue Samples

    PubMed Central

    Clair, Geremy; Piehowski, Paul D.; Nicola, Teodora; Kitzmiller, Joseph A.; Huang, Eric L.; Zink, Erika M.; Sontag, Ryan L.; Orton, Daniel J.; Moore, Ronald J.; Carson, James P.; Smith, Richard D.; Whitsett, Jeffrey A.; Corley, Richard A.; Ambalavanan, Namasivayam; Ansong, Charles

    2016-01-01

    Laser capture microdissection (LCM)-enabled region-specific tissue analyses are critical to better understand complex multicellular processes. However, current proteomics workflows entail several manual sample preparation steps and are challenged by the microscopic mass-limited samples generated by LCM, impacting measurement robustness, quantification and throughput. Here, we coupled LCM with a proteomics workflow that provides fully automated analysis of proteomes from microdissected tissues. Benchmarking against the current state-of-the-art in ultrasensitive global proteomics (FASP workflow), our approach demonstrated significant improvements in quantification (~2-fold lower variance) and throughput (>5 times faster). Using our approach we for the first time characterized, to a depth of >3,400 proteins, the ontogeny of protein changes during normal lung development in microdissected alveolar tissue containing only 4,000 cells. Our analysis revealed seven defined modules of coordinated transcription factor-signaling molecule expression patterns, suggesting a complex network of temporal regulatory control directs normal lung development with epigenetic regulation fine-tuning pre-natal developmental processes. PMID:28004771

  12. A Quantitative Proteomics Approach to Clinical Research with Non-Traditional Samples.

    PubMed

    Licier, Rígel; Miranda, Eric; Serrano, Horacio

    2016-10-17

    The proper handling of samples to be analyzed by mass spectrometry (MS) can guarantee excellent results and a greater depth of analysis when working in quantitative proteomics. This is critical when trying to assess non-traditional sources such as ear wax, saliva, vitreous humor, aqueous humor, tears, nipple aspirate fluid, breast milk/colostrum, cervical-vaginal fluid, nasal secretions, bronco-alveolar lavage fluid, and stools. We intend to provide the investigator with relevant aspects of quantitative proteomics and to recognize the most recent clinical research work conducted with atypical samples and analyzed by quantitative proteomics. Having as reference the most recent and different approaches used with non-traditional sources allows us to compare new strategies in the development of novel experimental models. On the other hand, these references help us to contribute significantly to the understanding of the proportions of proteins in different proteomes of clinical interest and may lead to potential advances in the emerging field of precision medicine.

  13. A Quantitative Proteomics Approach to Clinical Research with Non-Traditional Samples

    PubMed Central

    Licier, Rígel; Miranda, Eric; Serrano, Horacio

    2016-01-01

    The proper handling of samples to be analyzed by mass spectrometry (MS) can guarantee excellent results and a greater depth of analysis when working in quantitative proteomics. This is critical when trying to assess non-traditional sources such as ear wax, saliva, vitreous humor, aqueous humor, tears, nipple aspirate fluid, breast milk/colostrum, cervical-vaginal fluid, nasal secretions, bronco-alveolar lavage fluid, and stools. We intend to provide the investigator with relevant aspects of quantitative proteomics and to recognize the most recent clinical research work conducted with atypical samples and analyzed by quantitative proteomics. Having as reference the most recent and different approaches used with non-traditional sources allows us to compare new strategies in the development of novel experimental models. On the other hand, these references help us to contribute significantly to the understanding of the proportions of proteins in different proteomes of clinical interest and may lead to potential advances in the emerging field of precision medicine. PMID:28248241

  14. Proteomic tools for environmental microbiology--a roadmap from sample preparation to protein identification and quantification.

    PubMed

    Wöhlbrand, Lars; Trautwein, Kathleen; Rabus, Ralf

    2013-10-01

    The steadily increasing amount of (meta-)genomic sequence information of diverse organisms and habitats has a strong impact on research in microbial physiology and ecology. In-depth functional understanding of metabolic processes and overall physiological adaptation to environmental changes, however, requires application of proteomics, as the context specific proteome constitutes the true functional output of a cell. Considering the enormous structural and functional diversity of proteins, only rational combinations of various analytical approaches allow a holistic view on the overall state of the cell. Within the past decade, proteomic methods became increasingly accessible to microbiologists mainly due to the robustness of analytical methods (e.g. 2DE), and affordability of mass spectrometers and their relative ease of use. This review provides an overview on the complex portfolio of state-of-the-art proteomics and highlights the basic principles of key methods, ranging from sample preparation of laboratory or environmental samples, via protein/peptide separation (gel-based or gel-free) and different types of mass spectrometric protein/peptide analyses, to protein identification and abundance determination.

  15. Proteomic protease specificity profiling of clostridial collagenases reveals their intrinsic nature as dedicated degraders of collagen.

    PubMed

    Eckhard, Ulrich; Huesgen, Pitter F; Brandstetter, Hans; Overall, Christopher M

    2014-04-04

    Clostridial collagenases are among the most efficient degraders of collagen. Most clostridia are saprophytes and secrete proteases to utilize proteins in their environment as carbon sources; during anaerobic infections, collagenases play a crucial role in host colonization. Several medical and biotechnological applications have emerged utilizing their high collagenolytic efficiency. However, the contribution of the functionally most important peptidase domain to substrate specificity remains unresolved. We investigated the active site sequence specificity of the peptidase domains of collagenase G and H from Clostridium histolyticum and collagenase T from Clostridium tetani. Both prime and non-prime cleavage site specificity were simultaneously profiled using Proteomic Identification of protease Cleavage Sites (PICS), a mass spectrometry-based method utilizing database searchable proteome-derived peptide libraries. For each enzyme we identified >100 unique-cleaved peptides, resulting in robust cleavage logos revealing collagen-like specificity patterns: a strong preference for glycine in P3 and P1', proline at P2 and P2', and a slightly looser specificity at P1, which in collagen is typically occupied by hydroxyproline. This specificity for the classic collagen motifs Gly-Pro-X and Gly-X-Hyp represents a remarkable adaptation considering the complex requirements for substrate unfolding and presentation that need to be fulfilled before a single collagen strand becomes accessible for cleavage. We demonstrate the striking sequence specificity of a family of clostridial collagenases using proteome derived peptide libraries and PICS, Proteomic Identification of protease Cleavage Sites. In combination with the previously published crystal structures of these proteases, our results represent an important piece of the puzzle in understanding the complex mechanism underlying collagen hydrolysis, and pave the way for the rational design of specific test substrates and

  16. Classification of fish samples via an integrated proteomics and bioinformatics approach.

    PubMed

    Bellgard, Matthew; Taplin, Ross; Chapman, Brett; Livk, Andreja; Wellington, Crispin; Hunter, Adam; Lipscombe, Richard

    2013-11-01

    There is an increasing demand to develop cost-effective and accurate approaches to analyzing biological tissue samples. This is especially relevant in the fishing industry where closely related fish samples can be mislabeled, and the high market value of certain fish leads to the use of alternative species as substitutes, for example, Barramundi and Nile Perch (belonging to the same genus, Lates). There is a need to combine selective proteomic datasets with sophisticated computational analysis to devise a robust classification approach. This paper describes an integrated MS-based proteomics and bioinformatics approach to classifying a range of fish samples. A classifier is developed using training data that successfully discriminates between Barramundi and Nile Perch samples using a selected protein subset of the proteome. Additionally, the classifier is shown to successfully discriminate between test samples not used to develop the classifier, including samples that have been cooked, and to classify other fish species as neither Barramundi nor Nile Perch. This approach has applications to truth in labeling for fishmongers and restaurants, monitoring fish catches, and for scientific research into distances between species. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. Proteomic profile of Ortleppascaris sp.: A helminth parasite of Rhinella marina in the Amazonian region.

    PubMed

    E Silva, Jefferson Pereira; Furtado, Adriano Penha; Dos Santos, Jeannie Nascimento

    2014-08-01

    Ortleppascaris sp. is a helminth that, in its larval stage, infects the liver parenchyma of the amphibian Rhinella marina, resulting in severe physiological and pathological changes. This study used a proteomic approach to determine the overall profile of proteins expressed in a somatic extract from the nematodes to investigate the relationship between the parasite and its host. A total of 60 abundant proteins were selected from the two-dimensional electrophoresis, identified by peptide mass fingerprinting, and grouped based on their Gene Ontology by the biological processes in which they are potentially involved. Important helminthic derivatives, such as the immunoreactive As37 antigen, guanylyl cyclases, proteolytic enzymes, and other proteins conserved among different parasites, were identified through homology. This study represents a new approach to helminth-related proteomic studies using an amphibian animal model. Furthermore, this study identified protein markers that are important to the host-parasite relationship and the viability, development, infectivity, and virulence of helminths.

  18. Proteomic profile of Ortleppascaris sp.: A helminth parasite of Rhinella marina in the Amazonian region

    PubMed Central

    e Silva, Jefferson Pereira; Furtado, Adriano Penha; dos Santos, Jeannie Nascimento

    2014-01-01

    Ortleppascaris sp. is a helminth that, in its larval stage, infects the liver parenchyma of the amphibian Rhinella marina, resulting in severe physiological and pathological changes. This study used a proteomic approach to determine the overall profile of proteins expressed in a somatic extract from the nematodes to investigate the relationship between the parasite and its host. A total of 60 abundant proteins were selected from the two-dimensional electrophoresis, identified by peptide mass fingerprinting, and grouped based on their Gene Ontology by the biological processes in which they are potentially involved. Important helminthic derivatives, such as the immunoreactive As37 antigen, guanylyl cyclases, proteolytic enzymes, and other proteins conserved among different parasites, were identified through homology. This study represents a new approach to helminth-related proteomic studies using an amphibian animal model. Furthermore, this study identified protein markers that are important to the host–parasite relationship and the viability, development, infectivity, and virulence of helminths. PMID:25161903

  19. PrePhyloPro: phylogenetic profile-based prediction of whole proteome linkages

    PubMed Central

    Niu, Yulong; Liu, Chengcheng; Moghimyfiroozabad, Shayan; Yang, Yi

    2017-01-01

    Direct and indirect functional links between proteins as well as their interactions as part of larger protein complexes or common signaling pathways may be predicted by analyzing the correlation of their evolutionary patterns. Based on phylogenetic profiling, here we present a highly scalable and time-efficient computational framework for predicting linkages within the whole human proteome. We have validated this method through analysis of 3,697 human pathways and molecular complexes and a comparison of our results with the prediction outcomes of previously published co-occurrency model-based and normalization methods. Here we also introduce PrePhyloPro, a web-based software that uses our method for accurately predicting proteome-wide linkages. We present data on interactions of human mitochondrial proteins, verifying the performance of this software. PrePhyloPro is freely available at http://prephylopro.org/phyloprofile/. PMID:28875072

  20. Proteomics profile of cellular response to chiral drugs: prospects for pharmaceutical applications.

    PubMed

    Bun Ching, Chi; Zhang, Jianhua; Sui, Jianjun; Ning Chen, Wei

    2010-02-01

    Chiral drugs account for a large proportion of drugs available in the market. There is increasing awareness of the importance of drug chirality and the role it plays in explaining the oftentimes dramatic differences in biological activities in the current drug development portfolio. Using recently developed chiral drugs-cell interaction system, several examples of protein profiles induced by chiral drugs were illustrated in detail on the platform of 2-D LC interfaced with MS/MS system. In addition, the background of chiral drug investigation from which contemporary drug chirality research has emerged, the techniques involved in proteomics technology, the application of proteomics in this exciting area, and the perspectives in future applications are also discussed.

  1. PrePhyloPro: phylogenetic profile-based prediction of whole proteome linkages.

    PubMed

    Niu, Yulong; Liu, Chengcheng; Moghimyfiroozabad, Shayan; Yang, Yi; Alavian, Kambiz N

    2017-01-01

    Direct and indirect functional links between proteins as well as their interactions as part of larger protein complexes or common signaling pathways may be predicted by analyzing the correlation of their evolutionary patterns. Based on phylogenetic profiling, here we present a highly scalable and time-efficient computational framework for predicting linkages within the whole human proteome. We have validated this method through analysis of 3,697 human pathways and molecular complexes and a comparison of our results with the prediction outcomes of previously published co-occurrency model-based and normalization methods. Here we also introduce PrePhyloPro, a web-based software that uses our method for accurately predicting proteome-wide linkages. We present data on interactions of human mitochondrial proteins, verifying the performance of this software. PrePhyloPro is freely available at http://prephylopro.org/phyloprofile/.

  2. Proteomic profile evolution during steatosis development in ducks.

    PubMed

    Bax, M L; Chambon, C; Marty-Gasset, N; Remignon, H; Fernandez, X; Molette, C

    2012-01-01

    We investigated a protein profile evolution during steatosis in ducks using 2-dimensional electrophoresis gels to better understand the mechanisms underlying liver steatosis at the level of hepatic proteins in waterfowl. Two-dimensional electrophoresis gels were performed in the liver at different stages of steatosis in the duck. Mule ducks were slaughtered after 0, 14, or 23 meals of overfeeding, according to commercial conditions. Thirty-one proteic spots were differentially expressed between 3 or 2 durations of overfeeding: 3 spots were differentially expressed between the 3 times and 28 spots were differentially expressed between 2 times. The identified proteins (14) could be regrouped into 5 categories: enzymes, translation factors, proteins involved in cell structure, proteins with antioxidant properties, and proteins that can link calcium. This study opens new research areas in the understanding of steatosis in waterfowl, such as cell structure and oxidative stress.

  3. A Proteomics Sample Preparation Method for Mature, Recalcitrant Leaves of Perennial Plants

    PubMed Central

    Na, Zhang; Chengying, Lao; Bo, Wang; Dingxiang, Peng; Lijun, Liu

    2014-01-01

    Sample preparation is key to the success of proteomics studies. In the present study, two sample preparation methods were tested for their suitability on the mature, recalcitrant leaves of six representative perennial plants (grape, plum, pear, peach, orange, and ramie). An improved sample preparation method was obtained: Tris and Triton X-100 were added together instead of CHAPS to the lysis buffer, and a 20% TCA-water solution and 100% precooled acetone were added after the protein extraction for the further purification of protein. This method effectively eliminates nonprotein impurities and obtains a clear two-dimensional gel electrophoresis array. The method facilitates the separation of high-molecular-weight proteins and increases the resolution of low-abundance proteins. This method provides a widely applicable and economically feasible technology for the proteomic study of the mature, recalcitrant leaves of perennial plants. PMID:25028960

  4. Fermentation and proteome profiles of Lactobacillus plantarum strains during growth under food-like conditions.

    PubMed

    Siragusa, Sonya; De Angelis, Maria; Calasso, Maria; Campanella, Daniela; Minervini, Fabio; Di Cagno, Raffaella; Gobbetti, Marco

    2014-01-16

    This study aimed at investigating the proteomic adaptation of Lactobacillus plantarum strains. Cultivation of L. plantarum strains under food-like conditions (wheat flour hydrolyzed, whey milk, tomato juice) affected some metabolic traits (e.g., consumption of carbohydrates and synthesis of organic acids) compared to de Man, Rogosa and Sharpe (MRS) broth. The analysis of the fermentation profile showed that the highest number of carbon sources metabolized by L. plantarum strains was found using cells cultivated in media containing low concentration of glucose or no glucose at all. The proteomic maps of the strains were comparatively determined after growth on MRS broth and under food-like conditions. The amount of proteins depended on strain and, especially, on culture conditions. Proteins showing decreased or increased amounts under food-like conditions were identified using MALDI-TOF-MS/MS or LC-nano-ESI-MS/MS. Changes of the proteome concerned proteins that are involved in carbohydrate transport and metabolism, energy metabolism, Sec-dependent secretion system, stress response, nucleotide metabolism, regulation of nitrogen metabolism, and protein biosynthesis. A catabolic repression by glucose on carbohydrate transport and metabolism was also found. The characterization of the proteomes in response to changing environmental conditions could be useful to get L. plantarum strains adapted for specific applications. Microbial cell performance during food biotechnological processes has become one of the greatest concerns all over the world. L. plantarum is a lactic acid bacterium with a large industrial application for fermented foods or functional foods (e.g., probiotics). The present study compared the fermentation and proteomic profiling of L. plantarum strains during growth under food-like conditions and under optimal laboratory conditions (MRS broth). This study provides specific mechanisms of proteomic adaptation involved in the microbial performances

  5. Comparative proteomic study for profiling differentially expressed proteins between Chinese left- and right-sided colon cancers.

    PubMed

    Shen, Hong; Huang, Jinlin; Pei, Haiping; Zeng, Shan; Tao, Yiming; Shen, Liangfang; Zeng, Liang; Zhu, Hong

    2013-01-01

    The aim of the present study is to profile differentially expressed protein markers between left-sided colon cancer (LSCC) and right-sided colon cancer (RSCC). Fresh tumor tissue samples from LSCC (n = 7) and RSCC (n = 7) groups were analyzed by two-dimensional electrophoresis coupled with MALDI-TOF-MS, followed by Western blotting. In 50 paraffin embedded samples from each group, levels of four differentially expressed proteins (identified by proteomics analysis) were measured by tissue microarray with immunohistochemistry staining to compare the different protein markers between LSCC and RSCC. Sixteen proteins were found to be differentially expressed between LSCC and RSCC. Ten proteins including HSP-60 and PDIA1 were identified to be highly expressed in LSCC (P < 0.01 or P < 0.05), while the expression of six proteins including EEF1D and HSP-27 were higher in RSCC (P < 0.01 or P < 0.05). Virtually all of the indentified proteins were involved in cellular energy metabolism, protein folding/unfolding, and/or oxidative stress. Human colon tumors at various locations have different proteomic biomarkers. Differentially expressed proteins associated with energy metabolism, protein folding/unfolding and oxidative stress contribute to different tumorigenesis, tumor progression, and prognosis between left- and right-sided colon cancer.

  6. CSF profiling of the human brain enriched proteome reveals associations of neuromodulin and neurogranin to Alzheimer's disease

    PubMed Central

    Remnestål, Julia; Just, David; Mitsios, Nicholas; Fredolini, Claudia; Mulder, Jan; Schwenk, Jochen M; Uhlén, Mathias; Kultima, Kim; Ingelsson, Martin; Kilander, Lena; Lannfelt, Lars; Svenningsson, Per; Nellgård, Bengt; Zetterberg, Henrik; Blennow, Kaj; Häggmark‐Månberg, Anna

    2016-01-01

    1 Purpose This study is part of a larger effort aiming to expand the knowledge of brain‐enriched proteins in human cerebrospinal fluid (CSF) and to provide novel insight into the relation between such proteins and different neurodegenerative diseases. 2 Experimental design Here 280 brain‐enriched proteins in CSF from patients with Alzheimer's disease (AD), Parkinson's disease (PD) and dementia with Lewy bodies (DLB) are profiled. In total, 441 human samples of ventricular CSF collected post mortem and lumbar CSF collected ante mortem are analyzed using 376 antibodies in a suspension bead array setup, utilizing a direct labelling approach. 3 Results Among several proteins displaying differentiated profiles between sample groups, we focus here on two synaptic proteins, neuromodulin (GAP43) and neurogranin (NRGN). They are both found at elevated levels in CSF from AD patients in two independent cohorts, providing disease‐associated profiles in addition to verifying and strengthening previously observed patterns. Increased levels are also observed for patients for whom the AD diagnosis was not established at the time of sampling. 4 Conclusions and clinical relevance These findings indicate that analyzing the brain‐enriched proteins in CSF is of particular interest to increase the understanding of the CSF proteome and its relation to neurodegenerative disorders. In addition, this study lends support to the notion that measurements of these synaptic proteins could potentially be of great relevance in future diagnostic tests for AD. PMID:27604409

  7. Search for novel circulating cancer chemopreventive biomarkers of dietary rice bran intervention in Apc(Min) mice model of colorectal carcinogenesis, using proteomic and metabolic profiling strategies.

    PubMed

    Norris, Leonie; Malkar, Aditya; Horner-Glister, Emma; Hakimi, Amirmansoor; Ng, Leong L; Gescher, Andreas J; Creaser, Colin; Sale, Stewart; Jones, Donald J L

    2015-09-01

    There is strong epidemiological evidence indicating that consumption by humans of whole-grain foods including rice bran may be associated with a low incidence of cancer, especially in the colorectum. Molecular processes associated with cancer development may be retarded by fiber consumption. Consequently, intervention with dietary fiber might be suitable as a cancer chemoprevention strategy in high-risk populations. Here, we searched for putative molecular mechanism-based efficacy biomarkers of rice fiber consumption in the plasma of mice characterized by a genetic propensity to develop gastrointestinal adenomas. The hypothesis was tested that metabolic and proteomic changes in blood reflect the chemopreventive activity of rice bran. Apc(Min) mice received diet supplemented with rice bran at 5, 15, and 30%. Blood and tissue samples were taken. Plasma was subjected to MS-based proteomic and metabolic profiling analyses as well as assessment of hematocrit values. Gastrointestinal tracts were removed and adenomas were counted and their size was measured so that total tumor burden could be calculated. The hypothesis was tested that metabolic and proteomic changes in blood reflect chemopreventive activity. Rice bran consumption reduced adenoma burden and number in a dose-related fashion when compared to controls. Metabolic profiling data demonstrated strong clustering of the groups indicating that metabolic pathways are perturbed. Proteomic analysis identified adiponectin as a molecule that was significantly altered, which may play a role in tumor suppression. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. Development stage-specific proteomic profiling uncovers small, lineage specific proteins most abundant in the Aspergillus Fumigatus conidial proteome.

    PubMed

    Suh, Moo-Jin; Fedorova, Natalie D; Cagas, Steven E; Hastings, Susan; Fleischmann, Robert D; Peterson, Scott N; Perlin, David S; Nierman, William C; Pieper, Rembert; Momany, Michelle

    2012-04-30

    The pathogenic mold Aspergillus fumigatus is the most frequent infectious cause of death in severely immunocompromised individuals such as leukemia and bone marrow transplant patients. Germination of inhaled conidia (asexual spores) in the host is critical for the initiation of infection, but little is known about the underlying mechanisms of this process. To gain insights into early germination events and facilitate the identification of potential stage-specific biomarkers and vaccine candidates, we have used quantitative shotgun proteomics to elucidate patterns of protein abundance changes during early fungal development. Four different stages were examined: dormant conidia, isotropically expanding conidia, hyphae in which germ tube emergence has just begun, and pre-septation hyphae. To enrich for glycan-linked cell wall proteins we used an alkaline cell extraction method. Shotgun proteomic resulted in the identification of 375 unique gene products with high confidence, with no evidence for enrichment of cell wall-immobilized and secreted proteins. The most interesting discovery was the identification of 52 proteins enriched in dormant conidia including 28 proteins that have never been detected in the A. fumigatus conidial proteome such as signaling protein Pil1, chaperones BipA and calnexin, and transcription factor HapB. Additionally we found many small, Aspergillus specific proteins of unknown function including 17 hypothetical proteins. Thus, the most abundant protein, Grg1 (AFUA_5G14210), was also one of the smallest proteins detected in this study (M.W. 7,367). Among previously characterized proteins were melanin pigment and pseurotin A biosynthesis enzymes, histones H3 and H4.1, and other proteins involved in conidiation and response to oxidative or hypoxic stress. In contrast, expanding conidia, hyphae with early germ tubes, and pre-septation hyphae samples were enriched for proteins responsible for housekeeping functions, particularly translation

  9. Whole-proteome phylogeny of prokaryotes by feature frequency profiles: An alignment-free method with optimal feature resolution.

    PubMed

    Jun, Se-Ran; Sims, Gregory E; Wu, Guohong A; Kim, Sung-Hou

    2010-01-05

    We present a whole-proteome phylogeny of prokaryotes constructed by comparing feature frequency profiles (FFPs) of whole proteomes. Features are l-mers of amino acids, and each organism is represented by a profile of frequencies of all features. The selection of feature length is critical in the FFP method, and we have developed a procedure for identifying the optimal feature lengths for inferring the phylogeny of prokaryotes, strictly speaking, a proteome phylogeny. Our FFP trees are constructed with whole proteomes of 884 prokaryotes, 16 unicellular eukaryotes, and 2 random sequences. To highlight the branching order of major groups, we present a simplified proteome FFP tree of monophyletic class or phylum with branch support. In our whole-proteome FFP trees (i) Archaea, Bacteria, Eukaryota, and a random sequence outgroup are clearly separated; (ii) Archaea and Bacteria form a sister group when rooted with random sequences; (iii) Planctomycetes, which possesses an intracellular membrane compartment, is placed at the basal position of the Bacteria domain; (iv) almost all groups are monophyletic in prokaryotes at most taxonomic levels, but many differences in the branching order of major groups are observed between our proteome FFP tree and trees built with other methods; and (v) previously "unclassified" genomes may be assigned to the most likely taxa. We describe notable similarities and differences between our FFP trees and those based on other methods in grouping and phylogeny of prokaryotes.

  10. Proteome Profiles of Digested Products of Commercial Meat Sources

    PubMed Central

    Li, Li; Liu, Yuan; Zhou, Guanghong; Xu, Xinglian; Li, Chunbao

    2017-01-01

    This study was designed to characterize in vitro-digested products of proteins from four commercial meat products, including dry-cured ham, cooked ham, emulsion-type sausage, and dry-cured sausage. The samples were homogenized and incubated with pepsin and trypsin. The digestibility and particle sizes of digested products were measured. Nano-LC–MS/MS was applied to characterize peptides. The results showed the highest digestibility and the lowest particle size in dry-cured ham (P < 0.05), while the opposite was for cooked ham (P < 0.05). Nano-LC–MS/MS analysis revealed that dry-cured ham samples had the greatest number of 750–3,500 Da Mw peptides in pepsin-digested products. In the digested products of cooked ham and emulsion-type sausage, a lot of peptides were matched with soy protein that was added in the formulations. In addition, protein oxidation was also observed in different meat products. Our findings give an insight into nutritional values of different meat products. PMID:28396857

  11. Feeding Low or Pharmacological Concentrations of Zinc Oxide Changes the Hepatic Proteome Profiles in Weaned Piglets

    PubMed Central

    Bondzio, Angelika; Pieper, Robert; Gabler, Christoph; Weise, Christoph; Schulze, Petra; Zentek, Juergen; Einspanier, Ralf

    2013-01-01

    Pharmacological levels of zinc oxide can promote growth and health of weaning piglets, but the underlying molecular mechanisms are yet not fully understood. The aim of this study was to determine changes in the global hepatic protein expression in response to dietary zinc oxide in weaned piglets. Nine half-sib piglets were allocated to three dietary zinc treatment groups (50, 150, 2500 mg/kg dry matter). After 14 d, pigs were euthanized and liver samples taken. The increase in hepatic zinc concentration following dietary supplementation of zinc was accompanied by up-regulation of metallothionein mRNA and protein expression. Global hepatic protein profiles were obtained by two-dimensional difference gel electrophoresis following matrix-assisted laser desorption ionization/time-of-flight mass spectrometry. A total of 15 proteins were differentially (P<0.05) expressed between groups receiving control (150 mg/kg) or pharmacological levels of zinc (2500 mg/kg) with 7 down- (e.g. arginase1, thiosulfate sulfurtransferase, HSP70) and 8 up-regulated (e.g. apolipoprotein AI, transferrin, C1-tetrahydrofolate synthase) proteins. Additionally, three proteins were differentially expressed with low zinc supply (50 mg/kg Zn) in comparison to the control diet. The identified proteins were mainly associated with functions related to cellular stress, transport, metabolism, and signal transduction. The differential regulation was evaluated at the mRNA level and a subset of three proteins of different functional groups was selected for confirmation by western blotting. The results of this proteomic study suggest that zinc affects important liver functions such as blood protein secretion, protein metabolism, detoxification and redox homeostasis, thus supporting the hypothesis of intermediary effects of pharmacological levels of zinc oxide fed to pigs. PMID:24282572

  12. Proteomic profiling of inflammatory signaling molecules in the tears of patients on chronic glaucoma medication.

    PubMed

    Wong, Tina T; Zhou, Lei; Li, Jing; Tong, Louis; Zhao, Shao Zhen; Li, Xiao Rong; Yu, Shang Juan; Koh, Siew Kwan; Beuerman, Roger W

    2011-09-22

    To identify the tear proteins associated with the long-term use of glaucoma medication by using proteomic analysis and to compare these proteins to those previously reported in primary dry eye disease. Eighteen patients treated with topical antiglaucoma medications and 10 normal age-matched subjects with no prior topical treatment were recruited for the study. Tears were collected by using Schirmer's strip and analyzed by iTRAQ (isobaric tag for relative and absolute quantitation) for tear proteins by mass spectrometry. Conjunctival samples were collected and RNA expression determined by PCR. Of the 124 identified tear proteins (99% confidence, ProtScore ≥ 2.0), we found that the tear levels of S100-A8, S100-A9, mammaglobin B, and 14-3-3 ζ/δ were significantly increased in the medicated group compared with levels in the nonmedicated group (P < 0.05). For S100-A9, mammaglobin B, and 14-3-3 ζ/δ, use of topical medication for less than 1 year did not reach statistical significance compared with that in the nonmedicated group. Eyes on topical medication for less than 1 year showed a decrease in proline-rich 4 protein tear level (P = 0.0049) compared to nonmedicated group. The tear proteins detected in the medicated group differed from those in the primary dry eye group. Treatment with topical antiglaucoma medications for longer than 1 year may start to induce ocular surface inflammation. The inflammatory tear protein profile present in chronically medicated glaucomatous eyes appears to be different from that found in primary dry eye. Identification of tear proteins specific to medicated glaucomatous eyes will help to specifically develop targeted screening modalities and therapeutic agents different from current conventional dry eye management.

  13. Beyond the Western front: Targeted proteomics and organelle abundance profiling

    SciTech Connect

    Parsons, Harriet T.; Heazlewood, Joshua L.

    2015-05-05

    The application of westerns or immunoblotting techniques for assessing the composition, dynamics, and purity of protein extracts from plant material has become common practice. While the approach is reproducible, can be readily applied and is generally considered robust, the field of plant science suffers from a lack of antibody variety against plant proteins. The development of approaches that employ mass spectrometry to enable both relative and absolute quantification of many hundreds of proteins in a single sample from a single analysis provides a mechanism to overcome the expensive impediment in having to develop antibodies in plant science. Here, we consider it an opportune moment to consider and better develop the adoption of multiple reaction monitoring (MRM)-based analyses in plant biochemistry.

  14. Beyond the Western front: Targeted proteomics and organelle abundance profiling

    DOE PAGES

    Parsons, Harriet T.; Heazlewood, Joshua L.

    2015-05-05

    The application of westerns or immunoblotting techniques for assessing the composition, dynamics, and purity of protein extracts from plant material has become common practice. While the approach is reproducible, can be readily applied and is generally considered robust, the field of plant science suffers from a lack of antibody variety against plant proteins. The development of approaches that employ mass spectrometry to enable both relative and absolute quantification of many hundreds of proteins in a single sample from a single analysis provides a mechanism to overcome the expensive impediment in having to develop antibodies in plant science. Here, we considermore » it an opportune moment to consider and better develop the adoption of multiple reaction monitoring (MRM)-based analyses in plant biochemistry.« less

  15. The human liver-specific proteome defined by transcriptomics and antibody-based profiling.

    PubMed

    Kampf, Caroline; Mardinoglu, Adil; Fagerberg, Linn; Hallström, Björn M; Edlund, Karolina; Lundberg, Emma; Pontén, Fredrik; Nielsen, Jens; Uhlen, Mathias

    2014-07-01

    Human liver physiology and the genetic etiology of the liver diseases can potentially be elucidated through the identification of proteins with enriched expression in the liver. Here, we combined data from RNA sequencing (RNA-Seq) and antibody-based immunohistochemistry across all major human tissues to explore the human liver proteome with enriched expression, as well as the cell type-enriched expression in hepatocyte and bile duct cells. We identified in total 477 protein-coding genes with elevated expression in the liver: 179 genes have higher expression as compared to all the other analyzed tissues; 164 genes have elevated transcript levels in the liver shared with at least one other tissue type; and an additional 134 genes have a mild level of increased expression in the liver. We identified the precise localization of these proteins through antibody-based protein profiling and the subcellular localization of these proteins through immunofluorescent-based profiling. We also identified the biological processes and metabolic functions associated with these proteins, investigated their contribution in the occurrence of liver diseases, and identified potential targets for their treatment. Our study demonstrates the use of RNA-Seq and antibody-based immunohistochemistry for characterizing the human liver proteome, as well as the use of tissue-specific proteins in identification of novel drug targets and discovery of biomarkers.-Kampf, C., Mardinoglu, A., Fagerberg, L., Hallström, B. M., Edlund, K., Lundberg, E., Pontén, F., Nielsen, J., Uhlen, M. The human liver-specific proteome defined by transcriptomics and antibody-based profiling. © FASEB.

  16. Human monocyte-derived macrophages are heterogenous: Proteomic profile of different phenotypes.

    PubMed

    Eligini, S; Brioschi, M; Fiorelli, S; Tremoli, E; Banfi, C; Colli, S

    2015-06-21

    Tissue macrophages play a key role in many aspects of human physiology and pathology. These cells are heterogeneous both in term of morphology and function. As an example, heterogeneity has been reported within the atherosclerotic lesions where distinct populations exert opposite functions driving plaque progression or stability. Tissue macrophages are not easily obtained and differentiated blood-derived monocytes are largely used as surrogate model. We previously reported that human macrophages spontaneously differentiated from adherent monocytes show two dominant subsets, distinct for morphology (spindle and round) and functions. The aim of this study was to evaluate the intracellular proteome of these two macrophage subsets by means of a microproteomic workflow properly set up to simultaneously identify and quantify proteins from a minimal number of morphotypically heterogeneous cells in culture. We report two distinct proteomic profiles that distinguish round from spindle macrophages. In particular, differential abundances were observed for proteins involved in membrane traffic regulation, lipid handling, efferocytosis, and protection against stress conditions. Results reinforce and extend previous data on the functional and antigenic profile of these macrophage phenotypes strengthening the suitability of our model to focus on macrophage heterogeneity. Tissue macrophages patrol homeostatic functions, immune surveillance, and resolution of inflammation. The spectrum of macrophage activation states is, therefore, wide and gives ground for the heterogeneity of these cells, documented in health and disease. This study provides knowledge of the distinct proteome that characterises the two dominant morphotypes (round and spindle) of human macrophages that, in our culture condition, are generated by spontaneous differentiation from blood-derived monocytes. Results extend previous data about the different antigenic, transcriptional, and functional profiles of these

  17. Different Proteome Profiles between Male and Female Populus cathayana Exposed to UV-B Radiation

    PubMed Central

    Zhang, Yunxiang; Feng, Lihua; Jiang, Hao; Zhang, Yuanbin; Zhang, Sheng

    2017-01-01

    With increasing altitude, solar UV-B radiation is enhanced. Based on the phenomenon of male-biased sex ratio of Populus cathayana Rehder in high altitude alpine area, we hypothesized that males have a faster and more sophisticated responsive mechanism to high UV-B radiation than that of females. Our previous studies have shown sexually different responses to high UV-B radiation were existed in P. cathayana at the morphological, physiological, and transcriptomic levels. However, the responses at the proteomic level remain unclear. In this study, an isobaric tag for relative and absolute quantification (iTRAQ)-based quantitative proteome analysis was performed in P. cathayana females and males. A total of 2,405 proteins were identified, with 331 proteins defined as differentially expressed proteins (DEPs). Among of these, 79 and 138 DEPs were decreased and 47 and 107 DEPs were increased under high solar UV-B radiation in females and males, respectively. A bioinformatics analysis categorized the common responsive proteins in the sexes as related to carbohydrate and energy metabolism, translation/transcription/post-transcriptional modification, photosynthesis, and redox reactions. The responsive proteins that showed differences in sex were mainly those involved in amino acid metabolism, stress response, and translation/transcription/post-transcriptional modification. This study provides proteomic profiles that poplars responding to solar UV-B radiation, and it also provides new insights into differentially sex-related responses to UV-B radiation. PMID:28326097

  18. S-Nitrosylation Proteome Profile of Peripheral Blood Mononuclear Cells in Human Heart Failure

    PubMed Central

    Spratt, Heidi M.; Gupta, Shivali; Petersen, John R.; Kuyumcu-Martinez, Muge N.

    2016-01-01

    Nitric oxide (NO) protects the heart against ischemic injury; however, NO- and superoxide-dependent S-nitrosylation (S-NO) of cysteines can affect function of target proteins and play a role in disease outcome. We employed 2D-GE with thiol-labeling FL-maleimide dye and MALDI-TOF MS/MS to capture the quantitative changes in abundance and S-NO proteome of HF patients (versus healthy controls, n = 30/group). We identified 93 differentially abundant (59-increased/34-decreased) and 111 S-NO-modified (63-increased/48-decreased) protein spots, respectively, in HF subjects (versus controls, fold-change | ≥1.5|, p ≤ 0.05). Ingenuity pathway analysis of proteome datasets suggested that the pathways involved in phagocytes' migration, free radical production, and cell death were activated and fatty acid metabolism was decreased in HF subjects. Multivariate adaptive regression splines modeling of datasets identified a panel of proteins that will provide >90% prediction success in classifying HF subjects. Proteomic profiling identified ATP-synthase, thrombospondin-1 (THBS1), and vinculin (VCL) as top differentially abundant and S-NO-modified proteins, and these proteins were verified by Western blotting and ELISA in different set of HF subjects. We conclude that differential abundance and S-NO modification of proteins serve as a mechanism in regulating cell viability and free radical production, and THBS1 and VCL evaluation will potentially be useful in the prediction of heart failure. PMID:27635260

  19. Profiling the secretome and extracellular proteome of the potato late blight pathogen Phytophthora infestans.

    PubMed

    Meijer, Harold J G; Mancuso, Francesco M; Espadas, Guadalupe; Seidl, Michael F; Chiva, Cristina; Govers, Francine; Sabidó, Eduard

    2014-08-01

    Oomycetes are filamentous organisms that cause notorious diseases, several of which have a high economic impact. Well known is Phytophthora infestans, the causal agent of potato late blight. Previously, in silico analyses of the genome and transcriptome of P. infestans resulted in the annotation of a large number of genes encoding proteins with an N-terminal signal peptide. This set is collectively referred to as the secretome and comprises proteins involved in, for example, cell wall growth and modification, proteolytic processes, and the promotion of successful invasion of plant cells. So far, proteomic profiling in oomycetes was primarily focused on subcellular, intracellular or cell wall fractions; the extracellular proteome has not been studied systematically. Here we present the first comprehensive characterization of the in vivo secretome and extracellular proteome of P. infestans. We have used mass spectrometry to analyze P. infestans proteins present in seven different growth media with mycelial cultures and this resulted in the consistent identification of over two hundred proteins. Gene ontology classification pinpointed proteins involved in cell wall modifications, pathogenesis, defense responses, and proteolytic processes. Moreover, we found members of the RXLR and CRN effector families as well as several proteins lacking an obvious signal peptide. The latter were confirmed to be bona fide extracellular proteins and this suggests that, similar to other organisms, oomycetes exploit non-conventional secretion mechanisms to transfer certain proteins to the extracellular environment.

  20. Proteomic Profiling of Sugar Beet (Beta vulgaris) Leaves during Rhizomania Compatible Interactions

    PubMed Central

    Webb, Kimberly M.; Broccardo, Carolyn J.; Prenni, Jessica E.; Wintermantel, William M.

    2014-01-01

    Rhizomania, caused by Beet necrotic yellow vein virus (BNYVV), severely impacts sugar beet (Beta vulgaris) production throughout the world, and is widely prevalent in most production regions. Initial efforts to characterize proteome changes focused primarily on identifying putative host factors that elicit resistant interactions with BNYVV, but as resistance breaking strains become more prevalent, effective disease control strategies will require the application of novel methods based on better understanding of disease susceptibility and symptom development. Herein, proteomic profiling was conducted on susceptible sugar beet, infected with two strains of BNYVV, to clarify the types of proteins prevalent during compatible virus-host plant interactions. Total protein was extracted from sugar beet leaf tissue infected with BNYVV, quantified, and analyzed by mass spectrometry. A total of 203 proteins were confidently identified, with a predominance of proteins associated with photosynthesis and energy, metabolism, and response to stimulus. Many proteins identified in this study are typically associated with systemic acquired resistance and general plant defense responses. These results expand on relatively limited proteomic data available for sugar beet and provide the ground work for additional studies focused on understanding the interaction of BNYVV with sugar beet. PMID:28250378

  1. Different Proteome Profiles between Male and Female Populus cathayana Exposed to UV-B Radiation.

    PubMed

    Zhang, Yunxiang; Feng, Lihua; Jiang, Hao; Zhang, Yuanbin; Zhang, Sheng

    2017-01-01

    With increasing altitude, solar UV-B radiation is enhanced. Based on the phenomenon of male-biased sex ratio of Populus cathayana Rehder in high altitude alpine area, we hypothesized that males have a faster and more sophisticated responsive mechanism to high UV-B radiation than that of females. Our previous studies have shown sexually different responses to high UV-B radiation were existed in P. cathayana at the morphological, physiological, and transcriptomic levels. However, the responses at the proteomic level remain unclear. In this study, an isobaric tag for relative and absolute quantification (iTRAQ)-based quantitative proteome analysis was performed in P. cathayana females and males. A total of 2,405 proteins were identified, with 331 proteins defined as differentially expressed proteins (DEPs). Among of these, 79 and 138 DEPs were decreased and 47 and 107 DEPs were increased under high solar UV-B radiation in females and males, respectively. A bioinformatics analysis categorized the common responsive proteins in the sexes as related to carbohydrate and energy metabolism, translation/transcription/post-transcriptional modification, photosynthesis, and redox reactions. The responsive proteins that showed differences in sex were mainly those involved in amino acid metabolism, stress response, and translation/transcription/post-transcriptional modification. This study provides proteomic profiles that poplars responding to solar UV-B radiation, and it also provides new insights into differentially sex-related responses to UV-B radiation.

  2. Proteomic Profiling of Sugar Beet (Beta vulgaris) Leaves during Rhizomania Compatible Interactions.

    PubMed

    Webb, Kimberly M; Broccardo, Carolyn J; Prenni, Jessica E; Wintermantel, William M

    2014-04-09

    Rhizomania, caused by Beet necrotic yellow vein virus (BNYVV), severely impacts sugar beet (Beta vulgaris) production throughout the world, and is widely prevalent in most production regions. Initial efforts to characterize proteome changes focused primarily on identifying putative host factors that elicit resistant interactions with BNYVV, but as resistance breaking strains become more prevalent, effective disease control strategies will require the application of novel methods based on better understanding of disease susceptibility and symptom development. Herein, proteomic profiling was conducted on susceptible sugar beet, infected with two strains of BNYVV, to clarify the types of proteins prevalent during compatible virus-host plant interactions. Total protein was extracted from sugar beet leaf tissue infected with BNYVV, quantified, and analyzed by mass spectrometry. A total of 203 proteins were confidently identified, with a predominance of proteins associated with photosynthesis and energy, metabolism, and response to stimulus. Many proteins identified in this study are typically associated with systemic acquired resistance and general plant defense responses. These results expand on relatively limited proteomic data available for sugar beet and provide the ground work for additional studies focused on understanding the interaction of BNYVV with sugar beet.

  3. Profiling the Secretome and Extracellular Proteome of the Potato Late Blight Pathogen Phytophthora infestans

    PubMed Central

    Meijer, Harold J. G.; Mancuso, Francesco M.; Espadas, Guadalupe; Seidl, Michael F.; Chiva, Cristina; Govers, Francine; Sabidó, Eduard

    2014-01-01

    Oomycetes are filamentous organisms that cause notorious diseases, several of which have a high economic impact. Well known is Phytophthora infestans, the causal agent of potato late blight. Previously, in silico analyses of the genome and transcriptome of P. infestans resulted in the annotation of a large number of genes encoding proteins with an N-terminal signal peptide. This set is collectively referred to as the secretome and comprises proteins involved in, for example, cell wall growth and modification, proteolytic processes, and the promotion of successful invasion of plant cells. So far, proteomic profiling in oomycetes was primarily focused on subcellular, intracellular or cell wall fractions; the extracellular proteome has not been studied systematically. Here we present the first comprehensive characterization of the in vivo secretome and extracellular proteome of P. infestans. We have used mass spectrometry to analyze P. infestans proteins present in seven different growth media with mycelial cultures and this resulted in the consistent identification of over two hundred proteins. Gene ontology classification pinpointed proteins involved in cell wall modifications, pathogenesis, defense responses, and proteolytic processes. Moreover, we found members of the RXLR and CRN effector families as well as several proteins lacking an obvious signal peptide. The latter were confirmed to be bona fide extracellular proteins and this suggests that, similar to other organisms, oomycetes exploit non-conventional secretion mechanisms to transfer certain proteins to the extracellular environment. PMID:24872595

  4. Proteomic Profile in Glomeruli of Type-2 Diabetic KKAy Mice using 2-Dimensional Differential Gel Electrophoresis

    PubMed Central

    Liu, Xiaodan; Yang, Gang; Fan, Qiuling; Wang, Lining

    2014-01-01

    Background Diabetic nephropathy (DN) is a leading cause of end-stage renal disease. To search for glomerular proteins associated with early-stage DN, glomeruli of spontaneous type 2 diabetic KKAy mice were analyzed by 2-dimensional differential gel electrophoresis (2D-DIGE). Material/Methods Glomeruli of 20-week spontaneous type 2 diabetic KKAy mice and age-matched C57BL/6 mice were isolated by kidney perfusion with magnetic beads. Proteomic profiles of glomeruli were investigated by using 2D-DIGE and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Western blot analysis was used to confirm the results of proteomics. Immunohistochemical and semi-quantitative analysis were used to confirm the differential expression of prohibitin and annexin A2 in glomeruli. Results We identified 19 differentially expressed proteins – 17 proteins were significantly up-regulated and 2 proteins were significantly down-regulated in glomeruli of diabetic KKAy mice. Among them, prohibitin and annexin A2 were up-regulated and Western blot analysis validated the same result in proteomics. Immunohistochemical analysis also revealed up-regulation of prohibitin and annexin A2 in glomeruli of KKAy mice. Conclusions Our findings suggest that prohibitin and annexin A2 may be associated with early-stage DN. Further functional research might help to reveal the pathogenesis of DN. PMID:25515740

  5. Proteomic profile of circulating immune complexes in chronic Chagas disease.

    PubMed

    Ohyama, K; Huy, N T; Yoshimi, H; Kishikawa, N; Nishizawa, J E; Roca, Y; Revollo Guzmán, R J; Velarde, F U G; Kuroda, N; Hirayama, K

    2016-10-01

    Immune complexes (ICs) are the direct and real-time products of humoral immune responses. The identification of constituent foreign or autoantigens within ICs might bring new insights into the pathology of infectious diseases. We applied immune complexome analysis of plasma to the study of Chagas disease caused by Trypanosoma cruzi. Twenty seropositive plasma samples including cardiac and/or megacolon determinate patients (n = 11) and indeterminate (n = 9) were analysed along with 10 seronegative individuals to characterize the antigens bound to circulating ICs. We identified 39 T. cruzi antigens and 114 human autoantigens specific to patients with Chagas. Among those antigens, two T. cruzi antigens (surface protease GP63, glucose-6-isomerase) and six human autoantigens (CD180 antigen, ceruloplasmin, fibrinogen beta chain, fibrinogen beta chain isoform 2 preprotein, isoform gamma-A of fibrinogen γ-chain, serum paraoxonase) were detected in more than 50% of the patients tested. Human isoform short of complement factor H-related protein 2 and trans-sialidase of T. cruzi were more frequently found in the indeterminate (5/9 for both) compared with in the determinate Chagas (0/11, P = 0·046 for human, 1/11, P = 0·0498 for T. cruzi). The immune complexome could illustrate the difference of immune status between clinical forms of chronic Chagas disease. © 2016 John Wiley & Sons Ltd.

  6. Proteomic profiling of sea bass muscle by two-dimensional gel electrophoresis and tandem mass spectrometry.

    PubMed

    Terova, Genciana; Pisanu, Salvatore; Roggio, Tonina; Preziosa, Elena; Saroglia, Marco; Addis, Maria Filippa

    2014-02-01

    In this study, the proteome profile of European sea bass (Dicentrarchus labrax) muscle was analyzed using two-dimensional electrophoresis (2-DE) and tandem mass spectrometry with the aim of providing a more detailed characterization of its specific protein expression profile. A highly populated and well-resolved 2-DE map of the sea bass muscle tissue was generated, and the corresponding protein identity was provided for a total of 49 abundant protein spots. Upon Ingenuity Pathway Analysis, the proteins mapped in the sea bass muscle profile were mostly related to glycolysis and to the muscle myofibril structure, together with other biological activities crucial to fish muscle metabolism and contraction, and therefore to fish locomotor performance. The data presented in this work provide important and novel information on the sea bass muscle tissue-specific protein expression, which can be useful for future studies aimed to improve seafood traceability, food safety/risk management and authentication analysis. This work is also important for understanding the proteome map of the sea bass toward establishing the animal as a potential model for muscular studies.

  7. Profiling of antioxidative enzyme expression induced by various food components using targeted proteome analysis.

    PubMed

    Bartenbacher, Sven; Östreicher, Christiane; Pischetsrieder, Monika

    2017-09-01

    A method was developed for targeted proteome analysis of the expression profile of a set of antioxidative enzymes in rat macrophages and applied to screen the antioxidative potential of several food components/foods. Expression profiles of heme oxygenase 1, peroxiredoxin 1, thioredoxin reductase 1, glutathione reductase, glutathione-S transferase P1, and superoxide dismutase 1 were analyzed by nanoLC-MS/MS in selected scheduled reaction monitoring (sSRM) mode monitoring two to three peptides per protein and three transitions per peptide. Relative quantification was performed by metabolic labeling. The validated method was used to profile the activity of capsaicin, carnosol, diallyl trisulfide, maslinic acid, quercetin, sulforaphane, cinnamaldehyde and coffee extract to modulate the expression levels of antioxidative enzymes. Carnosol and sulforaphane most effectively induced protein expression, leading to upregulation of at least five out of the six antioxidative enzymes by a maximum factor of 22.80 ± 6.71 (heme oxygenase 1 by carnosol). Heme oxygenase 1 was most susceptible to nutritive modulation, whereas glutathione reductase expression rates were hardly affected. Targeted mass proteome analysis allows comprehensive evaluation of antioxidative effects by food ingredients. Simultaneous expression analysis of a set of proteins provided valuable insights how various enzymes were differently affected by food components. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. Development and Evaluation of a Micro- and Nanoscale Proteomic Sample Preparation Method

    SciTech Connect

    Wang, Haixing H.; Qian, Weijun; Mottaz, Heather M.; Clauss, Therese R.W.; Anderson, David J.; Moore, Ronald J.; Camp, David G.; Khan, Arshad H.; Sforza, Daniel M.; Pallavicini, Maria; Smith, Desmond J.; Smith, Richard D.

    2005-10-05

    Efficient and effective sample preparation of micro- and nano-scale (micro- and nano-gram) clinical specimens for proteomic applications is often difficult due to losses during the processing steps. Herein we describe a simple “single-tube” preparation protocol appropriate for small proteomic samples using the organic co-solvent, trifluoroethanol (TFE). TFE facilitates both protein extraction and protein denaturation without requiring a separate cleanup step, thus minimizing sample loss. The performance of the TFE method was initially evaluated by comparing to traditional detergent-based methods on relatively large scale sample processing using human breast cancer cells and mouse brain tissue. The results demonstrated that the TFE protocol provided comparable results to the traditional detergent-based protocols for larger samples (milligrams), based on both sample recovery and peptide/protein identification. The effectiveness of this protocol for micro- and nano-scale sample processing was then evaluated for the extraction of proteins/peptides and shown effective for small mouse brain tissue samples (~ 20 μg total protein content) and also for samples of ~ 5 000 human breast cancer MCF-7 cells (~ 500 ng total protein content), where the detergent-based methods were ineffective due to losses during cleanup and transfer steps.

  9. Differential proteomic profiling to study the mechanism of cardiac pharmacological preconditioning by resveratrol

    PubMed Central

    Bezstarosti, Karel; Das, Samarjit; Lamers, Jos M J; Das, Dipak K

    2006-01-01

    Recent studies demonstrated that resveratrol, a grape-derived polyphenolic phytoalexin, provides pharmacological preconditioning of the heart through a NO-dependent mechanism. To further explore the molecular mechanisms involved in resveratrol-mediated cardioprotection, we monitored the effects of resveratrol treatment after ischemia-reperfusion on the protein profile by implementation of proteomic analysis. Two groups of rats were studied; one group of animals was fed resveratrol for 7 days, while the other group was given vehicle only. The rats were sacrificed for the isolated working heart preparation and for isolation of cytoplasmic fraction from left ventricle homogenates to carry out the proteomic as well as immunoblot at baseline and at the end of 30 min ischemia/2-h perfusion. The results demonstrate significant cardiopro-tection with resveratrol evidenced by improved ventricular recovery and reduced infarct size and cardiomyocyte apopto-sis. The left ventricular cytoplasmic fractions were separated by two-dimensional electrophoresis (2-DE). Differentially regulated proteins were detected with quantitative computer analysis of the Coomassie blue stained 2-DE images and identified by MALDI-TOF (MS) and nanoLC-ESI-Q-TOF mass spectrometry (MS/MS). Five redox-regulated and precondi-tioning-related proteins were identified that were all upregulated by resveratrol: MAPKK, two different aB-crystallin species, HSP 27 and PE binding protein. Another HSP27 species and aldose reductase were downregulated and peroxire-doxin-2 remained constant. The results of the immunoblot analysis of phosphorylated MAPKK, -HSP27 and -aB-crys-tallin and PE binding protein were consistent with the proteomic findings, but not with peroxiredoxin-2. The proteomic analysis showed also downregulation of some proteins in the mitochondrial respiratory chain and matrix and the myofila-ment regulating protein MLC kinase-2. The results of the present study demonstrate that proteomic profiling

  10. Proteomic peptide profiling for preemptive diagnosis of acute graft-versus-host disease after allogeneic stem cell transplantation.

    PubMed

    Weissinger, E M; Metzger, J; Dobbelstein, C; Wolff, D; Schleuning, M; Kuzmina, Z; Greinix, H; Dickinson, A M; Mullen, W; Kreipe, H; Hamwi, I; Morgan, M; Krons, A; Tchebotarenko, I; Ihlenburg-Schwarz, D; Dammann, E; Collin, M; Ehrlich, S; Diedrich, H; Stadler, M; Eder, M; Holler, E; Mischak, H; Krauter, J; Ganser, A

    2014-04-01

    Allogeneic hematopoietic stem cell transplantation is one curative treatment for hematological malignancies, but is compromised by life-threatening complications, such as severe acute graft-versus-host disease (aGvHD). Prediction of severe aGvHD as early as possible is crucial to allow timely initiation of treatment. Here we report on a multicentre validation of an aGvHD-specific urinary proteomic classifier (aGvHD_MS17) in 423 patients. Samples (n=1106) were collected prospectively between day +7 and day +130 and analyzed using capillary electrophoresis coupled on-line to mass spectrometry. Integration of aGvHD_MS17 analysis with demographic and clinical variables using a logistic regression model led to correct classification of patients developing severe aGvHD 14 days before any clinical signs with 82.4% sensitivity and 77.3% specificity. Multivariate regression analysis showed that aGvHD_MS17 positivity was the only strong predictor for aGvHD grade III or IV (P<0.0001). The classifier consists of 17 peptides derived from albumin, β2-microglobulin, CD99, fibronectin and various collagen α-chains, indicating inflammation, activation of T cells and changes in the extracellular matrix as early signs of GvHD-induced organ damage. This study is currently the largest demonstration of accurate and investigator-independent prediction of patients at risk for severe aGvHD, thus allowing preemptive therapy based on proteomic profiling.

  11. Deep proteome profiling of circulating granulocytes reveals bactericidal/permeability-increasing protein as a biomarker for severe atherosclerotic coronary stenosis.

    PubMed

    Bleijerveld, Onno B; Wijten, Patrick; Cappadona, Salvatore; McClellan, Elizabeth A; Polat, Ayse N; Raijmakers, Reinout; Sels, Jan-Willem; Colle, Loes; Grasso, Simona; van den Toorn, Henk W; van Breukelen, Bas; Stubbs, Andrew; Pasterkamp, Gerard; Heck, Albert J R; Hoefer, Imo E; Scholten, Arjen

    2012-11-02

    Coronary atherosclerosis represents the major cause of death in Western societies. As atherosclerosis typically progresses over years without giving rise to clinical symptoms, biomarkers are urgently needed to identify patients at risk. Over the past decade, evidence has accumulated suggesting cross-talk between the diseased vasculature and cells of the innate immune system. We therefore employed proteomics to search for biomarkers associated with severe atherosclerotic coronary lumen stenosis in circulating leukocytes. In a two-phase approach, we first performed in-depth quantitative profiling of the granulocyte proteome on a small pooled cohort of patients suffering from chronic (sub)total coronary occlusion and matched control patients using stable isotope peptide labeling, two-dimensional LC-MS/MS and data-dependent decision tree fragmentation. Over 3000 proteins were quantified, among which 57 candidate biomarker proteins remained after stringent filtering. The most promising biomarker candidates were subsequently verified in the individual samples of the discovery cohort using label-free, single-run LC-MS/MS analysis, as well as in an independent verification cohort of 25 patients with total coronary occlusion (CTO) and 19 matched controls. Our data reveal bactericidal/permeability-increasing protein (BPI) as a promising biomarker for severe atherosclerotic coronary stenosis, being down-regulated in circulating granulocytes of CTO patients.

  12. Proteomic and metabolomic profiles demonstrate variation among free-living and symbiotic vibrio fischeri biofilms.

    PubMed

    Chavez-Dozal, Alba; Gorman, Clayton; Nishiguchi, Michele K

    2015-10-23

    A number of bacterial species are capable of growing in various life history modes that enable their survival and persistence in both planktonic free-living stages as well as in biofilm communities. Mechanisms contributing to either planktonic cell or biofilm persistence and survival can be carefully delineated using multiple differential techniques (e.g., genomics and transcriptomics). In this study, we present both proteomic and metabolomic analyses of Vibrio fischeri biofilms, demonstrating the potential for combined differential studies for elucidating life-history switches important for establishing the mutualism through biofilm formation and host colonization. The study used a metabolomics/proteomics or "meta-proteomics" approach, referring to the combined protein and metabolic data analysis that bridges the gap between phenotypic changes (planktonic cell to biofilm formation) with genotypic changes (reflected in protein/metabolic profiles). Our methods used protein shotgun construction, followed by liquid chromatography coupled with mass spectrometry (LC-MS) detection and quantification for both free-living and biofilm forming V. fischeri. We present a time-resolved picture of approximately 100 proteins (2D-PAGE and shotgun proteomics) and 200 metabolites that are present during the transition from planktonic growth to community biofilm formation. Proteins involved in stress response, DNA repair damage, and transport appeared to be highly expressed during the biofilm state. In addition, metabolites detected in biofilms correspond to components of the exopolysaccharide (EPS) matrix (sugars and glycerol-derived). Alterations in metabolic enzymes were paralleled by more pronounced changes in concentration of intermediates from the glycolysis pathway as well as several amino acids. This combined analysis of both types of information (proteins, metabolites) has provided a more complete picture of the biochemical processes of biofilm formation and what determines

  13. A single lysis solution for the analysis of tissue samples by different proteomic technologies.

    PubMed

    Gromov, Pavel; Celis, Julio E; Gromova, Irina; Rank, Fritz; Timmermans-Wielenga, Vera; Moreira, José M A

    2008-12-01

    Cancer, being a major healthcare concern worldwide, is one of the main targets for the application of emerging proteomic technologies and these tools promise to revolutionize the way cancer will be diagnosed and treated in the near future. Today, as a result of the unprecedented advances that have taken place in molecular biology, cell biology and genomics there is a pressing need to accelerate the translation of basic discoveries into clinical applications. This need, compounded by mounting evidence that cellular model systems are unable to fully recapitulate all biological aspects of human dissease, is driving scientists to increasingly use clinically relevant samples for biomarker and target discovery. Tissues are heterogeneous and as a result optimization of sample preparation is critical for generating accurate, representative, and highly reproducible quantitative data. Although a large number of protocols for preparation of tissue lysates has been published, so far no single recipe is able to provide a "one-size fits all" solubilization procedure that can be used to analyse the same lysate using different proteomics technologies. Here we present evidence showing that cell lysis buffer 1 (CLB1), a lysis solution commercialized by Zeptosens [a division of Bayer (Schweiz) AG], provides excellent sample solubilization and very high 2D PAGE protein resolution both when using carrier ampholytes and immobilized pH gradient strips. Moreover, this buffer can also be used for array-based proteomics (reverse-phase lysate arrays or direct antibody arrays), allowing the direct comparison of qualitative and quantitative data yielded by these technologies when applied to the same samples. The usefulness of the CLB1 solution for gel-based proteomics was further established by 2D PAGE analysis of a number of technically demanding specimens such as breast carcinoma core needle biopsies and problematic tissues such as brain cortex, cerebellum, skeletal muscle, kidney cortex and

  14. Global Cell Proteome Profiling, Phospho-signaling and Quantitative 
Proteomics for Identification of New Biomarkers in Acute Myeloid 
Leukemia Patients

    PubMed Central

    Aasebø, Elise; Forthun, Rakel B.; Berven, Frode; Selheim, Frode; Hernandez-Valladares, Maria

    2016-01-01

    The identification of protein biomarkers for acute myeloid leukemia (AML) that could find applications in AML diagnosis and prognosis, treatment and the selection for bone marrow transplant requires substantial comparative analyses of the proteomes from AML patients. In the past years, several studies have suggested some biomarkers for AML diagnosis or AML classification using methods for sample preparation with low proteome coverage and low resolution mass spectrometers. However, most of the studies did not follow up, confirm or validate their candidates with more patient samples. Current proteomics methods, new high resolution and fast mass spectrometers allow the identification and quantification of several thousands of proteins obtained from few tens of μg of AML cell lysate. Enrichment methods for posttranslational modifications (PTM), such as phosphorylation, can isolate several thousands of site-specific phosphorylated peptides from AML patient samples, which subsequently can be quantified with high confidence in new mass spectrometers. While recent reports aiming to propose proteomic or phosphoproteomic biomarkers on the studied AML patient samples have taken advantage of the technological progress, the access to large cohorts of AML patients to sample from and the availability of appropriate control samples still remain challenging. PMID:26306748

  15. Profiling of Protein N-Termini and Their Modifications in Complex Samples.

    PubMed

    Demir, Fatih; Niedermaier, Stefan; Kizhakkedathu, Jayachandran N; Huesgen, Pitter F

    2017-01-01

    Protein N termini are a unique window to the functional state of the proteome, revealing translation initiation sites, co-translation truncation and modification, posttranslational maturation, and further proteolytic processing into different proteoforms with distinct functions. As a direct readout of proteolytic activity, protein N termini further reveal proteolytic regulation of diverse biological processes and provide a route to determine specific substrates and hence the physiological functions for any protease of interest. Here, we describe our current protocol of the successful Terminal Amine Isotope Labeling of Substrates (TAILS) technique, which enriches protein N-terminal peptides from complex proteome samples by negative selection. Genome-encoded N termini, protease-generated neo-N termini, and endogenously modified N termini are all enriched simultaneously. Subsequent mass spectrometric analysis therefore profiles all protein N termini and their modifications present in a complex sample in a single experiment. We further provide a detailed protocol for the TAILS-compatible proteome preparation from plant material and discuss specific considerations for N terminome data analysis and annotation.

  16. Dynamic proteomic profiling of a unicellular cyanobacterium Cyanothece ATCC51142 across light-dark diurnal cycles

    PubMed Central

    2011-01-01

    Background Unicellular cyanobacteria of the genus Cyanothece are recognized for their ability to execute nitrogen (N2)-fixation in the dark and photosynthesis in the light. An understanding of these mechanistic processes in an integrated systems context should provide insights into how Cyanothece might be optimized for specialized environments and/or industrial purposes. Systems-wide dynamic proteomic profiling with mass spectrometry (MS) analysis should reveal fundamental insights into the control and regulation of these functions. Results To expand upon the current knowledge of protein expression patterns in Cyanothece ATCC51142, we performed quantitative proteomic analysis using partial ("unsaturated") metabolic labeling and high mass accuracy LC-MS analysis. This dynamic proteomic profiling identified 721 actively synthesized proteins with significant temporal changes in expression throughout the light-dark cycles, of which 425 proteins matched with previously characterized cycling transcripts. The remaining 296 proteins contained a cluster of proteins uniquely involved in DNA replication and repair, protein degradation, tRNA synthesis and modification, transport and binding, and regulatory functions. Functional classification of labeled proteins suggested that proteins involved in respiration and glycogen metabolism showed increased expression in the dark cycle together with nitrogenase, suggesting that N2-fixation is mediated by higher respiration and glycogen metabolism. Results indicated that Cyanothece ATCC51142 might utilize alternative pathways for carbon (C) and nitrogen (N) acquisition, particularly, aspartic acid and glutamate as substrates of C and N, respectively. Utilization of phosphoketolase (PHK) pathway for the conversion of xylulose-5P to pyruvate and acetyl-P likely constitutes an alternative strategy to compensate higher ATP and NADPH demand. Conclusion This study provides a deeper systems level insight into how Cyanothece ATCC51142

  17. Comparison of proteomic profiles in the zebrafish retina during experimental degeneration and regeneration

    PubMed Central

    Eastlake, Karen; Heywood, Wendy E.; Tracey-White, Dhani; Aquino, Erika; Bliss, Emily; Vasta, Gerardo R.; Mills, Kevin; Khaw, Peng T.; Moosajee, Mariya; Limb, G. Astrid

    2017-01-01

    Zebrafish spontaneously regenerate the retina after injury. Although the gene expression profile has been extensively studied in this species during regeneration, this does not reflect protein function. To further understand the regenerative process in the zebrafish, we compared the proteomic profile of the retina during injury and upon regeneration. Using two-dimensional difference gel electrophoresis (2D-DIGE) and label-free quantitative proteomics (quadrupole time of flight LC-MS/MS), we analysed the retina of adult longfin wildtype zebrafish at 0, 3 and 18 days after Ouabain injection. Gene ontology analysis indicates reduced metabolic processing, and increase in fibrin clot formation, with significant upregulation of fibrinogen gamma polypeptide, apolipoproteins A-Ib and A-II, galectin-1, and vitellogenin-6 during degeneration when compared to normal retina. In addition, cytoskeleton and membrane transport proteins were considerably altered during regeneration, with the highest fold upregulation observed for tubulin beta 2 A, histone H2B and brain type fatty acid binding protein. Key proteins identified in this study may play an important role in the regeneration of the zebrafish retina and investigations on the potential regulation of these proteins may lead to the design of protocols to promote endogenous regeneration of the mammalian retina following retinal degenerative disease. PMID:28300160

  18. Comparative proteomic analysis of four Bacillus clausii strains: proteomic expression signature distinguishes protein profile of the strains.

    PubMed

    Lippolis, Rosa; Gnoni, Antonio; Abbrescia, Anna; Panelli, Damiano; Maiorano, Stefania; Paternoster, Maria Stefania; Sardanelli, Anna Maria; Papa, Sergio; Gaballo, Antonio

    2011-11-18

    A comparative proteomic approach, using two dimensional gel electrophoresis and mass spectrometry, has been developed to compare and elucidate the differences among the cellular proteomes of four closely related isogenic O/C, SIN, N/R and T, B. clausii strains during both exponential and stationary phases of growth. Image analysis of the electropherograms reveals a high degree of concordance among the four proteomes, some proteins result, however, differently expressed. The proteins spots exhibiting high different expression level were identified, by mass-spectrometry analysis, as alcohol dehydrogenase (ADHA, EC1.2.1.3; ABC0046 isoform) aldehyde dehydrogenase (DHAS, EC 1.2.1.3; ABC0047 isoform) and flagellin-protein of B. clausii KSM-k16. The different expression levels of the two dehydrogenases were confirmed by quantitative RT-PCR and dehydrogenases enzymatic activity. The different patterns of protein expression can be considered as cell proteome signatures of the different strains.

  19. A high-throughput sample preparation method for cellular proteomics using 96-well filter plates.

    PubMed

    Switzar, Linda; van Angeren, Jordy; Pinkse, Martijn; Kool, Jeroen; Niessen, Wilfried M A

    2013-10-01

    A high-throughput sample preparation protocol based on the use of 96-well molecular weight cutoff (MWCO) filter plates was developed for shotgun proteomics of cell lysates. All sample preparation steps, including cell lysis, buffer exchange, protein denaturation, reduction, alkylation and proteolytic digestion are performed in a 96-well plate format, making the platform extremely well suited for processing large numbers of samples and directly compatible with functional assays for cellular proteomics. In addition, the usage of a single plate for all sample preparation steps following cell lysis reduces potential samples losses and allows for automation. The MWCO filter also enables sample concentration, thereby increasing the overall sensitivity, and implementation of washing steps involving organic solvents, for example, to remove cell membranes constituents. The optimized protocol allowed for higher throughput with improved sensitivity in terms of the number of identified cellular proteins when compared to an established protocol employing gel-filtration columns. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  20. One-dimensional proteomic profiling of Danio rerio embryo vitellogenin to estimate quantum dot toxicity.

    PubMed

    Petushkova, Natalia A; Kuznetsova, Galina P; Larina, Olesya V; Kisrieva, Yulia S; Samenkova, Natalia F; Trifonova, Oxana P; Miroshnichenko, Yuliana V; Zolotarev, Konstantin V; Karuzina, Irina I; Ipatova, Olga M; Lisitsa, Andrey V

    2015-01-01

    Vitellogenin (Vtg) is the major egg yolk protein (YP) in most oviparous species and may be useful as an indicator in ecotoxicological testing at the biochemical level. In this study, we obtained detailed information about the Vtgs of Danio rerio embryos by cutting SDS-PAGE gel lanes into thin slices, and analyzing them slice-by-slice with (MALDI-TOF) mass spectrometry. We conducted three proteomic analyses, comparing embryonic Danio rerio Vtg cleavage products after exposure for 48 h to CdSecore/ZnSshell quantum dots (QDs), after exposure to a mixture of the components used for quantum dot synthesis (MCS-QDs), and in untreated embryos. The Vtg mass spectrometric profiles of the QDs-treated embryos differed from those of the unexposed or MCS-QDs-treated embryos. This study demonstrates the possible utility of Vtg profiling in D. rerio embryos as a sensitive diagnostic tool to estimate nanoparticle toxicity.

  1. Lung Cancer Signatures in Plasma Based on Proteome Profiling of Mouse Tumor Models

    PubMed Central

    Taguchi, Ayumu; Politi, Katerina; Pitteri, Sharon J.; Lockwood, William W.; Faça, Vitor M.; Kelly-Spratt, Karen; Wong, Chee-Hong; Zhang, Qing; Chin, Alice; Park, Kwon-Sik; Goodman, Gary; Gazdar, Adi F.; Sage, Julien; Dinulescu, Daniela M.; Kucherlapati, Raju; DePinho, Ronald A.; Kemp, Christopher J.; Varmus, Harold E.; Hanash, Samir M.

    2012-01-01

    SUMMARY We investigated the potential of in-depth quantitative proteomics to reveal plasma protein signatures that reflect lung tumor biology. We compared plasma protein profiles of four mouse models of lung cancer with profiles of models of pancreatic, ovarian, colon, prostate, and breast cancer and two models of inflammation. A protein signature for Titf1/Nkx2-1, a known lineage-survival oncogene in lung cancer, was found in plasmas of mouse models of lung adenocarcinoma. An EGFR signature was found in plasma of an EGFR mutant model, and a distinct plasma signature related to neuroendocrine development was uncovered in the small-cell lung cancer model. We demonstrate relevance to human lung cancer of the protein signatures identified on the basis of mouse models. PMID:21907921

  2. In vivo Host-Pathogen Interaction as Revealed by Global Proteomic Profiling of Zebrafish Larvae

    PubMed Central

    Díaz-Pascual, Francisco; Ortíz-Severín, Javiera; Varas, Macarena A.; Allende, Miguel L.; Chávez, Francisco P.

    2017-01-01

    demonstrated the suitability of zebrafish embryos as a model for in vivo host-pathogen based proteomic studies in P. aeruginosa. Our global proteomic profiling identifies novel molecular signatures that give systematic insight into zebrafish-Pseudomonas interaction. PMID:28791256

  3. A Targeted Quantitative Proteomics Strategy for Global Kinome Profiling of Cancer Cells and Tissues*

    PubMed Central

    Xiao, Yongsheng; Guo, Lei; Wang, Yinsheng

    2014-01-01

    Kinases are among the most intensively pursued enzyme superfamilies as targets for anti-cancer drugs. Large data sets on inhibitor potency and selectivity for more than 400 human kinases became available recently, offering the opportunity to design rationally novel kinase-based anti-cancer therapies. However, the expression levels and activities of kinases are highly heterogeneous among different types of cancer and even among different stages of the same cancer. The lack of effective strategy for profiling the global kinome hampers the development of kinase-targeted cancer chemotherapy. Here, we introduced a novel global kinome profiling method, based on our recently developed isotope-coded ATP-affinity probe and a targeted proteomic method using multiple-reaction monitoring (MRM), for assessing simultaneously the expression of more than 300 kinases in human cells and tissues. This MRM-based assay displayed much better sensitivity, reproducibility, and accuracy than the discovery-based shotgun proteomic method. Approximately 250 kinases could be routinely detected in the lysate of a single cell line. Additionally, the incorporation of iRT into MRM kinome library rendered our MRM kinome assay easily transferrable across different instrument platforms and laboratories. We further employed this approach for profiling kinase expression in two melanoma cell lines, which revealed substantial kinome reprogramming during cancer progression and demonstrated an excellent correlation between the anti-proliferative effects of kinase inhibitors and the expression levels of their target kinases. Therefore, this facile and accurate kinome profiling assay, together with the kinome-inhibitor interaction map, could provide invaluable knowledge to predict the effectiveness of kinase inhibitor drugs and offer the opportunity for individualized cancer chemotherapy. PMID:24520089

  4. A targeted quantitative proteomics strategy for global kinome profiling of cancer cells and tissues.

    PubMed

    Xiao, Yongsheng; Guo, Lei; Wang, Yinsheng

    2014-04-01

    Kinases are among the most intensively pursued enzyme superfamilies as targets for anti-cancer drugs. Large data sets on inhibitor potency and selectivity for more than 400 human kinases became available recently, offering the opportunity to design rationally novel kinase-based anti-cancer therapies. However, the expression levels and activities of kinases are highly heterogeneous among different types of cancer and even among different stages of the same cancer. The lack of effective strategy for profiling the global kinome hampers the development of kinase-targeted cancer chemotherapy. Here, we introduced a novel global kinome profiling method, based on our recently developed isotope-coded ATP-affinity probe and a targeted proteomic method using multiple-reaction monitoring (MRM), for assessing simultaneously the expression of more than 300 kinases in human cells and tissues. This MRM-based assay displayed much better sensitivity, reproducibility, and accuracy than the discovery-based shotgun proteomic method. Approximately 250 kinases could be routinely detected in the lysate of a single cell line. Additionally, the incorporation of iRT into MRM kinome library rendered our MRM kinome assay easily transferrable across different instrument platforms and laboratories. We further employed this approach for profiling kinase expression in two melanoma cell lines, which revealed substantial kinome reprogramming during cancer progression and demonstrated an excellent correlation between the anti-proliferative effects of kinase inhibitors and the expression levels of their target kinases. Therefore, this facile and accurate kinome profiling assay, together with the kinome-inhibitor interaction map, could provide invaluable knowledge to predict the effectiveness of kinase inhibitor drugs and offer the opportunity for individualized cancer chemotherapy.

  5. MPLEx: a Robust and Universal Protocol for Single-Sample Integrative Proteomic, Metabolomic, and Lipidomic Analyses

    SciTech Connect

    Nakayasu, Ernesto S.; Nicora, Carrie D.; Sims, Amy C.; Burnum-Johnson, Kristin E.; Kim, Young-Mo; Kyle, Jennifer E.; Matzke, Melissa M.; Shukla, Anil K.; Chu, Rosalie K.; Schepmoes, Athena A.; Jacobs, Jon M.; Baric, Ralph S.; Webb-Robertson, Bobbie-Jo; Smith, Richard D.; Metz, Thomas O.; Chia, Nicholas

    2016-05-03

    ABSTRACT

    Integrative multi-omics analyses can empower more effective investigation and complete understanding of complex biological systems. Despite recent advances in a range of omics analyses, multi-omic measurements of the same sample are still challenging and current methods have not been well evaluated in terms of reproducibility and broad applicability. Here we adapted a solvent-based method, widely applied for extracting lipids and metabolites, to add proteomics to mass spectrometry-based multi-omics measurements. Themetabolite,protein, andlipidextraction (MPLEx) protocol proved to be robust and applicable to a diverse set of sample types, including cell cultures, microbial communities, and tissues. To illustrate the utility of this protocol, an integrative multi-omics analysis was performed using a lung epithelial cell line infected with Middle East respiratory syndrome coronavirus, which showed the impact of this virus on the host glycolytic pathway and also suggested a role for lipids during infection. The MPLEx method is a simple, fast, and robust protocol that can be applied for integrative multi-omic measurements from diverse sample types (e.g., environmental,in vitro, and clinical).

    IMPORTANCEIn systems biology studies, the integration of multiple omics measurements (i.e., genomics, transcriptomics, proteomics, metabolomics, and lipidomics) has been shown to provide a more complete and informative view of biological pathways. Thus, the prospect of extracting different types of molecules (e.g., DNAs, RNAs, proteins, and metabolites) and performing multiple omics measurements on single samples is very attractive, but such studies are challenging due to the fact that the extraction conditions differ according to the molecule type. Here, we adapted an organic solvent-based extraction method that demonstrated

  6. MPLEx: a Robust and Universal Protocol for Single-Sample Integrative Proteomic, Metabolomic, and Lipidomic Analyses

    PubMed Central

    Nakayasu, Ernesto S.; Nicora, Carrie D.; Sims, Amy C.; Burnum-Johnson, Kristin E.; Kim, Young-Mo; Kyle, Jennifer E.; Matzke, Melissa M.; Shukla, Anil K.; Chu, Rosalie K.; Schepmoes, Athena A.; Jacobs, Jon M.; Baric, Ralph S.; Webb-Robertson, Bobbie-Jo; Smith, Richard D.

    2016-01-01

    ABSTRACT Integrative multi-omics analyses can empower more effective investigation and complete understanding of complex biological systems. Despite recent advances in a range of omics analyses, multi-omic measurements of the same sample are still challenging and current methods have not been well evaluated in terms of reproducibility and broad applicability. Here we adapted a solvent-based method, widely applied for extracting lipids and metabolites, to add proteomics to mass spectrometry-based multi-omics measurements. The metabolite, protein, and lipid extraction (MPLEx) protocol proved to be robust and applicable to a diverse set of sample types, including cell cultures, microbial communities, and tissues. To illustrate the utility of this protocol, an integrative multi-omics analysis was performed using a lung epithelial cell line infected with Middle East respiratory syndrome coronavirus, which showed the impact of this virus on the host glycolytic pathway and also suggested a role for lipids during infection. The MPLEx method is a simple, fast, and robust protocol that can be applied for integrative multi-omic measurements from diverse sample types (e.g., environmental, in vitro, and clinical). IMPORTANCE In systems biology studies, the integration of multiple omics measurements (i.e., genomics, transcriptomics, proteomics, metabolomics, and lipidomics) has been shown to provide a more complete and informative view of biological pathways. Thus, the prospect of extracting different types of molecules (e.g., DNAs, RNAs, proteins, and metabolites) and performing multiple omics measurements on single samples is very attractive, but such studies are challenging due to the fact that the extraction conditions differ according to the molecule type. Here, we adapted an organic solvent-based extraction method that demonstrated broad applicability and robustness, which enabled comprehensive proteomics, metabolomics, and lipidomics analyses from the same sample. Author

  7. Proteomics applied to the authentication of fish glue: application to a 17th century artwork sample.

    PubMed

    Dallongeville, Sophie; Richter, Mark; Schäfer, Stephan; Kühlenthal, Michael; Garnier, Nicolas; Rolando, Christian; Tokarski, Caroline

    2013-09-21

    This work provides the first identification of fish glue from a few micrograms of a 17(th) century artwork sample using an adapted proteomics approach. Fish glue has been widely used as a binder in various art objects such as paintings, manuscripts or polychrome objects however its authentication remains particularly challenging. The lack of information on fish species in genomic and proteomic databases represents a major drawback. A supplementary difficulty is provided by the historical sample features, i.e. a few micrograms of a 17(th) century polychrome object with a multilayered structure. SYPRO® Ruby staining was used as a screening technique to probe the presence of proteins in the sample cross-section. Results revealed the presence of several layers containing proteins among which a thin proteinaceous layer located between the silver leaf and the glaze. This thin layer is described as fish glue coating by historical sources but its composition has not been identified yet. The optimized methodology, based on high resolution mass spectrometry and adapted bioinformatic tools, was successfully applied to 50 μg of a polychromy sample and resulted in the identification of several collagen proteins. Extensive interpretation of data generated by tandem mass spectrometry allowed the identification of proteins from different biological origins. In particular, seven peptides specific to fish collagen proteins were identified for the first time proving the presence of fish glue in the sample and corroborating information found in historical texts dealing with the polychromy technique.

  8. Investigation of urine proteomic profile of cosmonauts after long-term space flight

    NASA Astrophysics Data System (ADS)

    Obraztcova, Olga; Liudmila Pastushkova, MRS.; Larina, Irina; Dobrokhotov, Igor; Kononikhin, Alexey; Nikolaev, Eugene

    The main interest is the study of changes in the protein composition of urine caused by aggressive factors of space flight. To analyze these changes, we investigated the proteome of urine obtained from cosmonauts after long-term spaceflight. We studied the protein composition of the second morning urine fractions obtained from six Russian cosmonauts aged 35 to 51 years, whose mission at the International Space Station continued from 169 to 199 days. Were used proteomic data acquisition technology and advanced bioinformatics analysis approaches. Collection of biomaterial was held within the space experiment "Proteome" before the flight, on the first and seventh day after landing. Urine protein was not detected spectrophotometrically in the majority of the urine samples before the flight, but on the first day after landing it was detected in four cosmonauts, and later - in two cosmonauts. By liquid chromatography (Agilent Technologies Inc., USA) - mass-spectrometry (Thermo, Germany) technic, proteins in urine samples were detected in all periods of observation. As a result of our analysis, we have determined that the detected proteins had different origin. There were identified proteins synthesized in the kidney, liver and prostate. There was observed the drift of the protein composition in urine. One of the hallmarks of this drift was the disappearance of the five proteins in urine samples during the first day after the flight, despite their presence in the samples pre-flight period. They were: receptor tyrosine kinases, cytoskeletal keratin-1, G-protein-coupled receptors, inter-alpha (globulin) inhibitor H4. Such changes could be explained by the influence of factors of space flight, as well as the individual response of each cosmonaut’ organism when they return to the Earth conditions. Also, there was detected the trend to activate proteolysis of proteins in post-flight period, based on the identified secretory proteins with protease activity (cystatin M

  9. Examination of Microbial Proteome Preservation Techniques Applicable to Autonomous Environmental Sample Collection

    PubMed Central

    Saito, Mak A.; Bulygin, Vladimir V.; Moran, Dawn M.; Taylor, Craig; Scholin, Chris

    2011-01-01

    Improvements in temporal and spatial sampling frequency have the potential to open new windows into the understanding of marine microbial dynamics. In recent years, efforts have been made to allow automated samplers to collect microbial biomass for DNA/RNA analyses from moored observatories and autonomous underwater vehicles. Measurements of microbial proteins are also of significant interest given their biogeochemical importance as enzymes that catalyze reactions and transporters that interface with the environment. We examined the influence of five preservatives solutions (SDS-extraction buffer, ethanol, trichloroacetic acid, B-PER, and RNAlater) on the proteome integrity of the marine cyanobacterium Synechococcus WH8102 after 4 weeks of storage at room temperature. Four approaches were used to assess degradation: total protein recovery, band integrity on an SDS detergent polyacrylamide electrophoresis (SDS-PAGE) gel, and number of protein identifications and relative abundances by 1-dimensional LC–MS/MS proteomic analyses. Total protein recoveries from the preserved samples were lower than the frozen control due to processing losses, which could be corrected for with internal standardization. The trichloroacetic acid preserved sample showed significant loss of protein band integrity on the SDS-PAGE gel. The RNAlater preserved sample showed the highest number of protein identifications (103% relative to the control; 520 ± 31 identifications in RNAlater versus 504 ± 4 in the control), equivalent to the frozen control. Relative abundances of individual proteins in the RNAlater treatment were quite similar to that of the frozen control (average ratio of 1.01 ± 0.27 for the 50 most abundant proteins), while the SDS-extraction buffer, ethanol, and B-PER all showed significant decreases in both number of identifications and relative abundances of individual proteins. Based on these findings, RNAlater was an effective proteome preservative, although

  10. Examination of microbial proteome preservation techniques applicable to autonomous environmental sample collection.

    PubMed

    Saito, Mak A; Bulygin, Vladimir V; Moran, Dawn M; Taylor, Craig; Scholin, Chris

    2011-01-01

    Improvements in temporal and spatial sampling frequency have the potential to open new windows into the understanding of marine microbial dynamics. In recent years, efforts have been made to allow automated samplers to collect microbial biomass for DNA/RNA analyses from moored observatories and autonomous underwater vehicles. Measurements of microbial proteins are also of significant interest given their biogeochemical importance as enzymes that catalyze reactions and transporters that interface with the environment. We examined the influence of five preservatives solutions (SDS-extraction buffer, ethanol, trichloroacetic acid, B-PER, and RNAlater) on the proteome integrity of the marine cyanobacterium Synechococcus WH8102 after 4 weeks of storage at room temperature. Four approaches were used to assess degradation: total protein recovery, band integrity on an SDS detergent polyacrylamide electrophoresis (SDS-PAGE) gel, and number of protein identifications and relative abundances by 1-dimensional LC-MS/MS proteomic analyses. Total protein recoveries from the preserved samples were lower than the frozen control due to processing losses, which could be corrected for with internal standardization. The trichloroacetic acid preserved sample showed significant loss of protein band integrity on the SDS-PAGE gel. The RNAlater preserved sample showed the highest number of protein identifications (103% relative to the control; 520 ± 31 identifications in RNAlater versus 504 ± 4 in the control), equivalent to the frozen control. Relative abundances of individual proteins in the RNAlater treatment were quite similar to that of the frozen control (average ratio of 1.01 ± 0.27 for the 50 most abundant proteins), while the SDS-extraction buffer, ethanol, and B-PER all showed significant decreases in both number of identifications and relative abundances of individual proteins. Based on these findings, RNAlater was an effective proteome preservative, although

  11. Universal Solid-Phase Reversible Sample-Prep for Concurrent Proteome and N-Glycome Characterization.

    PubMed

    Zhou, Hui; Morley, Samantha; Kostel, Stephen; Freeman, Michael R; Joshi, Vivek; Brewster, David; Lee, Richard S

    2016-03-04

    We describe a novel solid-phase reversible sample-prep (SRS) platform that enables rapid sample preparation for concurrent proteome and N-glycome characterization for nearly all protein samples. SRS utilizes a uniquely functionalized, silica-based bead that has strong affinity toward proteins with minimal to no affinity for peptides and other small molecules. By leveraging this inherent size difference between proteins and peptides, SRS permits high-capacity binding of proteins, rapid removal of small molecules (detergents, metabolites, salts, peptides, etc.), extensive manipulation including enzymatic and chemical treatments on bead-bound proteins, and easy recovery of N-glycans and peptides. SRS was evaluated in a wide range of samples including glycoproteins, cell lysate, murine tissues, and human urine. SRS was also coupled to a quantitative strategy to investigate the differences between DU145 prostate cancer cells and its DIAPH3-silenced counterpart. Previous studies suggested that DIAPH3 silencing in DU145 induced transition to an amoeboid phenotype that correlated with tumor progression and metastasis. In this pilot study we identified distinct proteomic and N-glycomic alterations between them. A metastasis-associated tyrosine kinase receptor ephrin-type-A receptor (EPHA2) was highly up-regulated in DIAPH3-silenced cells, indicating a possible connection between EPHA2 and DIAPH3. Moreover, distinct alterations in the N-glycome were identified, suggesting cross-links between DIAPH3 and glycosyltransferase networks.

  12. Comparative proteomic analysis using samples obtained with laser microdissection and saturation dye labelling.

    PubMed

    Wilson, Kate E; Marouga, Rita; Prime, John E; Pashby, D Paul; Orange, Paul R; Crosier, Steven; Keith, Alexander B; Lathe, Richard; Mullins, John; Estibeiro, Peter; Bergling, Helene; Hawkins, Edward; Morris, Christopher M

    2005-10-01

    Comparative proteomic methods are rapidly being applied to many different biological systems including complex tissues. One pitfall of these methods is that in some cases, such as oncology and neuroscience, tissue complexity requires isolation of specific cell types and sample is limited. Laser microdissection (LMD) is commonly used for obtaining such samples for proteomic studies. We have combined LMD with sensitive thiol-reactive saturation dye labelling of protein samples and 2-D DIGE to identify protein changes in a test system, the isolated CA1 pyramidal neurone layer of a transgenic (Tg) rat carrying a human amyloid precursor protein transgene. Saturation dye labelling proved to be extremely sensitive with a spot map of over 5,000 proteins being readily produced from 5 mug total protein, with over 100 proteins being significantly altered at p < 0.0005. Of the proteins identified, all showed coherent changes associated with transgene expression. It was, however, difficult to identify significantly different proteins using PMF and MALDI-TOF on gels containing less than 500 mug total protein. The use of saturation dye labelling of limiting samples will therefore require the use of highly sensitive MS techniques to identify the significantly altered proteins isolated using methods such as LMD.

  13. A HUPO test sample study reveals common problems in mass spectrometry-based proteomics

    PubMed Central

    Bell, Alexander W.; Deutsch, Eric W.; Au, Catherine E.; Kearney, Robert E.; Beavis, Ron; Sechi, Salvatore; Nilsson, Tommy; Bergeron, John J.M.

    2009-01-01

    We carried out a test sample study to try to identify errors leading to irreproducibility, including incompleteness of peptide sampling, in LC-MS-based proteomics. We distributed a test sample consisting of an equimolar mix of 20 highly purified recombinant human proteins, to 27 laboratories for identification. Each protein contained one or more unique tryptic peptides of 1250 Da to also test for ion selection and sampling in the mass spectrometer. Of the 27 labs, initially only 7 labs reported all 20 proteins correctly, and only 1 lab reported all the tryptic peptides of 1250 Da. Nevertheless, a subsequent centralized analysis of the raw data revealed that all 20 proteins and most of the 1250 Da peptides had in fact been detected by all 27 labs. The centralized analysis allowed us to determine sources of problems encountered in the study, which include missed identifications (false negatives), environmental contamination, database matching, and curation of protein identifications. Improved search engines and databases are likely to increase the fidelity of mass spectrometry-based proteomics. PMID:19448641

  14. Serum Proteome Profiles in Stricturing Crohn’s Disease: A pilot study.

    SciTech Connect

    Townsend, Peter; Zhang, Qibin; Shapiro, Jason; Webb-Robertson, Bobbie-Jo M.; Bramer, Lisa M.; Schepmoes, Athena A.; Weitz, Karl K.; Mallette, Meaghan; Moniz, Heather; Bright, Renee; Merrick, Marjorie; Shah, Samir A.; Sands, Bruce E.; Leleiko, Neal

    2015-08-01

    Background: Crohn’s disease (CD) is a form of inflammatory bowel disease (IBD) with different described behaviors, including stricture. At present, there are no laboratory studies that can differentiate stricturing CD from other phenotypes of IBD. We performed a pilot study to examine differences in the proteome among patients with stricturing Crohn’s disease, non-stricturing Crohn’s disease, and ulcerative colitis (UC). Methods: Serum samples were selected from the Ocean State Crohn’s and Colitis Area Registry (OSCCAR), an established cohort of patients with IBD. Crohn’s disease patients with surgically-resected stricture were matched with similar patients with Crohn’s disease without known stricture, and with UC. Serum samples from each patient were digested and analyzed using liquid chromatography-mass spectrometry to characterize the proteome. Statistical analyses were performed to identify peptides and proteins that can differentiate CD with stricture. Results: Samples from 9 patients in each group (27 total patients) were analyzed. Baseline demographic characteristics were similar among the three groups. We quantified 7668 peptides and 897 proteins for analysis. ROC analysis identified a subset of peptides with an area under the curve greater than 0.9, indicating greater separation potential. Partial least squares discriminant analysis was able to distinguish among the three groups with up to 70% accuracy by peptides, and up to 80% accuracy by proteins. We identified the significantly different proteins and peptides, and determined their function based on previously published literature. Conclusions: The serum of patients with stricturing CD, non-stricturing CD, and UC are distinguishable via proteomic analysis. Some of the proteins that differentiate the stricturing phenotype have been implicated in complement activation, fibrinolytic pathways, and lymphocyte adhesion.

  15. Automation of nanoflow liquid chromatography-tandem mass spectrometry for proteome and peptide profiling analysis by using a monolithic analytical capillary column.

    PubMed

    Jiang, Xiaogang; Dong, Jing; Wang, Fangjun; Feng, Shun; Ye, Mingliang; Zou, Hanfa

    2008-04-01

    An automated nano-LC-MS/MS platform without trap column was established, which only used a 20 cm lauryl methacrylate-ethylene dimethacrylate (LMA-EDMA) monolithic capillary column to allow preconcentration and separation of peptides. The monolithic column had the advantages of good permeability and low backpressure resulting in higher flow rates for capillary columns. Tryptic digests of bovine albumin and yeast protein extract were tested using the monolithic column system. High proteomic coverage using this approach were demonstrated in this study. Furthermore, peptide samples extracted from mouse liver were separated by using the monolithic column system combined with size-exclusion chromatography prefractionation. This monolithic column system might be a promising alternative for the automated system previously using a trap column for routine proteome and peptide profiling analysis.

  16. Proteomic profiling of the brain of mice with experimental cerebral malaria.

    PubMed

    Moussa, Ehab; Huang, Honglei; Ahras, Malika; Lall, Amar; Thezenas, Marie L; Fischer, Roman; Kessler, Benedikt M; Pain, Arnab; Billker, Oliver; Casals-Pascual, Climent

    2017-06-05

    Cerebral malaria (CM) is a severe neurological complication of malaria infection in both adults and children. In pursuit of effective treatment of CM, clinical studies, postmortem analysis and animal models have been employed to understand the pathology and identify effective interventions. In this study, a shotgun proteomics analysis was conducted to profile the proteomic signature of the brain tissue of mice with experimental cerebral malaria (ECM) in order to further understand the underlying pathology. To identify CM-associated response, proteomic signatures of the brains of C57/Bl6N mice infected with P. berghei ANKA that developed neurological syndrome were compared to those of mice infected with P. berghei NK65 that developed equally high parasite burdens without neurological signs, and to those of non-infected mice. The results show that the CM-associated response in mice that developed neurological signs comprise mainly acute-phase reaction and coagulation cascade activation, and indicate the leakage of plasma proteins into the brain parenchyma. Cerebral malaria (CM) remains a major cause of death in children. The majority of these deaths occur in sub-Saharan Africa. Even with adequate access to treatment, mortality remains high and neurological sequelae can be found in up to 20% of survivors. No adjuvant treatment to date has been shown to reduce mortality and the pathophysiology of CM is largely unknown. Experimental cerebral malaria (ECM) is a well-established model that may contribute to identify and test druggable targets. In this study we have identified the disruption of the blood-brain barrier following inflammatory and vascular injury as a mechanism of disease. In this study we report a number of proteins that could be validated as potential biomarkers of ECM. Further studies, will be required to validate the clinical relevance of these biomarkers in human CM. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. Urinary proteomic profiling reveals diclofenac-induced renal injury and hepatic regeneration in mice

    SciTech Connect

    Swelm, Rachel P.L. van; Laarakkers, Coby M.M.; Pertijs, Jeanne C.L.M.; Verweij, Vivienne; Masereeuw, Rosalinde; Russel, Frans G.M.

    2013-06-01

    Diclofenac (DF) is a widely used non-steroidal anti-inflammatory drug for the treatment of rheumatic disorders, but is often associated with liver injury. We applied urinary proteomic profiling using MALDI-TOF MS to identify biomarkers for DF-induced hepatotoxicity in mice. Female CH3/HeOUJIco mice were treated with 75 mg/kg bw DF by oral gavage and 24 h urine was collected. Proteins identified in urine of DF-treated mice included epidermal growth factor, transthyretin, kallikrein, clusterin, fatty acid binding protein 1 and urokinase, which are related to liver regeneration but also to kidney injury. Both organs showed enhanced levels of oxidative stress (TBARS, p < 0.01). Kidney injury was confirmed by histology and increased Kim1 and Il-6 mRNA expression levels (p < 0.001 and p < 0.01). Liver histology and plasma ALT levels in DF-treated mice were not different from control, but mRNA expression of Stat3 (p < 0.001) and protein expression of PCNA (p < 0.05) were increased, indicating liver regeneration. In conclusion, urinary proteome analysis revealed that DF treatment in mice induced kidney and liver injury. Within 24 h, however, the liver was able to recover by activating tissue regeneration processes. Hence, the proteins found in urine of DF-treated mice represent kidney damage rather than hepatic injury. - Highlights: • The urinary proteome shows biological processes involved in adverse drug reactions. • Urine proteins of DF-treated mice relate to kidney injury rather than liver injury. • Liver regeneration, not liver injury, is apparent 24h after oral DF administration. • Pretreatment with LPS does not enhance DF-induced liver injury in mice.

  18. Temporal profiling of the adipocyte proteome during differentiation using a 5-plex SILAC based strategy

    PubMed Central

    Ruch, Travis; Kim, Jae-Woo; Mortensen, Peter; Otto, Tamara; Nalli, Anuradha; Tang, Qi-Qun; Lane, M. Daniel; Chaerkady, Raghothama; Pandey, Akhilesh

    2008-01-01

    The adipose tissue has important secretory and endocrine functions in humans. The regulation of adipocyte differentiation has been actively pursued using transcriptomic methods over the last several years. Quantitative proteomics has emerged as a promising approach to obtain temporal profiles of biological processes such as differentiation. Stable isotope labeling with amino acids in cell culture (SILAC) is a simple and robust method for labeling proteins in vivo. Here, we describe the development and application of a five-plex SILAC experiment using four different heavy stable isotopic forms of arginine to study the nuclear proteome and the secretome during the course of adipocyte differentiation. Tandem mass spectrometry analysis using a quadrupole time-of-flight instrument resulted in identification of a total 882 proteins from these two proteomes. Of these proteins, 427 were identified on the basis of one or more arginine containing peptides that allowed quantitation. In addition to previously reported molecules that are differentially expressed during the process of adipogenesis (e.g. adiponectin and lipoprotein lipase), we identified several proteins whose differential expression during adipocyte differentiation has not been documented previously. For example, THO complex 4, a context-dependent transcriptional activator in the T-cell receptor alpha enhancer complex, showed highest expression at middle stage of adipogenesis while SNF2 alpha, a chromatin remodeling protein, was downregulated upon initiation of adipogenesis and remained so during subsequent time points. This study using a 5-plex SILAC to investigate dynamics illustrates the power of this approach to identify differentially expressed proteins in a temporal fashion. PMID:18947249

  19. The influence of iron on the proteomic profile of Chromobacterium violaceum.

    PubMed

    Lima, Daniel C; Duarte, Fábio T; Medeiros, Viviane K S; Lima, Diogo B; Carvalho, Paulo C; Bonatto, Diego; Batistuzzo de Medeiros, Silvia R

    2014-10-20

    Chromobacterium violaceum is a bacterium commonly found in tropical and subtropical regions and is associated with important pharmacological and industrial attributes such as producing substances with therapeutic properties and synthesizing biodegradable polymers. Its genome was sequenced, however, approximately 40% of its genes still remain with unknown functions. Although C. violaceum is known by its versatile capacity of living in a wide range of environments, little is known on how it achieves such success. Here, we investigated the proteomic profile of C. violaceum cultivated in the absence and presence of high iron concentration, describing some proteins of unknown function that might play an important role in iron homeostasis, amongst others. Briefly, C. violaceum was cultivated in the absence and in the presence of 9 mM of iron during four hours. Total proteins were identified by LC-MS and through the PatternLab pipeline. Our proteomic analysis indicates major changes in the energetic metabolism, and alterations in the synthesis of key transport and stress proteins. In addition, it may suggest the presence of a yet unidentified operon that could be related to oxidative stress, together with a set of other proteins with unknown function. The protein-protein interaction network also pinpointed the importance of energetic metabolism proteins to the acclimatation of C. violaceum in high concentration of iron. This is the first proteomic analysis of the opportunistic pathogen C. violaceum in the presence of high iron concentration. Our data allowed us to identify a yet undescribed operon that might have a role in oxidative stress defense. Our work provides new data that will contribute to understand how this bacterium achieve its capacity of surviving in harsh conditions as well as to open a way to explore the yet little availed biotechnological characteristics of this bacterium with the further exploring of the proteins of unknown function that we showed to be

  20. Efficient removal of DNA from proteomic samples prior to two-dimensional map analysis.

    PubMed

    Antonioli, Paolo; Bachi, Angela; Fasoli, Elisa; Righetti, Pier Giorgio

    2009-04-24

    Several methods have been described in the literature for removal of DNA from protein samples prior to proteome analysis. They in general involve protein precipitation techniques. In other protocols, DNAse treatment is suggested prior to precipitation of proteins in excess acetone. All these methods have been evaluated and found to perform poorly in DNA removal, as illustrated by two-dimensional (2D) maps where horizontal and vertical sample streaking are still substantial. Such removal is in general necessary in tissue lysates and especially when analysing sub-cellular organelles, such as nuclei, where the high DNA levels strongly interfere with proteome analysis. Another method is proposed here for efficient DNA removal: two-phase extraction of DNA in chloroform/phenol/isoamyl alcohol, a procedure commonly used to rid DNA samples of protein contaminants, but rarely applied to protein preparation. This extraction is not very efficient if performed at slightly acidic to neutral pH values, but it performs extremely well at pH values of 9.5 or higher. The 2D maps thus obtained of Escherichia coli lysates as well as extracts from purified nuclei of eukaryotic cells are not only devoid of any vertical or horizontal streaking, but exhibit many more spots, especially in the alkaline region of the 2D gels, suggesting that these basic proteins were in general lost to proteome analysis due to co-precipitation in tenacious protein-DNA complexes. It is hypothesized that the alkaline pH values adopted in the two-phase extraction help to fully disrupt any residual DNA-protein complexes, due to strong Coulombic repulsion.

  1. Optimized Extraction Method To Remove Humic Acid Interferences from Soil Samples Prior to Microbial Proteome Measurements.

    PubMed

    Qian, Chen; Hettich, Robert L

    2017-07-07

    The microbial composition and their activities in soil environments play a critical role in organic matter transformation and nutrient cycling. Liquid chromatography coupled to high-performance mass spectrometry provides a powerful approach to characterize soil microbiomes; however, the limited microbial biomass and the presence of abundant interferences in soil samples present major challenges to proteome extraction and subsequent MS measurement. To this end, we have designed an experimental method to improve microbial proteome measurement by removing the soil-borne humic substances coextraction from soils. Our approach employs an in situ detergent-based microbial lysis/TCA precipitation coupled to an additional cleanup step involving acidified precipitation and filtering at the peptide level to remove most of the humic acid interferences prior to proteolytic peptide measurement. The novelty of this approach is an integration to exploit two different characteristics of humic acids: (1) Humic acids are insoluble in acidic solution but should not be removed at the protein level, as undesirable protein removal may also occur. Rather it is better to leave the humics acids in the samples until the peptide level, at which point the significant differential solubility of humic acids versus peptides at low pH can be exploited very efficiently. (2) Most of the humic acids have larger molecule weights than the peptides. Therefore, filtering a pH 2 to 3 peptide solution with a 10 kDa filter will remove most of the humic acids. This method is easily interfaced with normal proteolytic processing approaches and provides a reliable and straightforward protein extraction method that efficiently removes soil-borne humic substances without inducing proteome sample loss or biasing protein identification in mass spectrometry. In general, this humic acid removal step is universal and can be adopted by any workflow to effectively remove humic acids to avoid them negatively competing

  2. Glycomic and Proteomic Profiling of Pancreatic Cyst Fluids Identifies Hyperfucosylated Lactosamines on the N-linked Glycans of Overexpressed Glycoproteins*

    PubMed Central

    Mann, Benjamin F.; Goetz, John A.; House, Michael G.; Schmidt, C. Max; Novotny, Milos V.

    2012-01-01

    Pancreatic cancer is now the fourth leading cause of cancer deaths in the United States, and it is associated with an alarmingly low 5-year survival rate of 5%. However, a patient's prognosis is considerably improved when the malignant lesions are identified at an early stage of the disease and removed by surgical resection. Unfortunately, the absence of a practical screening strategy and clinical diagnostic test for identifying premalignant lesions within the pancreas often prevents early detection of pancreatic cancer. To aid in the development of a molecular screening system for early detection of the disease, we have performed glycomic and glycoproteomic profiling experiments on 21 pancreatic cyst fluid samples, including fluids from mucinous cystic neoplasms and intraductal papillary mucinous neoplasms, two types of mucinous cysts that are considered high risk to undergo malignant transformation. A total of 80 asparagine-linked (N-linked) glycans, including high mannose and complex structures, were identified. Of special interest was a series of complex N-linked glycans containing two to six fucose residues, located predominantly as substituents on β-lactosamine extensions. Following the observation of these “hyperfucosylated” glycans, bottom-up proteomics experiments utilizing a label-free quantitative approach were applied to the investigation of two sets of tryptically digested proteins derived from the cyst fluids: 1) all soluble proteins in the raw samples and 2) a subproteome of the soluble cyst fluid proteins that were selectively enriched for fucosylation through the use of surface-immobilized Aleuria aurantia lectin. A comparative analysis of these two proteomic data sets identified glycoproteins that were significantly enriched by lectin affinity. Several candidate glycoproteins that appear hyperfucosylated were identified, including triacylglycerol lipase and pancreatic α-amylase, which were 20- and 22-fold more abundant, respectively

  3. Glycomic and proteomic profiling of pancreatic cyst fluids identifies hyperfucosylated lactosamines on the N-linked glycans of overexpressed glycoproteins.

    PubMed

    Mann, Benjamin F; Goetz, John A; House, Michael G; Schmidt, C Max; Novotny, Milos V

    2012-07-01

    Pancreatic cancer is now the fourth leading cause of cancer deaths in the United States, and it is associated with an alarmingly low 5-year survival rate of 5%. However, a patient's prognosis is considerably improved when the malignant lesions are identified at an early stage of the disease and removed by surgical resection. Unfortunately, the absence of a practical screening strategy and clinical diagnostic test for identifying premalignant lesions within the pancreas often prevents early detection of pancreatic cancer. To aid in the development of a molecular screening system for early detection of the disease, we have performed glycomic and glycoproteomic profiling experiments on 21 pancreatic cyst fluid samples, including fluids from mucinous cystic neoplasms and intraductal papillary mucinous neoplasms, two types of mucinous cysts that are considered high risk to undergo malignant transformation. A total of 80 asparagine-linked (N-linked) glycans, including high mannose and complex structures, were identified. Of special interest was a series of complex N-linked glycans containing two to six fucose residues, located predominantly as substituents on β-lactosamine extensions. Following the observation of these "hyperfucosylated" glycans, bottom-up proteomics experiments utilizing a label-free quantitative approach were applied to the investigation of two sets of tryptically digested proteins derived from the cyst fluids: 1) all soluble proteins in the raw samples and 2) a subproteome of the soluble cyst fluid proteins that were selectively enriched for fucosylation through the use of surface-immobilized Aleuria aurantia lectin. A comparative analysis of these two proteomic data sets identified glycoproteins that were significantly enriched by lectin affinity. Several candidate glycoproteins that appear hyperfucosylated were identified, including triacylglycerol lipase and pancreatic α-amylase, which were 20- and 22-fold more abundant, respectively, following A

  4. Proteomic profiling of the signal crayfish Pacifastacus leniusculus egg and spermatophore.

    PubMed

    Niksirat, Hamid; Andersson, Liselotte; James, Peter; Kouba, Antonín; Kozák, Pavel

    2014-10-01

    Proteins of the signal crayfish Pacifastacus leniusculus egg and spermatophore were identified using in-gel digestion, mass spectrometry, and Mascot search. Forty-one and one-hundred-fifty proteins were identified in egg and spermatophore, respectively. The proteins were classified into nine categories including cell defence, cell signaling, cytoskeleton, DNA related activity, metabolism and energy production, protease and protease inhibitor, respiration, transportation, and others and unknown. Twenty-two proteins were found in both egg and spermatophore. The respiration and cytoskeleton groups are the most diverse categories in the protein profiles of the egg and spermatophore, respectively. No protein was assigned to DNA related activity and cell defence categories in the protein profile of the crayfish egg. Differences between protein profiles of the crayfish egg and spermatophore show different functional priorities for each of gametes. Several proteins having possible roles in gametogenesis, capacitation, acrosome reaction, and fertilization were identified. This proteomic profile of signal crayfish gametes provides a basis for further investigation of functional roles of the identified proteins in aspects of reproduction such as capacitation and fertilization. Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.

  5. Proteomic and activity profiles of ascorbate-glutathione cycle enzymes in germinating barley embryo.

    PubMed

    Bønsager, Birgit C; Shahpiri, Azar; Finnie, Christine; Svensson, Birte

    2010-10-01

    Enzymes involved in redox control are important during seed germination and seedling growth. Ascorbate-glutathione cycle enzymes in barley embryo extracts were monitored both by 2D-gel electrophoresis and activity measurements from 4 to 144 h post imbibition (PI). Strikingly different activity profiles were observed. No ascorbate peroxidase (APX) activity was present in mature seeds but activity was detected after 24 h PI and increased 14-fold up to 144 h PI. In contrast, dehydroascorbate reductase (DHAR) activity was present at 4h PI and first decreased by 9-fold until 72 h PI followed by a 5-fold increase at 144 h PI. Glutathione reductase and monodehydroascorbate reductase activities were also detected at 4 h PI, and showed modest increases of 1.8- and 2.7-fold, respectively, by 144 h PI. The combination of functional analysis with the proteomics approach enabled correlation of the activity profiles and protein abundance. While gel spots containing APX showed intensity changes consistent with the activity profile from 0 to 72 h PI, DHAR spot intensities indicated that post-translational regulation may be responsible for the observed changes in activity. Transcript profiling, 2D-western blotting and mass spectrometric characterization of multiple APX spots demonstrated the presence of APX1 and minor amounts of APX2.

  6. Sampling From the Proteome to the Human Leukocyte Antigen-DR (HLA-DR) Ligandome Proceeds Via High Specificity.

    PubMed

    Mommen, Geert P M; Marino, Fabio; Meiring, Hugo D; Poelen, Martien C M; van Gaans-van den Brink, Jacqueline A M; Mohammed, Shabaz; Heck, Albert J R; van Els, Cécile A C M

    2016-04-01

    Comprehensive analysis of the complex nature of the Human Leukocyte Antigen (HLA) class II ligandome is of utmost importance to understand the basis for CD4(+)T cell mediated immunity and tolerance. Here, we implemented important improvements in the analysis of the repertoire of HLA-DR-presented peptides, using hybrid mass spectrometry-based peptide fragmentation techniques on a ligandome sample isolated from matured human monocyte-derived dendritic cells (DC). The reported data set constitutes nearly 14 thousand unique high-confident peptides,i.e.the largest single inventory of human DC derived HLA-DR ligands to date. From a technical viewpoint the most prominent finding is that no single peptide fragmentation technique could elucidate the majority of HLA-DR ligands, because of the wide range of physical chemical properties displayed by the HLA-DR ligandome. Our in-depth profiling allowed us to reveal a strikingly poor correlation between the source proteins identified in the HLA class II ligandome and the DC cellular proteome. Important selective sieving from the sampled proteome to the ligandome was evidenced by specificity in the sequences of the core regions both at their N- and C- termini, hence not only reflecting binding motifs but also dominant protease activity associated to the endolysosomal compartments. Moreover, we demonstrate that the HLA-DR ligandome reflects a surface representation of cell-compartments specific for biological events linked to the maturation of monocytes into antigen presenting cells. Our results present new perspectives into the complex nature of the HLA class II system and will aid future immunological studies in characterizing the full breadth of potential CD4(+)T cell epitopes relevant in health and disease.

  7. Sampling From the Proteome to the Human Leukocyte Antigen-DR (HLA-DR) Ligandome Proceeds Via High Specificity*

    PubMed Central

    Mommen, Geert P. M.; Marino, Fabio; Meiring, Hugo D.; Poelen, Martien C. M.; van Gaans-van den Brink, Jacqueline A. M.; Mohammed, Shabaz; Heck, Albert J. R.; van Els, Cécile A. C. M.

    2016-01-01

    Comprehensive analysis of the complex nature of the Human Leukocyte Antigen (HLA) class II ligandome is of utmost importance to understand the basis for CD4+ T cell mediated immunity and tolerance. Here, we implemented important improvements in the analysis of the repertoire of HLA-DR-presented peptides, using hybrid mass spectrometry-based peptide fragmentation techniques on a ligandome sample isolated from matured human monocyte-derived dendritic cells (DC). The reported data set constitutes nearly 14 thousand unique high-confident peptides, i.e. the largest single inventory of human DC derived HLA-DR ligands to date. From a technical viewpoint the most prominent finding is that no single peptide fragmentation technique could elucidate the majority of HLA-DR ligands, because of the wide range of physical chemical properties displayed by the HLA-DR ligandome. Our in-depth profiling allowed us to reveal a strikingly poor correlation between the source proteins identified in the HLA class II ligandome and the DC cellular proteome. Important selective sieving from the sampled proteome to the ligandome was evidenced by specificity in the sequences of the core regions both at their N- and C- termini, hence not only reflecting binding motifs but also dominant protease activity associated to the endolysosomal compartments. Moreover, we demonstrate that the HLA-DR ligandome reflects a surface representation of cell-compartments specific for biological events linked to the maturation of monocytes into antigen presenting cells. Our results present new perspectives into the complex nature of the HLA class II system and will aid future immunological studies in characterizing the full breadth of potential CD4+ T cell epitopes relevant in health and disease. PMID:26764012

  8. Proteomics profiling of epidermal mucus secretion of a cichlid (Symphysodon aequifasciata) demonstrating parental care behavior.

    PubMed

    Chong, Kenny; Joshi, Shashikant; Jin, Lam Toong; Shu-Chien, Alexander Chong

    2006-04-01

    The discus fish (Symphysodon aequifasciata) is a cichlid demonstrating advanced mode of parental care towards fry. Both male and female fish utilized epidermal mucus secreted from specialized epidermal cells to feed developing fry. We utilized proteomics to compare protein profile from parental and nonparental fish. Gel analysis revealed a total of 35 spots that were up-regulated in parental mucus. In tandem, another 18 spots were uniquely expressed in parental mucus. MS analysis of these spots identified proteins such as fructose biphosphate aldolase, nucleoside diphosphate kinase, and heat shock proteins, which are essential to support energy provision, cell repair and proliferation, stress mediation, and defense mechanism in parental fish during parental-care period. Concurrently, the detection of several antioxidant-related proteins such as thioredoxin peroxidase and hemopexin suggests a need to overcome oxidative stress during hypermucosal production in parental-care behavior. A C-type lectin was also found to be uniquely expressed in parental mucus and could have important role in providing antimicrobial defense to both parental fish and fry. In summary, our study shows that discus mucus proteome undergoes changes in protein expression during parental-care period.

  9. Biomarker discovery for ovine paratuberculosis (Johne's disease) by proteomic serum profiling.

    PubMed

    Zhong, L; Taylor, D; Begg, D J; Whittington, R J

    2011-07-01

    Paratuberculosis (Johne's disease) is a chronic granulomatous enteritis affecting ruminants and other species. It is caused by Mycobacterium avium subsp. paratuberculosis (MAP). In this study, surface enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI TOF-MS) was used as a platform to identify candidate biomarkers from sheep serum. Multivariate biomarker models which aimed to differentiate sheep with paratuberculosis and vaccinated-exposed sheep from unexposed animals were proposed based on classification and regression tree (CART) and linear discriminant analysis (LDA) algorithms from two array types. The accuracy of classification of sheep into unexposed or exposed groups ranged from 75 to 100% among models. SELDI was used to monitor protein profile changes over time during an experimental infection trial by examining sera collected at 4-, 8- and 13-months post infection. Although three different SELDI instruments were used, nine consistent proteomic features were observed associated with exposure to MAP. Two of the putative serum biomarkers were purified from serum using chromatographic methods and were identified as transthyretin and alpha haemoglobin by tandem mass spectrometry. They belong to highly abundant, acute phase reactants in the serum proteome and have also been discovered as serum biomarkers in human inflammatory conditions and cancer. Their relationship to the pathogenesis of Johne's disease remains to be elucidated.

  10. Identification of three immunodominant motifs with atypical isotype profile scattered over the Onchocerca volvulus proteome

    PubMed Central

    Van Dorst, Bieke; Stuyver, Lieven J.

    2017-01-01

    Understanding the immune response upon infection with the filarial nematode Onchocerca volvulus and the mechanisms that evolved in this parasite to evade immune mediated elimination is essential to expand the toolbox available for diagnostics, therapeutics and vaccines development. Using high-density peptide microarrays we scanned the proteome-wide linear epitope repertoire in Cameroonian onchocerciasis patients and healthy controls from Southern Africa which led to the identification of 249 immunodominant antigenic peptides. Motif analysis learned that 3 immunodominant motifs, encompassing 3 linear epitopes, are present in 70, 43, and 31 of these peptides, respectively and appear to be scattered over the entire proteome in seemingly non-related proteins. These linear epitopes are shown to have an atypical isotype profile dominated by IgG1, IgG3, IgE and IgM, in contrast to the commonly observed IgG4 response in chronic active helminth infections. The identification of these linear epitope motifs may lead to novel diagnostic development but further evaluation of cross-reactivity against common co-infecting human nematode infections will be needed. PMID:28125577

  11. Dynamic proteomic profile of potato tuber during its in vitro development.

    PubMed

    Yu, Jae Woong; Choi, Jong-Soon; Upadhyaya, Chandrama Prakash; Kwon, Sang Oh; Gururani, Mayank Anand; Nookaraju, Akula; Nam, Ju-Hyun; Choi, Chi-Won; Kim, Seung Il; Ajappala, Hemavathi; Kim, Hyun Soon; Jeon, Jae Heung; Park, Se Won

    2012-10-01

    Potato tuberization is a complicated biochemical process, which is dependent on external environmental factors. Tuber development in potato consists of a series of biochemical and morphological processes at the stolon tip. Signal transduction proteins are involved in the source-sink transition during potato tuberization. In the present study, we examined protein profiles under in vitro tuber-inducing conditions using a shotgun proteomic approach involving denaturing gel electrophoresis and liquid chromatography-mass spectrometry. A total of 251 proteins were identified and classified into 9 groups according to distinctive expression patterns during the tuberization stage. Stolon stage-specific proteins were primarily involved in the photosynthetic machinery. Proteins specific to the initial tuber stage included patatin. Proteins specific to the developing tuber stage included 6-fructokinase, phytoalexin-deficient 4-1, metallothionein II-like protein, and malate dehydrogenase. Novel stage-specific proteins identified during in vitro tuberization were ferredoxin-NADP reductase, 34 kDa porin, aquaporin, calmodulin, ripening-regulated protein, and starch synthase. Superoxide dismutase, dehydroascorbate reductase, and catalase I were most abundantly expressed in the stolon; however, the enzyme activities of these proteins were most activated at the initial tuber. The present shotgun proteomic study provides insights into the proteins that show altered expression during in vitro potato tuberization. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  12. Modulation of proteomic profile in H295R adrenocortical cell line induced by mitotane.

    PubMed

    Stigliano, A; Cerquetti, L; Borro, M; Gentile, G; Bucci, B; Misiti, S; Piergrossi, P; Brunetti, E; Simmaco, M; Toscano, V

    2008-03-01

    Mitotane, 1,1-dichloro-2-(o-chlorophenyl)-2-(p-chloro-phenyl) ethane (o,p'-DDD), is a compound that represents the effective agent in the treatment of the adrenocortical carcinoma (ACC), able to block cortisol synthesis. In this type of cancer, the biological mechanism induced by this treatment remains still unknown. In this study, we have already shown a greater impairment in the first steps of the steroidogenesis and recognized a little effect on cell cycle. We also evaluated the variation of proteomic profile of the H295R ACC cell line, either in total cell extract or in mitochondria-enriched fraction after treatment with mitotane. In total cell extracts, triose phosphate isomerase, alpha-enolase, D-3-phosphoglycerate dehydrogenase, peroxiredoxin II and VI, heat shock protein 27, prohibitin, histidine triad nucleotide-binding protein, and profilin-1 showed a different expression. In the mitochondrial fraction, the following proteins appeared to be down regulated: aldolase A, peroxiredoxin I, heterogenous nuclear ribonucleoprotein A2/B1, tubulin-beta isoform II, heat shock cognate 71 kDa protein, and nucleotide diphosphate kinase, whereas adrenodoxin reductase, cathepsin D, and heat shock 70 kDa protein 1A were positively up-regulated. This study represents the first proteomic study on the mitotane effects on ACC. It permits to identify some protein classes affected by the drug involved in energetic metabolism, stress response, cytoskeleton structure, and tumorigenesis.

  13. Immunostimulatory potential and proteome profiling of Leishmania donovani soluble exogenous antigens.

    PubMed

    Kumar, A; Samant, M; Misra, P; Khare, P; Sundar, S; Garg, Ravendra; Dube, A

    2015-07-01

    Isolation of the soluble exogenous antigens (SEAgs), its immune response study and proteome profiling is an essential prerequisite for understanding the molecular pathogenesis of Leishmania donovani. The immunostimulatory potential of L. donovani SEAgs, purified from culture of L. donovani clinical isolate, was evaluated for their ability to induce cellular responses in treated/cured hamsters. SEAgs induced significant proliferative responses in lymphocytes (SI 5.6 ± 2.3; P < 0.01) isolated from cured hamster. In addition, significant NO production in response to SEAgs was also noticed in macrophages of hamsters, mouse and human cell lines (J774A-1 and THP1). Western blot analyses with antibodies against proteophosphoglycan (PPG; surface-expressed and secreted molecule) of L. donovani revealed that PPG molecules are also present in L. donovani SEAgs. Mass spectrometry (MS)-based proteome analysis of 12 protein bands of SEAgs through MALDI-TOF/TOF endorsed the identification of some Th1-stimulatory immunogenic proteins. These immunogenic proteins may offer increased hope for the discovery of new promising vaccine candidates against visceral leishmaniasis (VL). The overall results suggest that immunostimulatory molecules are present in the SEAgs, which may be further exploited, for developing a subunit vaccine against VL a fatal human disease.

  14. Gold-nanobeacons for gene therapy: evaluation of genotoxicity, cell toxicity and proteome profiling analysis.

    PubMed

    Conde, João; Larguinho, Miguel; Cordeiro, Ana; Raposo, Luís R; Costa, Pedro M; Santos, Susana; Diniz, Mário S; Fernandes, Alexandra R; Baptista, Pedro V

    2014-08-01

    Antisense therapy is a powerful tool for post-transcriptional gene silencing suitable for down-regulating target genes associated to disease. Gold nanoparticles have been described as effective intracellular delivery vehicles for antisense oligonucleotides providing increased protection against nucleases and targeting capability via simple surface modification. We constructed an antisense gold-nanobeacon consisting of a stem-looped oligonucleotide double-labelled with 3'-Cy3 and 5'-Thiol-C6 and tested for the effective blocking of gene expression in colorectal cancer cells. Due to the beacon conformation, gene silencing was directly detected as fluorescence increases with hybridisation to target, which can be used to assess the level of silencing. Moreover, this system was extensively evaluated for the genotoxic, cytotoxic and proteomic effects of gold-nanobeacon exposure to cancer cells. The exposure was evaluated by two-dimensional protein electrophoresis followed by mass spectrometry to perform a proteomic profile and 3-(4,5-Dimethylthiazol-2-Yl)-2,5-Diphenyltetrazolium Bromide (MTT) assay, glutathione-S-transferase assay, micronucleus test and comet assay to assess the genotoxicity. This integrated toxicology evaluation showed that the proposed nanotheranostics strategy does not exhibit significant toxicity, which is extremely relevant when translating into in vivo systems.

  15. Streptococcus iniae SF1: Complete Genome Sequence, Proteomic Profile, and Immunoprotective Antigens

    PubMed Central

    Zhang, Bao-cun; Zhang, Jian; Sun, Li

    2014-01-01

    Streptococcus iniae is a Gram-positive bacterium that is reckoned one of the most severe aquaculture pathogens. It has a broad host range among farmed marine and freshwater fish and can also cause zoonotic infection in humans. Here we report for the first time the complete genome sequence as well as the host factor-induced proteomic profile of a pathogenic S. iniae strain, SF1, a serotype I isolate from diseased fish. SF1 possesses a single chromosome of 2,149,844 base pairs, which contains 2,125 predicted protein coding sequences (CDS), 12 rRNA genes, and 45 tRNA genes. Among the protein-encoding CDS are genes involved in resource acquisition and utilization, signal sensing and transduction, carbohydrate metabolism, and defense against host immune response. Potential virulence genes include those encoding adhesins, autolysins, toxins, exoenzymes, and proteases. In addition, two putative prophages and a CRISPR-Cas system were found in the genome, the latter containing a CRISPR locus and four cas genes. Proteomic analysis detected 21 secreted proteins whose expressions were induced by host serum. Five of the serum-responsive proteins were subjected to immunoprotective analysis, which revealed that two of the proteins were highly protective against lethal S. iniae challenge when used as purified recombinant subunit vaccines. Taken together, these results provide an important molecular basis for future study of S. iniae in various aspects, in particular those related to pathogenesis and disease control. PMID:24621602

  16. Time- and compartment-resolved proteome profiling of the extracellular niche in lung injury and repair

    PubMed Central

    Schiller, Herbert B; Fernandez, Isis E; Burgstaller, Gerald; Schaab, Christoph; Scheltema, Richard A; Schwarzmayr, Thomas; Strom, Tim M; Eickelberg, Oliver; Mann, Matthias

    2015-01-01

    The extracellular matrix (ECM) is a key regulator of tissue morphogenesis and repair. However, its composition and architecture are not well characterized. Here, we monitor remodeling of the extracellular niche in tissue repair in the bleomycin-induced lung injury mouse model. Mass spectrometry quantified 8,366 proteins from total tissue and bronchoalveolar lavage fluid (BALF) over the course of 8 weeks, surveying tissue composition from the onset of inflammation and fibrosis to its full recovery. Combined analysis of proteome, secretome, and transcriptome highlighted post-transcriptional events during tissue fibrogenesis and defined the composition of airway epithelial lining fluid. To comprehensively characterize the ECM, we developed a quantitative detergent solubility profiling (QDSP) method, which identified Emilin-2 and collagen-XXVIII as novel constituents of the provisional repair matrix. QDSP revealed which secreted proteins interact with the ECM, and showed drastically altered association of morphogens to the insoluble matrix upon injury. Thus, our proteomic systems biology study assigns proteins to tissue compartments and uncovers their dynamic regulation upon lung injury and repair, potentially contributing to the development of anti-fibrotic strategies. PMID:26174933

  17. Identification of three immunodominant motifs with atypical isotype profile scattered over the Onchocerca volvulus proteome.

    PubMed

    Lagatie, Ole; Van Dorst, Bieke; Stuyver, Lieven J

    2017-01-01

    Understanding the immune response upon infection with the filarial nematode Onchocerca volvulus and the mechanisms that evolved in this parasite to evade immune mediated elimination is essential to expand the toolbox available for diagnostics, therapeutics and vaccines development. Using high-density peptide microarrays we scanned the proteome-wide linear epitope repertoire in Cameroonian onchocerciasis patients and healthy controls from Southern Africa which led to the identification of 249 immunodominant antigenic peptides. Motif analysis learned that 3 immunodominant motifs, encompassing 3 linear epitopes, are present in 70, 43, and 31 of these peptides, respectively and appear to be scattered over the entire proteome in seemingly non-related proteins. These linear epitopes are shown to have an atypical isotype profile dominated by IgG1, IgG3, IgE and IgM, in contrast to the commonly observed IgG4 response in chronic active helminth infections. The identification of these linear epitope motifs may lead to novel diagnostic development but further evaluation of cross-reactivity against common co-infecting human nematode infections will be needed.

  18. [The proteomic profiling of blood serum of children with gastroesophageal reflux disease].

    PubMed

    Korkotashvili, L V; Kolesov, S A; Jukova, E A; Vidmanova, T A; Kankova, N Yu; Bashurova, I A; Sidorova, A M; Kulakova, E V

    2015-03-01

    The mass-spectra of proteome of blood serum from healthy children and children with gastroesophageal reflux disease were received. The technology platform including direct proteome mass-spectrometer profiling after pre-fractional rectification using magnetic particles MB WCX was applied. The significant differences in mass-spectra were established manifesting in detection of more mass-spectrometer peaks and higher indicators of their intensity and area in group of healthy children. The study detected 39 particular peptides and low-molecular proteins predominantly intrinsic to healthy or ill children. It was established that two peptides with molecular mass 925 and 909 Da. are registered only in healthy patients and have no traces in group ofpatients with gastroesophageal reflux disease. The peptide 1564 Da is detected only in blood of children with gastroesophageal reflux disease and totally is absent in healthy children. The research data permitted to reveal specific patterns (signatures) of low-molecular proteins and peptides specific for blood serum of healthy children and patients with gastroesophageal reflux disease. The results testify the availability of singularities in metabolism of low-molecular proteins and can be used as a basis for development of minimally invasive mass-spectrometer system for its diagnostic.

  19. Profiling the erythrocyte membrane proteome isolated from patients diagnosed with chronic obstructive pulmonary disease.

    PubMed

    Alexandre, Bruno M; Charro, Nuno; Blonder, Josip; Lopes, Carlos; Azevedo, Pilar; Bugalho de Almeida, António; Chan, King C; Prieto, DaRue A; Issaq, Haleem; Veenstra, Timothy D; Penque, Deborah

    2012-12-05

    Structural and metabolic alterations in erythrocytes play an important role in the pathophysiology of Chronic Obstructive Pulmonary Disease (COPD). Whether these dysfunctions are related to the modulation of erythrocyte membrane proteins in patients diagnosed with COPD remains to be determined. Herein, a comparative proteomic profiling of the erythrocyte membrane fraction isolated from peripheral blood of smokers diagnosed with COPD and smokers with no COPD was performed using differential (16)O/(18)O stable isotope labeling. A total of 219 proteins were quantified as being significantly differentially expressed within the erythrocyte membrane proteomes of smokers with COPD and healthy smokers. Functional pathway analysis showed that the most enriched biofunctions were related to cell-to-cell signaling and interaction, hematological system development, immune response, oxidative stress and cytoskeleton. Chorein (VPS13A), a cytoskeleton related protein whose defects had been associated with the presence of cell membrane deformation of circulating erythrocytes was found to be down-regulated in the membrane fraction of erythrocytes obtained from COPD patients. Methemoglobin reductase (CYB5R3) was also found to be underexpressed in these cells, suggesting that COPD patients may be at higher risk for developing methemoglobinemia. This article is part of a Special Issue entitled: Integrated omics.

  20. Proteomic Profiling and Functional Characterization of Multiple Post-Translational Modifications of Tubulin.

    PubMed

    Liu, Ningning; Xiong, Yun; Ren, Yiran; Zhang, Linlin; He, Xianfei; Wang, Xincheng; Liu, Min; Li, Dengwen; Shui, Wenqing; Zhou, Jun

    2015-08-07

    Tubulin is known to undergo unique post-translational modifications (PTMs), such as detyrosination and polyglutamylation, particularly in the unstructured carboxy-terminal tails (CTTs). However, more conventional PTMs of tubulin and their roles in the regulation of microtubule properties and functions remain poorly defined. Here, we report the comprehensive profiling of tubulin phosphorylation, acetylation, ubiquitylation, and O-GlcNAcylation in HeLa cells with a proteomic approach. Our tubulin-targeted analysis has identified 80 residues bearing single or multiple conventional PTMs including 24 novel PTM sites not covered in previous global proteomic surveys. By using a series of PTM-deficient or PTM-mimicking mutants, we further find that tubulin phosphorylation and acetylation play important roles in the control of microtubule assembly and stability. In addition, these tubulin PTMs have distinct effects on the retrograde transport of adenoviruses along microtubules. These findings thus enlarge the repertoire of tubulin PTMs and foster our understanding of their versatile roles in the regulation of microtubule dynamics and cellular functions.

  1. Proteomic profiling of nipple aspirate fluid (NAF): Exploring the complementarity of different peptide fractionation strategies.

    PubMed

    Brunoro, Giselle Villa Flor; Carvalho, Paulo Costa; Ferreira, André Teixeira da Silva; Perales, Jonas; Valente, Richard Hemmi; de Moura Gallo, Claudia Vitória; Pagnoncelli, Dante; Neves-Ferreira, Ana Gisele da Costa

    2015-03-18

    NAF is a breast fluid that is closely related to the tumor microenvironment and a valuable sample for studying breast cancer. To perform an in-depth proteomic analysis of this sample, aliquots of a single NAF digest were analyzed by the following peptide-centric fractionation strategies: a) 30-cm reversed-phase (RP) column on-line with an LTQ-Orbitrap XL; b) off-line strong cation-exchange (SCX) column; and c) pI-based OFFGEL fractionation. All fractions from approaches (b) and (c) were further analyzed on a 10-cm RP column hyphenated to the same mass spectrometer. The RP-30cm, SCX/RP-10cm, and OFFGEL/RP-10cm approaches identified 1676, 2930, and 3240 peptides, which corresponded to 193, 390 and 528 proteins, respectively. In our cumulative dataset, 4466 distinct NAF peptides corresponded to a total of 557 proteins, of which only 34% were identified by all three approaches. No exclusive protein identification was associated to the RP-30cm approach, while SCX/RP-10cm and OFFGEL/RP-10cm contributed to 28 and 166 exclusive identifications, respectively. Each approach provided additional information related to energy metabolism (fermentation process/carbohydrate biosynthesis). In conclusion, the pre-fractionation platforms used were complementary for the comprehensive characterization of NAF and our work provides methodological information for future quantitative cancer-related NAF sample studies. High-resolution peptide separation is a sine qua non condition for achieving extensive proteome coverage. Various techniques have been employed to improve peptide fractionation prior to LC-MS/MS, thus allowing a comprehensive characterization of complex biological samples. Although fractionation efficiency is very sample-dependent, this issue is commonly overlooked, and a "cookbook" approach is routinely used during this critical step. The present study provides a systematic comparison of analytical information needed for the successful large-scale differential proteomic

  2. Protein probabilities in shotgun proteomics: evaluating different estimation methods using a semi-random sampling model.

    PubMed

    Xue, Xiaofang; Wu, Songfeng; Wang, Zhongsheng; Zhu, Yunping; He, Fuchu

    2006-12-01

    The calculation of protein probabilities is one of the most intractable problems in large-scale proteomic research. Current available estimating methods, for example, ProteinProphet, PROT_PROBE, Poisson model and two-peptide hits, employ different models trying to resolve this problem. Until now, no efficient method is used for comparative evaluation of the above methods in large-scale datasets. In order to evaluate these various methods, we developed a semi-random sampling model to simulate large-scale proteomic data. In this model, the identified peptides were sampled from the designed proteins and their cross-correlation scores were simulated according to the results from reverse database searching. The simulated result of 18 control proteins was consistent with the experimental one, demonstrating the efficiency of our model. According to the simulated results of human liver sample, ProteinProphet returned slightly higher probabilities and lower specificity than real cases. PROT_PROBE was a more efficient method with higher specificity. Predicted results from a Poisson model roughly coincide with real datasets, and the method of two-peptide hits seems solid but imprecise. However, the probabilities of identified proteins are strongly correlated with several experimental factors including spectra number, database size and protein abundance distribution.

  3. Profiling the proteomics in honeybee worker brains submitted to the proboscis extension reflex.

    PubMed

    da Silva Menegasso, Anally Ribeiro; Pratavieira, Marcel; de Saldanha da Gama Fischer, Juliana; Carvalho, Paulo Costa; Roat, Thaisa Cristina; Malaspina, Osmar; Palma, Mario Sergio

    2017-01-16

    The proboscis extension reflex (PER) is an unconditioned stimulus (US) widely used to access the ability of honeybees to correlate it with a conditioned stimulus (CS) during learning and memory acquisition. However, little is known about the biochemical/genetic changes in worker honeybee brains induced by the PER alone. The present investigation profiled the proteomic complement associated with the PER to further the understanding of the major molecular transformations in the honeybee brain during the execution of a US. In the present study, a quantitative shotgun proteomic approach was employed to assign the proteomic complement of the honeybee brain. The results were analyzed under the view of protein networking for different processes involved in PER behavior. In the brains of PER-stimulated individuals, the metabolism of cyclic/heterocyclic/aromatic compounds was activated in parallel with the metabolism of nitrogenated compounds, followed by the up-regulation of carbohydrate metabolism, the proteins involved with the anatomic and cytoskeleton; the down-regulation of the anatomic development and cell differentiation in other neurons also occurred. The assay of proboscis extension reflex is frequently used to access honeybees' ability to correlate an unconditioned stimulus with a conditioned stimulus (such as an odor) to establish learning and memory acquisition. The reflex behavior of proboscis extension was associated with various conditioned stimuli, and the biochemical/genetic evaluation of the changes occurring in honeybee brains under these conditions reflect the synergistic effects of both insect manipulations (training to answer to an unconditioned stimulus and training to respond to a conditioned stimulus). Little or no information is available regarding the biochemical changes stimulated by an unconditioned stimulus alone, such as the proboscis extension reflex. The present investigation characterizes the proteomic changes occurring in the brains of

  4. Gel-aided sample preparation (GASP)--a simplified method for gel-assisted proteomic sample generation from protein extracts and intact cells.

    PubMed

    Fischer, Roman; Kessler, Benedikt M

    2015-04-01

    We describe a "gel-assisted" proteomic sample preparation method for MS analysis. Solubilized protein extracts or intact cells are copolymerized with acrylamide, facilitating denaturation, reduction, quantitative cysteine alkylation, and matrix formation. Gel-aided sample preparation has been optimized to be highly flexible, scalable, and to allow reproducible sample generation from 50 cells to milligrams of protein extracts. This methodology is fast, sensitive, easy-to-use on a wide range of sample types, and accessible to nonspecialists. © 2014 The Authors. PROTEOMICS published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. DEVELOPMENTAL CIGARETTE SMOKE EXPOSURE: HIPPOCAMPUS PROTEOME AND METABOLOME PROFILES IN LOW BIRTH WEIGHT PUPS

    PubMed Central

    Neal, Rachel E.; Chen, Jing; Jagadapillai, Rekha; Jang, HyeJeong; Abomoelak, Bassam; Brock, Guy; Greene, Robert M.; Pisano, M. Michele

    2014-01-01

    Exposure to cigarette smoke during development is linked to neurodevelopmental delays and cognitive impairment including impulsivity, attention deficit disorder, and lower IQ. However, brain region specific biomolecular alterations induced by developmental cigarette smoke exposure (CSE) remain largely unexplored. In the current molecular phenotyping study, a mouse model of ‘active’ developmental CSE (serum cotinine>50 ng/mL) spanning pre-implantation through third trimester-equivalent brain development (gestational day (GD) 1 through postnatal day (PD) 21) was utilized. Hippocampus tissue collected at the time of cessation of exposure was processed for gel-based proteomic and non-targeted metabolomic profiling with Partial Least Squares-Discriminant Analysis (PLS-DA) for selection of features of interest. Ingenuity Pathway Analysis was utilized to identify candidate molecular and metabolic pathways impacted within the hippocampus. CSE impacted glycolysis, oxidative phosphorylation, fatty acid metabolism, and neurodevelopment pathways within the developing hippocampus. PMID:24486158

  6. Proteomic Profile of Brucella abortus-Infected Bovine Chorioallantoic Membrane Explants

    PubMed Central

    Mol, Juliana P. S.; Pires, Simone F.; Chapeaurouge, Alexander D.; Perales, Jonas; Santos, Renato L.; Andrade, Hélida M.; Lage, Andrey P.

    2016-01-01

    Brucella abortus is the etiological agent of bovine brucellosis, a zoonotic disease that causes significant economic losses worldwide. The differential proteomic profile of bovine chorioallantoic membrane (CAM) explants at early stages of infection with B. abortus (0.5, 2, 4, and 8 h) was determined. Analysis of CAM explants at 0.5 and 4 h showed the highest differences between uninfected and infected CAM explants, and therefore were used for the Differential Gel Electrophoresis (DIGE). A total of 103 spots were present in only one experimental group and were selected for identification by mass spectrometry (MALDI/ToF-ToF). Proteins only identified in extracts of CAM explants infected with B. abortus were related to recognition of PAMPs by TLR, production of reactive oxygen species, intracellular trafficking, and inflammation. PMID:27104343

  7. Proteomic profiling of the mitochondrial ribosome identifies Atp25 as a composite mitochondrial precursor protein.

    PubMed

    Woellhaf, Michael W; Sommer, Frederik; Schroda, Michael; Herrmann, Johannes M

    2016-10-15

    Whereas the structure and function of cytosolic ribosomes are well characterized, we only have a limited understanding of the mitochondrial translation apparatus. Using SILAC-based proteomic profiling, we identified 13 proteins that cofractionated with the mitochondrial ribosome, most of which play a role in translation or ribosomal biogenesis. One of these proteins is a homologue of the bacterial ribosome-silencing factor (Rsf). This protein is generated from the composite precursor protein Atp25 upon internal cleavage by the matrix processing peptidase MPP, and in this respect, it differs from all other characterized mitochondrial proteins of baker's yeast. We observed that cytosolic expression of Rsf, but not of noncleaved Atp25 protein, is toxic. Our results suggest that eukaryotic cells face the challenge of avoiding negative interference from the biogenesis of their two distinct translation machineries.

  8. In situ imaging and proteome profiling indicate andrographolide is a highly promiscuous compound

    PubMed Central

    Li, Lin; Wijaya, Hadhi; Samanta, Sanjay; Lam, Yulin; Yao, Shao Q.

    2015-01-01

    Natural products represent an enormous source of pharmacologically useful compounds, and are often used as the starting point in modern drug discovery. Many biologically interesting natural products are however not being pursued as potential drug candidates, partly due to a lack of well-defined mechanism-of-action. Traditional in vitro methods for target identification of natural products based on affinity protein enrichment from crude cellular lysates cannot faithfully recapitulate protein-drug interactions in living cells. Reported herein are dual-purpose probes inspired by the natural product andrographolide, capable of both reaction-based, real-time bioimaging and in situ proteome profiling/target identification in live mammalian cells. Our results confirm that andrographolide is a highly promiscuous compound and engaged in covalent interactions with numerous previously unknown cellular targets in cell type-specific manner. We caution its potential therapeutic effects should be further investigated in detail. PMID:26105662

  9. Proteomic Profile of Brucella abortus-Infected Bovine Chorioallantoic Membrane Explants.

    PubMed

    Mol, Juliana P S; Pires, Simone F; Chapeaurouge, Alexander D; Perales, Jonas; Santos, Renato L; Andrade, Hélida M; Lage, Andrey P

    2016-01-01

    Brucella abortus is the etiological agent of bovine brucellosis, a zoonotic disease that causes significant economic losses worldwide. The differential proteomic profile of bovine chorioallantoic membrane (CAM) explants at early stages of infection with B. abortus (0.5, 2, 4, and 8 h) was determined. Analysis of CAM explants at 0.5 and 4 h showed the highest differences between uninfected and infected CAM explants, and therefore were used for the Differential Gel Electrophoresis (DIGE). A total of 103 spots were present in only one experimental group and were selected for identification by mass spectrometry (MALDI/ToF-ToF). Proteins only identified in extracts of CAM explants infected with B. abortus were related to recognition of PAMPs by TLR, production of reactive oxygen species, intracellular trafficking, and inflammation.

  10. Enrichment of low-abundance proteins from bovine and porcine serum samples for proteomic studies.

    PubMed

    Marco-Ramell, Anna; Bassols, Anna

    2010-12-01

    One of the main applications of serum proteomics is the identification of new biomarkers for animal disease or animal production. However, potential obstacles to these studies are the poor performance of affinity serum depletion methods based on human antigens when using animal samples, and loss of minor serum components bound to albumin and other proteins. In the present study, we have analyzed the efficiency and reproducibility of the ProteoMiner® beads with bovine and porcine serum samples, and compared to a traditional immunoaffinity-based albumin and IgG depletion system specific for human samples. The ProteoMiner kit is based on the use of a combinatorial peptide binding library and intends to enrich low-abundance proteins.

  11. Yeast Interspecies Comparative Proteomics Reveals Divergence in Expression Profiles and Provides Insights into Proteome Resource Allocation and Evolutionary Roles of Gene Duplication.

    PubMed

    Kito, Keiji; Ito, Haruka; Nohara, Takehiro; Ohnishi, Mihoko; Ishibashi, Yuko; Takeda, Daisuke

    2016-01-01

    Omics analysis is a versatile approach for understanding the conservation and diversity of molecular systems across multiple taxa. In this study, we compared the proteome expression profiles of four yeast species (Saccharomyces cerevisiae, Saccharomyces mikatae, Kluyveromyces waltii, and Kluyveromyces lactis) grown on glucose- or glycerol-containing media. Conserved expression changes across all species were observed only for a small proportion of all proteins differentially expressed between the two growth conditions. Two Kluyveromyces species, both of which exhibited a high growth rate on glycerol, a nonfermentative carbon source, showed distinct species-specific expression profiles. In K. waltii grown on glycerol, proteins involved in the glyoxylate cycle and gluconeogenesis were expressed in high abundance. In K. lactis grown on glycerol, the expression of glycolytic and ethanol metabolic enzymes was unexpectedly low, whereas proteins involved in cytoplasmic translation, including ribosomal proteins and elongation factors, were highly expressed. These marked differences in the types of predominantly expressed proteins suggest that K. lactis optimizes the balance of proteome resource allocation between metabolism and protein synthesis giving priority to cellular growth. In S. cerevisiae, about 450 duplicate gene pairs were retained after whole-genome duplication. Intriguingly, we found that in the case of duplicates with conserved sequences, the total abundance of proteins encoded by a duplicate pair in S. cerevisiae was similar to that of protein encoded by nonduplicated ortholog in Kluyveromyces yeast. Given the frequency of haploinsufficiency, this observation suggests that conserved duplicate genes, even though minor cases of retained duplicates, do not exhibit a dosage effect in yeast, except for ribosomal proteins. Thus, comparative proteomic analyses across multiple species may reveal not only species-specific characteristics of metabolic processes under

  12. Yeast Interspecies Comparative Proteomics Reveals Divergence in Expression Profiles and Provides Insights into Proteome Resource Allocation and Evolutionary Roles of Gene Duplication*

    PubMed Central

    Kito, Keiji; Ito, Haruka; Nohara, Takehiro; Ohnishi, Mihoko; Ishibashi, Yuko; Takeda, Daisuke

    2016-01-01

    Omics analysis is a versatile approach for understanding the conservation and diversity of molecular systems across multiple taxa. In this study, we compared the proteome expression profiles of four yeast species (Saccharomyces cerevisiae, Saccharomyces mikatae, Kluyveromyces waltii, and Kluyveromyces lactis) grown on glucose- or glycerol-containing media. Conserved expression changes across all species were observed only for a small proportion of all proteins differentially expressed between the two growth conditions. Two Kluyveromyces species, both of which exhibited a high growth rate on glycerol, a nonfermentative carbon source, showed distinct species-specific expression profiles. In K. waltii grown on glycerol, proteins involved in the glyoxylate cycle and gluconeogenesis were expressed in high abundance. In K. lactis grown on glycerol, the expression of glycolytic and ethanol metabolic enzymes was unexpectedly low, whereas proteins involved in cytoplasmic translation, including ribosomal proteins and elongation factors, were highly expressed. These marked differences in the types of predominantly expressed proteins suggest that K. lactis optimizes the balance of proteome resource allocation between metabolism and protein synthesis giving priority to cellular growth. In S. cerevisiae, about 450 duplicate gene pairs were retained after whole-genome duplication. Intriguingly, we found that in the case of duplicates with conserved sequences, the total abundance of proteins encoded by a duplicate pair in S. cerevisiae was similar to that of protein encoded by nonduplicated ortholog in Kluyveromyces yeast. Given the frequency of haploinsufficiency, this observation suggests that conserved duplicate genes, even though minor cases of retained duplicates, do not exhibit a dosage effect in yeast, except for ribosomal proteins. Thus, comparative proteomic analyses across multiple species may reveal not only species-specific characteristics of metabolic processes under

  13. Comparison of in-gel protein separation techniques commonly used for fractionation in mass spectrometry-based proteomic profiling

    PubMed Central

    Jafari, Mohieddin; Primo, Vincent; Smejkal, Gary B.; Moskovets, Eugene V.; Kuo, Winston P.; Ivanov, Alexander R.

    2014-01-01

    Fractionation of complex samples at the cellular, subcellular, protein or peptide level is an indispensable strategy to improve the sensitivity in mass spectrometry-based proteomic profiling. This study revisits, evaluates, and compares the most common gel-based protein separation techniques i.e., 1-D SDS PAGE, preparative 1-D SDS PAGE, isoelectric focusing in immobilized pH gradients (IEF-IPG), and 2-D PAGE in their performance as fractionation approaches in nanoLC-ESI-MS/MS analysis of a mixture of protein standards and mitochondrial extracts isolated from rat liver. This work demonstrates that all the above techniques provide complementary protein identification results, but 1-D SDS PAGE and IEF-IPG had the highest number of identifications. The IEF-IPG technique resulted in the highest average number of detected peptides per protein. The 2-D PAGE was evaluated as a protein fractionation approach. This work shows that the recovery of proteins and resulting proteolytic digests is highly dependent on the total volume of the gel matrix. The performed comparison of the fractionation techniques demonstrates the potential of a combination of orthogonal 1-D SDS PAGE and IEF-IPG for the improved sensitivity of profiling without significant decrease in throughput. PMID:22899259

  14. PeptideManager: a peptide selection tool for targeted proteomic studies involving mixed samples from different species

    PubMed Central

    Demeure, Kevin; Duriez, Elodie; Domon, Bruno; Niclou, Simone P.

    2014-01-01

    The search for clinically useful protein biomarkers using advanced mass spectrometry approaches represents a major focus in cancer research. However, the direct analysis of human samples may be challenging due to limited availability, the absence of appropriate control samples, or the large background variability observed in patient material. As an alternative approach, human tumors orthotopically implanted into a different species (xenografts) are clinically relevant models that have proven their utility in pre-clinical research. Patient derived xenografts for glioblastoma have been extensively characterized in our laboratory and have been shown to retain the characteristics of the parental tumor at the phenotypic and genetic level. Such models were also found to adequately mimic the behavior and treatment response of human tumors. The reproducibility of such xenograft models, the possibility to identify their host background and perform tumor-host interaction studies, are major advantages over the direct analysis of human samples. At the proteome level, the analysis of xenograft samples is challenged by the presence of proteins from two different species which, depending on tumor size, type or location, often appear at variable ratios. Any proteomics approach aimed at quantifying proteins within such samples must consider the identification of species specific peptides in order to avoid biases introduced by the host proteome. Here, we present an in-house methodology and tool developed to select peptides used as surrogates for protein candidates from a defined proteome (e.g., human) in a host proteome background (e.g., mouse, rat) suited for a mass spectrometry analysis. The tools presented here are applicable to any species specific proteome, provided a protein database is available. By linking the information from both proteomes, PeptideManager significantly facilitates and expedites the selection of peptides used as surrogates to analyze proteins of interest

  15. Acclimation to different depths by the marine angiosperm Posidonia oceanica: transcriptomic and proteomic profiles

    PubMed Central

    Dattolo, Emanuela; Gu, Jenny; Bayer, Philipp E.; Mazzuca, Silvia; Serra, Ilia A.; Spadafora, Antonia; Bernardo, Letizia; Natali, Lucia; Cavallini, Andrea; Procaccini, Gabriele

    2013-01-01

    For seagrasses, seasonal and daily variations in light and temperature represent the mains factors driving their distribution along the bathymetric cline. Changes in these environmental factors, due to climatic and anthropogenic effects, can compromise their survival. In a framework of conservation and restoration, it becomes crucial to improve our knowledge about the physiological plasticity of seagrass species along environmental gradients. Here, we aimed to identify differences in transcriptomic and proteomic profiles, involved in the acclimation along the depth gradient in the seagrass Posidonia oceanica, and to improve the available molecular resources in this species, which is an important requisite for the application of eco-genomic approaches. To do that, from plant growing in shallow (−5 m) and deep (−25 m) portions of a single meadow, (i) we generated two reciprocal Expressed Sequences Tags (EST) libraries using a Suppressive Subtractive Hybridization (SSH) approach, to obtain depth/specific transcriptional profiles, and (ii) we identified proteins differentially expressed, using the highly innovative USIS mass spectrometry methodology, coupled with 1D-SDS electrophoresis and labeling free approach. Mass spectra were searched in the open source Global Proteome Machine (GPM) engine against plant databases and with the X!Tandem algorithm against a local database. Transcriptional analysis showed both quantitative and qualitative differences between depths. EST libraries had only the 3% of transcripts in common. A total of 315 peptides belonging to 64 proteins were identified by mass spectrometry. ATP synthase subunits were among the most abundant proteins in both conditions. Both approaches identified genes and proteins in pathways related to energy metabolism, transport and genetic information processing, that appear to be the most involved in depth acclimation in P. oceanica. Their putative rules in acclimation to depth were discussed. PMID:23785376

  16. Do cultural conditions induce differential protein expression: Profiling of extracellular proteome of Aspergillus terreus CM20.

    PubMed

    M, Saritha; Singh, Surender; Tiwari, Rameshwar; Goel, Renu; Nain, Lata

    2016-11-01

    The present study reports the diversity in extracellular proteins expressed by the filamentous fungus, Aspergillus terreus CM20 with respect to differential hydrolytic enzyme production profiles in submerged fermentation (SmF) and solid-state fermentation (SSF) conditions, and analysis of the extracellular proteome. The SSF method was superior in terms of increase in enzyme activities resulting in 1.5-3 fold enhancement as compared to SmF, which was explained by the difference in growth pattern of the fungus under the two culture conditions. As revealed by zymography, multiple isoforms of endo-β-glucanase, β-glucosidase and xylanase were expressed in SSF, but not in SmF. Extracellular proteome profiling of A. terreus CM20 under SSF condition using liquid chromatography coupled tandem mass spectrometry (LC-MS/MS) identified 63 proteins. Functional classification revealed the hydrolytic system to be composed of glycoside hydrolases (56%), proteases (16%), oxidases and dehydrogenases (6%), decarboxylases (3%), esterases (3%) and other proteins (16%). Twenty families of glycoside hydrolases (GH) (1, 3, 5, 7, 10, 11, 12, 15, 16, 28, 30, 32, 35, 43, 54, 62, 67, 72, 74 and 125), and one family each of auxiliary activities (AA7) and carbohydrate esterase (CE1) were detected, unveiling the vast diversity of synergistically acting biomass-cleaving enzymes expressed by the fungus. Saccharification of alkali-pretreated paddy straw with A. terreus CM20 proteins released high amounts of glucose (439.63±1.50mg/gds), xylose (121.04±1.25mg/gds) and arabinose (56.13±0.56mg/gds), thereby confirming the potential of the enzyme cocktail in bringing about considerable conversion of lignocellulosic polysaccharides to sugar monomers.

  17. Proteomic profile determination of autosomal aneuploidies by mass spectrometry on amniotic fluids

    PubMed Central

    Mange, Alain; Desmetz, Caroline; Bellet, Virginie; Molinari, Nicolas; Maudelonde, Thierry; Solassol, Jerome

    2008-01-01

    Background Prenatal diagnosis of chromosomal abnormalities by cytogenetic analysis is time-consuming, expensive, and requires highly qualified technicians. Rapid diagnosis of aneuploidies followed by reassurance of women with normal results can be performed by molecular analysis of uncultured foetal cells. In the present study, we developed a proteomic fingerprinting approach coupled with a statistical classification method to improve diagnosis of aneuploidies, including trisomies 13, 18, and 21, in amniotic fluid samples. Results The proteomic spectra obtained from 52 pregnant women were compiled, normalized, and mass peaks with mass-to-charge ratios between 2.5 and 50 kDa identified. Peak information was combined together and analysed using univariate statistics. Among the 208 expressed protein peaks, 40 differed significantly between aneuploid and non aneuploid samples, with AUC diagnostic values ranging from 0.71 to 0.91. Hierarchical clustering, principal component analysis and support vector machine (SVM) analysis were performed. Two class predictor models were defined from the training set, which resulted in a prediction accuracy of 92.3% and 96.43%, respectively. Using an external and independent validation set, diagnostic accuracies were maintained at 87.5% and 91.67%, respectively. Conclusion This pilot study demonstrates the potential interest of protein expression signature in the identification of new potential biological markers that might be helpful for the rapid clinical management of high-risk pregnancies. PMID:18190690

  18. Unraveling the proteomic profile of mice testis during the initiation of meiosis.

    PubMed

    Shao, Binbin; Guo, Yueshuai; Wang, Lei; Zhou, Quan; Gao, Tingting; Zheng, Bo; Zheng, Haoyu; Zhou, Tao; Zhou, Zuomin; Guo, Xuejiang; Huang, Xiaoyan; Sha, Jiahao

    2015-04-29

    In mice, once primordial germ cells (PGCs) are generated, they continue to proliferate and migrate to eventually reach the future gonads. They initiate sexual differentiation after their colonization of the gonads. During this process, retinoic acid (RA) induces meiosis in the female germ cells, which proceeds to the diplotene stage of meiotic prophase I, whereas the male germ cells initiate growth arrest. After birth, meiosis is initiated in mice spermatogonia by their conversion to preleptotene spermatocytes. There are evidences showing the roles of RA in the regulation of spermatogonial differentiation and meiosis initiation. However, it is still not well known on what responds to RA and how RA signaling engages meiosis. Thus, we constructed a proteomic profile of proteins associated with meiosis onset during testis development in mouse and identified 104 differentially expressed proteins (≥1.5 folds). Bioinformatic analysis showed proteins functioning in specific cell processes. The expression patterns of five selected proteins were verified via Western blot, of which we found that Tfrc gene was RA responsive, with a RA responsive element, and could be up regulated by RA in spermatogonial stem cell (SSC) line. Taken together, the results provide an important reference profile for further functional study of meiosis initiation. Spermatogenesis involves mitosis of spermatogonia, meiosis of spermatocytes and spermiogenesis, in which meiosis is a unique event to germ cells, and not in the somatic cells. Till now, the detailed molecular mechanisms of the transition from mitosis to meiosis are still not elucidated. With high-throughput proteomic technology, it is now possible to systemically identify proteins possibly involved. With TMT-6plex based quantification, we identified 104 proteins differentially between testes without meiosis (day 8.5) and those that were meiosis initiated (day 10.5). And a well-known protein essential for meiosis initiation, stra8, was

  19. Sample preparation for proteomic analysis using a GeLC-MS/MS strategy.

    PubMed

    Paulo, Joao A

    In-gel digestion coupled with mass spectrometric analysis (GeLC-MS/MS) is a cornerstone for protein identification and characterization. Here I review this versatile approach which combines classical and modern biochemistry strategies and allows for targeted and proteome-wide analyses. Starting with any protein sample, reduced and alkylated proteins are precipitated prior to fractionation by SDS-PAGE. Proteins are in-gel digested and the resulting peptides are extracted and desalted for downstream LC-MS/MS analysis. GeLC-MS/MS leverages the advantages of both traditional SDS-PAGE visualization and protein fractionation with the robust protein and post-translational modification identification and quantitation capabilities of state-of-the-art mass spectrometry-based technology. As such, this strategy allows for the visible assessment of protein amount and quality, prior to analysis via virtually any mass spectrometry platform. Moreover, gel extracted peptides may be derived from any sample type-e.g., from cell culture, tissue, body fluid, or recombinantly-expressed protein-and are fully compatible with isobaric tagging. GeLC-MS/MS is an invaluable technique for proteomic analyses.

  20. Suspension trapping (STrap) sample preparation method for bottom-up proteomics analysis.

    PubMed

    Zougman, Alexandre; Selby, Peter J; Banks, Rosamonde E

    2014-05-01

    Despite recent developments in bottom-up proteomics, the need still exists in a fast, uncomplicated, and robust method for comprehensive sample processing especially when applied to low protein amounts. The suspension trapping method combines the advantage of efficient SDS-based protein extraction with rapid detergent removal, reactor-type protein digestion, and peptide cleanup. Proteins are solubilized in SDS. The sample is acidified and introduced into the suspension trapping tip incorporating the depth filter and hydrophobic compartments, filled with the neutral pH methanolic solution. The instantly formed fine protein suspension is trapped in the depth filter stack-this crucial step is aimed at separating the particulate matter in space. SDS and other contaminants are removed in the flow-through, and a protease is introduced. Following the digestion, the peptides are cleaned up using the tip's hydrophobic part. The methodology allows processing of protein loads down to the low microgram/submicrogram levels. The detergent removal takes about 5 min, whereas the tryptic proteolysis of a cellular lysate is complete in as little as 30 min. We have successfully utilized the method for analysis of cellular lysates, enriched membrane preparations, and immunoprecipitates. We expect that due to its robustness and simplicity, the method will become an essential proteomics tool.

  1. Comprehensive and quantitative proteomic analyses of zebrafish plasma reveals conserved protein profiles between genders and between zebrafish and human.

    PubMed

    Li, Caixia; Tan, Xing Fei; Lim, Teck Kwang; Lin, Qingsong; Gong, Zhiyuan

    2016-04-13

    Omic approaches have been increasingly used in the zebrafish model for holistic understanding of molecular events and mechanisms of tissue functions. However, plasma is rarely used for omic profiling because of the technical challenges in collecting sufficient blood. In this study, we employed two mass spectrometric (MS) approaches for a comprehensive characterization of zebrafish plasma proteome, i.e. conventional shotgun liquid chromatography-tandem mass spectrometry (LC-MS/MS) for an overview study and quantitative SWATH (Sequential Window Acquisition of all THeoretical fragment-ion spectra) for comparison between genders. 959 proteins were identified in the shotgun profiling with estimated concentrations spanning almost five orders of magnitudes. Other than the presence of a few highly abundant female egg yolk precursor proteins (vitellogenins), the proteomic profiles of male and female plasmas were very similar in both number and abundance and there were basically no other highly gender-biased proteins. The types of plasma proteins based on IPA (Ingenuity Pathway Analysis) classification and tissue sources of production were also very similar. Furthermore, the zebrafish plasma proteome shares significant similarities with human plasma proteome, in particular in top abundant proteins including apolipoproteins and complements. Thus, the current study provided a valuable dataset for future evaluation of plasma proteins in zebrafish.

  2. Universal Solid-phase Reversible Sample-Prep for Concurrent Proteome and N-glycome Characterization

    PubMed Central

    Zhou, Hui; Morley, Samantha; Kostel, Stephen; Freeman, Michael R.; Joshi, Vivek; Brewster, David; Lee, Richard S.

    2017-01-01

    SUMMARY We describe a novel Solid-phase Reversible Sample-Prep (SRS) platform, which enables rapid sample preparation for concurrent proteome and N-glycome characterization by mass spectrometry. SRS utilizes a uniquely functionalized, silica-based bead that has strong affinity toward proteins with minimal-to-no affinity for peptides and other small molecules. By leveraging the inherent size difference between, SRS permits high-capacity binding of proteins, rapid removal of small molecules (detergents, metabolites, salts, etc.), extensive manipulation including enzymatic and chemical treatments on beads-bound proteins, and easy recovery of N-glycans and peptides. The efficacy of SRS was evaluated in a wide range of biological samples including single glycoprotein, whole cell lysate, murine tissues, and human urine. To further demonstrate the SRS platform, we coupled a quantitative strategy to SRS to investigate the differences between DU145 prostate cancer cells and its DIAPH3-silenced counterpart. Our previous studies suggested that DIAPH3 silencing in DU145 prostate cancer cells induced transition to an amoeboid phenotype that correlated with tumor progression and metastasis. In this analysis we identified distinct proteomic and N-glycomic alterations between the two cells. Intriguingly, a metastasis-associated tyrosine kinase receptor ephrin-type-A receptor (EPHA2) was highly upregulated in DIAPH3-silenced cells, indicating underling connection between EPHA2 and DIAPH3. Moreover, distinct alterations in the N-glycome were identified, suggesting a cross-link between DIAPH3 and glycosyltransferase networks. Overall, SRS is an enabling universal sample preparation strategy that is not size limited and has the capability to efficiently prepare and clean peptides and N-glycans concurrently from nearly all sample types. Conceptually, SRS can be utilized for the analysis of other posttranslational modifications, and the unique surface chemistry can be further

  3. Personalized Proteome Profiles of Healthy and Tumor Human Colon Organoids Reveal Both Individual Diversity and Basic Features of Colorectal Cancer.

    PubMed

    Cristobal, Alba; van den Toorn, Henk W P; van de Wetering, Marc; Clevers, Hans; Heck, Albert J R; Mohammed, Shabaz

    2017-01-03

    Diseases at the molecular level are complex and patient dependent, necessitating development of strategies that enable precision treatment to optimize clinical outcomes. Organoid technology has recently been shown to have the potential to recapitulate the in vivo characteristics of the original individual's tissue in a three-dimensional in vitro culture system. Here, we present a quantitative mass-spectrometry-based proteomic analysis and a comparative transcriptomic analysis of human colorectal tumor and healthy organoids derived, in parallel, from seven patients. Although gene and protein signatures can be derived to distinguish the tumor organoid population from healthy organoids, our data clearly reveal that each patient possesses a distinct organoid signature at the proteomic level. We demonstrate that a personalized patient-specific organoid proteome profile can be related to the diagnosis of a patient and with future development contribute to the generation of personalized therapies.

  4. Shotgun electroelution: a proteomic tool for simultaneous sample elution from whole SDS-polyacrylamide gel slabs.

    PubMed

    Antal, József; Bányász, Borbála; Buzás, Zsuzsanna

    2007-02-01

    A high-throughput device has been constructed which allows parallel electroelution of separated SDS-protein bands directly from intact unsectioned polyacrylamide gel slabs as well as single electroelution of certain protein spots into a 384-well standard flat-bottom multiwell plate. The prototype provides complete, quick elution for proteomics from 1-D or from 2-D gels without gel sectioning. Since the elution chamber matrix requires no assembly, sample handling can be easily carried out by existing robotic workstations. The current design is a good candidate for automation of spot elution since there are no moving liquid containing components in the apparatus. Eight SDS-proteins were eluted in test runs and an average 70% sample recovery was achieved by re-electrophoresis of the electro-eluates.

  5. Filter-Aided Sample Preparation: The Versatile and Efficient Method for Proteomic Analysis.

    PubMed

    Wiśniewski, J R

    2017-01-01

    Filter-aided sample preparation (FASP) is a versatile and efficient way of processing protein extracts for bottom-up proteomic analysis. The method repurposes centrifugal ultrafiltration concentrators for removal of detergents, protein cleavage, and isolation of pure peptide fractions. FASP can be used for protein cleavage with different proteinases either with single enzymes or in a mode of successive multienzyme digestion (MED)-FASP. The FASP methods are useful for processing of samples ranging in their sizes from submicrogram to several milligram amounts of total protein. They also allow peptide fractionation, and isolation and quantitation of total RNA and DNA acid contents. This chapter describes principles, limitations, and applications of FASP. Additionally detailed FASP and MED-FASP protocols are provided. © 2017 Elsevier Inc. All rights reserved.

  6. Reducing sample complexity in proteomics by chromatofocusing with simple buffer mixtures.

    PubMed

    Shen, Hong; Li, Xiang; Bieberich, Charles J; Frey, Douglas D

    2008-01-01

    Chromatofocusing has many potential applications in the field of proteomics, such as for the isolation and removal of major sample components to facilitate the analysis of low-abundance components, and for sample prefractionation prior to a subsequent separation using SDS-PAGE, narrow-pI-range 2D-PAGE, or additional chromatography steps. However, the chromatofocusing techniques that are most commonly used employ propriety polyampholyte elution buffers and highly specialized column packings, both of which limit the use of chromatofocusing in practice. To expand the range of application for this technique, this chapter considers chromatofocusing methods which employ common ion-exchange column packings and elution buffers which are simple mixtures of readily available buffering species. Of particular interest is the use of chromatofocusing with a multistep pH gradient for the fractionation of protein mixtures into narrow-pI-range fractions. The cross-contamination characteristics of these fractions using SDS-PAGE are also assessed.

  7. Optimization of proteomic sample preparation procedures for comprehensive protein characterization of pathogenic systems

    SciTech Connect

    Brewer, Heather M.; Norbeck, Angela D.; Adkins, Joshua N.; Manes, Nathan P.; Ansong, Charles; Shi, Liang; Rikihisa, Yasuko; Kikuchi, Takane; Wong, Scott; Estep, Ryan D.; Heffron, Fred; Pasa-Tolic, Ljiljana; Smith, Richard D.

    2008-12-19

    The elucidation of critical functional pathways employed by pathogens and hosts during an infectious cycle is both challenging and central to our understanding of infectious diseases. In recent years, mass spectrometry-based proteomics has been used as a powerful tool to identify key pathogenesis-related proteins and pathways. Despite the analytical power of mass spectrometry-based technologies, samples must be appropriately prepared to characterize the functions of interest (e.g. host-response to a pathogen or a pathogen-response to a host). The preparation of these protein samples requires multiple decisions about what aspect of infection is being studied, and it may require the isolation of either host and/or pathogen cellular material.

  8. Proteomic profiling of rice embryos from a hybrid rice cultivar and its parental lines.

    PubMed

    Wang, Weiwei; Meng, Bo; Ge, Xiaomeng; Song, Shuhui; Yang, Yue; Yu, Xiaomin; Wang, Liguo; Hu, Songnian; Liu, Siqi; Yu, Jun

    2008-11-01

    Elite rice hybrids, when compared to their parental lines, exhibit increased yield and other favorable agronomical traits, such as pathogen- and water-stress resistances, which are described as heterosis, and the molecular mechanism of heterosis remains to be elucidated. Since genomic sequences of the paternal (9311) and maternal lines (P64S) of a major rice hybrid variety LYP9 (Liang-You-Pei-Jiu) were acquired recently, we performed a proteomic study on mature embryos of this hybrid triad based on 2-DE and MALDI-TOF MS analyses, and identified 54 differentially expressed proteins involved in major biological processes including nutrient reservoir, response to stress, and metabolism. We observed that most of the storage proteins exhibit overdominance and stress-induced proteins display additivity. We compared proteomic results with transcriptomic data generated from the same embryo samples and found 28 candidate heterosis-associated genes shared by the two datasets. We further traced back to their genomic structures including protein-coding and regulatory regions and found that most of these genes have multiple copies in rice genomes as paralogous genes. Based on alignment of coding and regulation regions, we found that most of the differentially expressed genes at both protein and RNA levels are recent gene duplicates (paralogous genes) with relative little difference in protein-coding regions between orthologous genes (between genes of the two parental genomes) as compared to regulatory regions that harbor numerous indels and base substitutions.

  9. Proteome-wide quantitative multiplexed profiling of protein expression: carbon-source dependency in Saccharomyces cerevisiae

    PubMed Central

    Paulo, Joao A.; O’Connell, Jeremy D.; Gaun, Aleksandr; Gygi, Steven P.

    2015-01-01

    The global proteomic alterations in the budding yeast Saccharomyces cerevisiae due to differences in carbon sources can be comprehensively examined using mass spectrometry–based multiplexing strategies. In this study, we investigate changes in the S. cerevisiae proteome resulting from cultures grown in minimal media using galactose, glucose, or raffinose as the carbon source. We used a tandem mass tag 9-plex strategy to determine alterations in relative protein abundance due to a particular carbon source, in triplicate, thereby permitting subsequent statistical analyses. We quantified more than 4700 proteins across all nine samples; 1003 proteins demonstrated statistically significant differences in abundance in at least one condition. The majority of altered proteins were classified as functioning in metabolic processes and as having cellular origins of plasma membrane and mitochondria. In contrast, proteins remaining relatively unchanged in abundance included those having nucleic acid–related processes, such as transcription and RNA processing. In addition, the comprehensiveness of the data set enabled the analysis of subsets of functionally related proteins, such as phosphatases, kinases, and transcription factors. As a resource, these data can be mined further in efforts to understand better the roles of carbon source fermentation in yeast metabolic pathways and the alterations observed therein, potentially for industrial applications, such as biofuel feedstock production. PMID:26399295

  10. Temporal regulation of proteome profile in the fruit fly, Drosophila melanogaster.

    PubMed

    Subramanian, Perumal; Jayapalan, Jaime J; Abdul-Rahman, Puteri S; Arumugam, Manjula; Hashim, Onn H

    2016-01-01

    Background. Diurnal rhythms of protein synthesis controlled by the biological clock underlie the rhythmic physiology in the fruit fly, Drosophila melanogaster. In this study, we conducted a proteome-wide investigation of rhythmic protein accumulation in D. melanogaster. Materials and Methods. Total protein collected from fly samples harvested at 4 h intervals over the 24 h period were subjected to two-dimensional gel electrophoresis, trypsin digestion and MS/MS analysis. Protein spots/clusters were identified with MASCOT search engine and Swiss-Prot database. Expression of proteins was documented as percentage of volume contribution using the Image Master 2D Platinum software. Results. A total of 124 protein spots/clusters were identified using MS/MS analysis. Significant variation in the expression of 88 proteins over the 24-h period was observed. A relatively higher number of proteins was upregulated during the night compared to the daytime. The complexity of temporal regulation of the D. melanogaster proteome was further reflected from functional annotations of the differently expressed proteins, with those that were upregulated at night being restricted to the heat shock proteins and proteins involved in metabolism, muscle activity, protein synthesis/folding/degradation and apoptosis, whilst those that were overexpressed in the daytime were apparently involved in metabolism, muscle activity, ion-channel/cellular transport, protein synthesis/folding/degradation, redox homeostasis, development and transcription. Conclusion. Our data suggests that a wide range of proteins synthesized by the fruit fly, D. melanogaster, is under the regulation of the biological clock.

  11. Quantitative Proteomic Profiling of Low-Dose Ionizing Radiation Effects in a Human Skin Model

    PubMed Central

    Hengel, Shawna M.; Aldrich, Joshua T.; Waters, Katrina M.; Pasa-Tolic, Ljiljana; Stenoien, David L.

    2014-01-01

    To assess responses to low-dose ionizing radiation (LD-IR) exposures potentially encountered during medical diagnostic procedures, nuclear accidents or terrorist acts, a quantitative proteomic approach was used to identify changes in protein abundance in a reconstituted human skin tissue model treated with 0.1 Gy of ionizing radiation. To improve the dynamic range of the assay, subcellular fractionation was employed to remove highly abundant structural proteins and to provide insight into radiation-induced alterations in protein localization. Relative peptide quantification across cellular fractions, control and irradiated samples was performing using 8-plex iTRAQ labeling followed by online two-dimensional nano-scale liquid chromatography and high resolution MS/MS analysis. A total of 107 proteins were detected with statistically significant radiation-induced change in abundance (>1.5 fold) and/or subcellular localization compared to controls. The top biological pathways identified using bioinformatics include organ development, anatomical structure formation and the regulation of actin cytoskeleton. From the proteomic data, a change in proteolytic processing and subcellular localization of the skin barrier protein, filaggrin, was identified, and the results were confirmed by western blotting. This data indicate post-transcriptional regulation of protein abundance, localization and proteolytic processing playing an important role in regulating radiation response in human tissues. PMID:28250387

  12. Identification of proteomic biomarkers predicting prostate cancer aggressiveness and lethality despite biopsy-sampling error

    PubMed Central

    Shipitsin, M; Small, C; Choudhury, S; Giladi, E; Friedlander, S; Nardone, J; Hussain, S; Hurley, A D; Ernst, C; Huang, Y E; Chang, H; Nifong, T P; Rimm, D L; Dunyak, J; Loda, M; Berman, D M; Blume-Jensen, P

    2014-01-01

    Background: Key challenges of biopsy-based determination of prostate cancer aggressiveness include tumour heterogeneity, biopsy-sampling error, and variations in biopsy interpretation. The resulting uncertainty in risk assessment leads to significant overtreatment, with associated costs and morbidity. We developed a performance-based strategy to identify protein biomarkers predictive of prostate cancer aggressiveness and lethality regardless of biopsy-sampling variation. Methods: Prostatectomy samples from a large patient cohort with long follow-up were blindly assessed by expert pathologists who identified the tissue regions with the highest and lowest Gleason grade from each patient. To simulate biopsy-sampling error, a core from a high- and a low-Gleason area from each patient sample was used to generate a ‘high' and a ‘low' tumour microarray, respectively. Results: Using a quantitative proteomics approach, we identified from 160 candidates 12 biomarkers that predicted prostate cancer aggressiveness (surgical Gleason and TNM stage) and lethal outcome robustly in both high- and low-Gleason areas. Conversely, a previously reported lethal outcome-predictive marker signature for prostatectomy tissue was unable to perform under circumstances of maximal sampling error. Conclusions: Our results have important implications for cancer biomarker discovery in general and development of a sampling error-resistant clinical biopsy test for prediction of prostate cancer aggressiveness. PMID:25032733

  13. Proteomic profiling reveals dopaminergic regulation of progenitor cell functions of goldfish radial glial cells in vitro.

    PubMed

    Xing, Lei; Martyniuk, Christopher J; Esau, Crystal; Da Fonte, Dillon F; Trudeau, Vance L

    2016-07-20

    proteome on a large scale in a vertebrate species. These data provide novel insight into glial protein networks that are associated with neuroendocrine function and neurogenesis in the teleost brain. While the role of radial glial cells in organizing brain structure and neurogenesis has been well studied, protein profiling experiments in this unique cell type has not been conducted. This study is the first to profile the proteome of goldfish radial glial cells in culture and to study the regulation of progenitor functions of radial glial cells by the neurotransmitter dopamine. This study provides the foundation for molecular network analysis in fish radial glial cells, and identifies cellular processes and signaling pathways in these cells with roles in neurogenesis and neuroendocrine function. Lastly, this study begins to characterize signatures and biomarkers for specific neuroendocrine and neurogenesis disruptors. Copyright © 2016 Elsevier B.V. All rights reserved.

  14. Integrated Left Ventricular Global Transcriptome and Proteome Profiling in Human End-Stage Dilated Cardiomyopathy

    PubMed Central

    Kaya, Namik; Muiya, Nzioka P.; AlHarazi, Olfat; Shinwari, Zakia; Andres, Editha

    2016-01-01

    Aims The disease pathways leading to idiopathic dilated cardiomyopathy (DCM) are still elusive. The present study investigated integrated global transcriptional and translational changes in human DCM for disease biomarker discovery. Methods We used identical myocardial tissues from five DCM hearts compared to five non-failing (NF) donor hearts for both transcriptome profiling using the ABI high-density oligonucleotide microarrays and proteome expression with One-Dimensional Nano Acquity liquid chromatography coupled with tandem mass spectrometry on the Synapt G2 system. Results We identified 1262 differentially expressed genes (DEGs) and 269 proteins (DEPs) between DCM cases and healthy controls. Among the most significantly upregulated (>5-fold) proteins were GRK5, APOA2, IGHG3, ANXA6, HSP90AA1, and ATP5C1 (p< 0.01). On the other hand, the most significantly downregulated proteins were GSTM5, COX17, CAV1 and ANXA3. At least ten entities were concomitantly upregulated on the two analysis platforms: GOT1, ALDH4A1, PDHB, BDH1, SLC2A11, HSP90AA1, HSP90AB1, H2AFV, HSPA5 and NDUFV1. Gene ontology analyses of DEGs and DEPs revealed significant overlap with enrichment of genes/proteins related to metabolic process, biosynthetic process, cellular component organization, oxidative phosphorylation, alterations in glycolysis and ATP synthesis, Alzheimer’s disease, chemokine-mediated inflammation and cytokine signalling pathways. Conclusion The concomitant use of transcriptome and proteome expression to evaluate global changes in DCM has led to the identification of sixteen commonly altered entities as well as novel genes, proteins and pathways whose cardiac functions have yet to be deciphered. This data should contribute towards better management of the disease. PMID:27711126

  15. Proteomic profile of carbonylated proteins in rat liver: discovering possible mechanisms for tetracycline-induced steatosis.

    PubMed

    Deng, Zhenglu; Yan, Siyu; Hu, Hui; Duan, Zhigui; Yin, Lanxuan; Liao, Shenke; Sun, Yubai; Yin, Dazhong; Li, Guolin

    2015-01-01

    To investigate biochemical mechanisms for the tetracycline-induced steatosis in rats, targeted proteins of oxidative modification were profiled. The results showed that tetracycline induced lipid accumulation, oxidative stress, and cell viability decline in HepG2 cells only under the circumstances of palmitic acid overload. Tetracycline administration in rats led to significant decrement in blood lipids, while resulted in more than four times increment in intrahepatic triacylglycerol and typical microvesicular steatosis in the livers. The triacylglycerol levels were positively correlated with oxidative stress. Proteomic profiles of carbonylated proteins revealed 26 targeted proteins susceptible to oxidative modification and most of them located in mitochondria. Among them, the long-chain specific acyl-CoA dehydrogenase was one of the key enzymes regulating fatty acid β-oxidation. Oxidative modification of the enzyme in the tetracycline group depressed its enzymatic activity. In conclusion, the increased influx of lipid into the livers is the first hit of tetracycline-induced microvesicular steatosis. Oxidative stress is an essential part of the second hit, which may arise from the lipid overload and attack a series of functional proteins, aggravating the development of steatosis. The 26 targeted proteins revealed here provide a potential direct link between oxidative stress and tetracycline-induced steatosis. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. Quantitative transcriptome, proteome, and sulfur metabolite profiling of the Saccharomyces cerevisiae response to arsenite.

    PubMed

    Thorsen, Michael; Lagniel, Gilles; Kristiansson, Erik; Junot, Christophe; Nerman, Olle; Labarre, Jean; Tamás, Markus J

    2007-06-19

    Arsenic is ubiquitously present in nature, and various mechanisms have evolved enabling cells to evade toxicity and acquire tolerance. Herein, we explored how Saccharomyces cerevisiae (budding yeast) respond to trivalent arsenic (arsenite) by quantitative transcriptome, proteome, and sulfur metabolite profiling. Arsenite exposure affected transcription of genes encoding functions related to protein biosynthesis, arsenic detoxification, oxidative stress defense, redox maintenance, and proteolytic activity. Importantly, we observed that nearly all components of the sulfate assimilation and glutathione biosynthesis pathways were induced at both gene and protein levels. Kinetic metabolic profiling evidenced a significant increase in the pools of sulfur metabolites as well as elevated cellular glutathione levels. Moreover, the flux in the sulfur assimilation pathway as well as the glutathione synthesis rate strongly increased with a concomitant reduction of sulfur incorporation into proteins. By combining comparative genomics and molecular analyses, we pinpointed transcription factors that mediate the core of the transcriptional response to arsenite. Taken together, our data reveal that arsenite-exposed cells channel a large part of assimilated sulfur into glutathione biosynthesis, and we provide evidence that the transcriptional regulators Yap1p and Met4p control this response in concert.

  17. Differential lysine acetylation profiles of Erwinia amylovora strains revealed by proteomics.

    PubMed

    Wu, Xia; Vellaichamy, Adaikkalam; Wang, Dongping; Zamdborg, Leonid; Kelleher, Neil L; Huber, Steven C; Zhao, Youfu

    2013-02-21

    Protein lysine acetylation (LysAc) has recently been demonstrated to be widespread in E. coli and Salmonella, and to broadly regulate bacterial physiology and metabolism. However, LysAc in plant pathogenic bacteria is largely unknown. Here we first report the lysine acetylome of Erwinia amylovora, an enterobacterium causing serious fire blight disease of apples and pears. Immunoblots using generic anti-lysine acetylation antibodies demonstrated that growth conditions strongly affected the LysAc profiles in E. amylovora. Differential LysAc profiles were also observed for two E. amylovora strains, known to have differential virulence in plants, indicating translational modification of proteins may be important in determining virulence of bacterial strains. Proteomic analysis of LysAc in two E. amylovora strains identified 141 LysAc sites in 96 proteins that function in a wide range of biological pathways. Consistent with previous reports, 44% of the proteins are involved in metabolic processes, including central metabolism, lipopolysaccharide, nucleotide and amino acid metabolism. Interestingly, for the first time, several proteins involved in E. amylovora virulence, including exopolysaccharide amylovoran biosynthesis- and type III secretion-associated proteins, were found to be lysine acetylated, suggesting that LysAc may play a major role in bacterial virulence. Comparative analysis of LysAc sites in E. amylovora and E. coli further revealed the sequence and structural commonality for LysAc in the two organisms. Collectively, these results reinforce the notion that LysAc of proteins is widespread in bacterial metabolism and virulence.

  18. Comparative analysis of proteomic profiles between endometrial caruncular and intercaruncular areas in ewes during the peri-implantation period

    PubMed Central

    2013-01-01

    The endometrium of sheep consists of plenty of raised aglandular areas called caruncular (C), and intensely glandular intercaruncular areas (IC). In order to better understand the endometrium involved mechanisms of implantation, we used LC-MS/MS technique to profile the proteome of ovine endometrial C areas and IC areas separately during the peri-implantation period, and then compared the proteomic profiles between these two areas. We successfully detected 1740 and 1813 proteins in C areas and IC areas respectively. By comparing the proteome of these two areas, we found 170 differentially expressed proteins (DEPs) (P < 0.05), functional bioinformatics analysis showed these DEPs were mainly involved in growth and remodeling of endometrial tissue, cell adhesion and protein transport, and so on. Our study, for the first time, provided a proteomic reference for elucidating the differences between C and IC areas, as an integrated function unit respectively, during the peri-implantation period. The results could help us to better understand the implantation in the ewes. In addition, we established a relatively detailed protein database of ovine endometrium, which provide a unique reference for further studies. PMID:24093944

  19. Sample clean-up strategies for ESI mass spectrometry applications in bottom-up proteomics: Trends from 2012 to 2016.

    PubMed

    Tubaon, Ria Marni; Haddad, Paul R; Quirino, Joselito P

    2017-03-08

    Bottom-up proteomics is a mass spectrometric (MS)-based approach for the characterization of peptides obtained from in-solution protein digestion. MS is favored over other methods for peptide and protein analysis because of its better sensitivity and high throughput. Inorganic ions and surfactants present in the sample or produced during tryptic digestion are detrimental in MS analysis and affect the proteome data, thus sample preparation for removal of these unwanted components has become essential. Here, we review 48 research papers on strategies for removal of salts and surfactants (in particular, SDS) prior to ESI-MS analysis in bottom-up proteomics from 2012 to 2016. The strategies were mostly based on SPE and membrane-based filter-aided sample preparation for salt and SDS removal, respectively. Some known limitations of SPE and filter-aided sample preparation procedures are that they can be time consuming, laborious, and require the use of organic solvents before a concentrated extract suitable for analysis is obtained. The development of faster analytical methods by reducing the sample preparation time and thereby, increasing sample throughput, and in a solvent-less and membrane-less operation, is a significant contribution to proteome research. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  20. Open Tubular Lab-On-Column/Mass Spectrometry for Targeted Proteomics of Nanogram Sample Amounts

    PubMed Central

    Hustoft, Hanne Kolsrud; Vehus, Tore; Brandtzaeg, Ole Kristian; Krauss, Stefan; Greibrokk, Tyge; Wilson, Steven Ray; Lundanes, Elsa

    2014-01-01

    A novel open tubular nanoproteomic platform featuring accelerated on-line protein digestion and high-resolution nano liquid chromatography mass spectrometry (LC-MS) has been developed. The platform features very narrow open tubular columns, and is hence particularly suited for limited sample amounts. For enzymatic digestion of proteins, samples are passed through a 20 µm inner diameter (ID) trypsin + endoproteinase Lys-C immobilized open tubular enzyme reactor (OTER). Resulting peptides are subsequently trapped on a monolithic pre-column and transferred on-line to a 10 µm ID porous layer open tubular (PLOT) liquid chromatography LC separation column. Wnt/ß-catenein signaling pathway (Wnt-pathway) proteins of potentially diagnostic value were digested+detected in targeted-MS/MS mode in small cell samples and tumor tissues within 120 minutes. For example, a potential biomarker Axin1 was identifiable in just 10 ng of sample (protein extract of ∼1,000 HCT15 colon cancer cells). In comprehensive mode, the current OTER-PLOT set-up could be used to identify approximately 1500 proteins in HCT15 cells using a relatively short digestion+detection cycle (240 minutes), outperforming previously reported on-line digestion/separation systems. The platform is fully automated utilizing common commercial instrumentation and parts, while the reactor and columns are simple to produce and have low carry-over. These initial results point to automated solutions for fast and very sensitive MS based proteomics, especially for samples of limited size. PMID:25222838

  1. An off-line high pH reversed-phase fractionation and nano-liquid chromatography-mass spectrometry method for global proteomic profiling of cell lines.

    PubMed

    Wang, Hang; Sun, Shengnan; Zhang, Yi; Chen, Si; Liu, Ping; Liu, Bin

    2015-01-01

    Liquid chromatography coupled to tandem mass spectrometry (LC/MS/MS) and first-dimensional fractionation is widely used for reducing sample complexity in large-scale proteomic profiling experiments. However, the limited number of proteins identified and the relatively long running time are a barrier to the successful application of this approach. In this study, off-line high pH reversed-phase fractionation (RPF) was combined with nano-LC-MS/MS in order to develop an improved method for global proteomic profiling of different cell lines. In the first dimensional reverse phase HPLC separation, 300 μg of digested cell protein was separated into 78 fractions under high pH conditions and condensed into 26 fractions for the second nano-LC-MS/MS analysis at low pH. The chromatographic conditions for the first and second steps were optimized, and the accuracy and reproducibility of protein quantification were investigated with an average Pearson correlation coefficient of 0.94. The method was then applied in the identification of proteins in six common cell lines (DMS, MFM, HepG2, U2OS, 293T and yeast), which resulted in identification of 7300-8500 and 8956 proteins in heavy/light labeled and label-free cell samples, respectively, in 1.5 days. The performance of the developed method was compared with isoelectric focusing (IEF)-nano-LC-MS/MS and the previously reported method; and off-line high pH RPF-nano-LC-MS/MS proved advantageous in terms of the number of proteins identified and the analytical time needed to achieve a successful global proteomic profiling outcome. The RPF-nano-LC-MS/MS method identified more proteins from low abundance (150 μg) samples with an average sequence coverage for each cell line of 23.4-35.1%. RPF-nano-LC-MS/MS may therefore be an efficient alternative tool for achieving improved proteomic coverage of multiple cell lines. Copyright © 2014 Elsevier B.V. All rights reserved.

  2. Standardization of a sample preparation and analytical workflow for proteomics of archival endometrial cancer tissue.

    PubMed

    Alkhas, Addie; Hood, Brian L; Oliver, Kate; Teng, Pang-Ning; Oliver, Julie; Mitchell, David; Hamilton, Chad A; Maxwell, G Larry; Conrads, Thomas P

    2011-11-04

    The goal of the present study was to establish a standard operating procedure for mass spectrometry (MS)-based proteomic analysis of laser microdissected (LMD) formalin-fixed, paraffin-embedded (FFPE) uterine tissue. High resolution bioimage analysis of a large endometrial cancer tissue microarray immunostained for the breast cancer type 1 susceptibility protein enabled precise counting of cells to establish that there is an average of 600 cells/nL of endometrial cancer tissue. We sought to characterize the peptide recovery from various volumes of tissue gathered by LMD and processed/digested using the present methodology. We observed a nearly linear increase in peptide recovery amount with increasing tissue volume dissected. There was little discernible difference in the peptide recovery from stromal versus malignant epithelium, and there was no apparent difference in the day-to-day recovery. This methodology reproducibly results in 100 ng of digested peptides per nL of endometrial tissue, or ∼25 pg peptides/endometrial cancer cell. Results from liquid chromatography (LC)-MS/MS experiments to assess the impact of total peptide load on column on the total number of peptides and proteins identified from FFPE tissue digests prepared with the present methodology indicate a demonstrable increase in the total number of peptides identified up to 1000 ng, beyond which diminishing returns were observed. Furthermore, we observed no impact on the peptide identification rates from analyses of equivalent peptide amounts derived from lower volume LMD samples. These results show that this single-tube collection-to-injection proteomics (CTIP) workflow represents a straightforward, scalable, and highly reliable methodology for sample preparation to enable high throughput LMD-MS analysis of tissues derived from biopsy or surgery.

  3. Evaluation of FASP, SP3 and iST Protocols for Proteomic Sample Preparation in the Low Microgram Range.

    PubMed

    Sielaff, Malte; Kuharev, Jörg; Bohn, Toszka; Hahlbrock, Jennifer; Bopp, Tobias; Tenzer, Stefan; Distler, Ute

    2017-09-26

    Efficient and reproducible sample preparation is a prerequisite for any robust and sensitive quantitative bottom-up proteomics workflow. Here, we performed an independent comparison between single-pot solid-phase-enhanced sample preparation (SP3), filter-aided sample preparation (FASP) and a commercial kit based on the in-StageTip (iST) method. We assessed their performance for the processing of proteomic samples in the low μg-range using varying amounts of HeLa cell lysate (1 µg - 20 μg of total protein). All three workflows showed similar performance for 20 µg of starting material. When handling sample sizes below 10 µg, the number of identified proteins and peptides as well as the quantitative reproducibility and precision drastically dropped in case of FASP. In contrast, SP3 and iST provided high proteome coverage even in the low µg-range. Even when digesting 1 µg of starting material both methods still enabled the identification of over 3,000 proteins and between 25,000 to 30,000 peptides. On average, the quantitative reproducibility between experimental replicates was slightly higher in case of SP3 (R2= 0.97 (SP3); R2= 0.93 (iST)). Applying SP3 towards the characterization of the proteome of FACS-sorted tumor-associated macrophages in the B16 tumor model enabled the quantification of 2,965 proteins and revealed a "mixed" M1/M2 phenotype.

  4. From global proteome profiling to single targeted molecules of follicular fluid and oocyte: contribution to embryo development and IVF outcome.

    PubMed

    Benkhalifa, Moncef; Madkour, Aicha; Louanjli, Noureddine; Bouamoud, Nouzha; Saadani, Brahim; Kaarouch, Ismail; Chahine, Hikmat; Sefrioui, Omar; Merviel, Philippe; Copin, Henri

    2015-08-01

    The development of in vitro fertilization (IVF) techniques for infertility management has led to the investigation of the proteome of follicular fluid and oocyte. In addition, different markers contributing to oocyte maturation and embryo development potential have been reported in the literature. Different techniques were utilized to analyze whole proteome or single protein markers in follicular fluid and oocytes, particularly in animal models. Data from several studies have generated large amounts of information, however, an ideal profile to predict the best oocytes and embryos suitable for implantation are still to be uncovered. The identification of such profiles and markers from follicular fluid, oocytes and endometrium should help scientists and clinicians develop better strategies to improving clinical outcome of IVF cycles.

  5. Genomic and Proteomic Profiling Reveals Reduced Mitochondrial Function and Disruption of the Neuromuscular Junction Driving Rat Sarcopenia

    PubMed Central

    Ibebunjo, Chikwendu; Chick, Joel M.; Kendall, Tracee; Eash, John K.; Li, Christine; Zhang, Yunyu; Vickers, Chad; Wu, Zhidan; Clarke, Brian A.; Shi, Jun; Cruz, Joseph; Fournier, Brigitte; Brachat, Sophie; Gutzwiller, Sabine; Ma, QiCheng; Markovits, Judit; Broome, Michelle; Steinkrauss, Michelle; Skuba, Elizabeth; Galarneau, Jean-Rene; Gygi, Steven P.

    2013-01-01

    Molecular mechanisms underlying sarcopenia, the age-related loss of skeletal muscle mass and function, remain unclear. To identify molecular changes that correlated best with sarcopenia and might contribute to its pathogenesis, we determined global gene expression profiles in muscles of rats aged 6, 12, 18, 21, 24, and 27 months. These rats exhibit sarcopenia beginning at 21 months. Correlation of the gene expression versus muscle mass or age changes, and functional annotation analysis identified gene signatures of sarcopenia distinct from gene signatures of aging. Specifically, mitochondrial energy metabolism (e.g., tricarboxylic acid cycle and oxidative phosphorylation) pathway genes were the most downregulated and most significantly correlated with sarcopenia. Also, perturbed were genes/pathways associated with neuromuscular junction patency (providing molecular evidence of sarcopenia-related functional denervation and neuromuscular junction remodeling), protein degradation, and inflammation. Proteomic analysis of samples at 6, 18, and 27 months confirmed the depletion of mitochondrial energy metabolism proteins and neuromuscular junction proteins. Together, these findings suggest that therapeutic approaches that simultaneously stimulate mitochondrogenesis and reduce muscle proteolysis and inflammation have potential for treating sarcopenia. PMID:23109432

  6. Comparing ancient DNA survival and proteome content in 69 archaeological cattle tooth and bone samples from multiple European sites.

    PubMed

    Wadsworth, Caroline; Procopio, Noemi; Anderung, Cecilia; Carretero, José-Miguel; Iriarte, Eneko; Valdiosera, Cristina; Elburg, Rengert; Penkman, Kirsty; Buckley, Michael

    2017-03-31

    Ancient DNA (aDNA) is the most informative biomolecule extracted from skeletal remains at archaeological sites, but its survival is unpredictable and its extraction and analysis is time consuming, expensive and often fails. Several proposed methods for better understanding aDNA survival are based upon the characterisation of some aspect of protein survival, but these are typically non-specific; proteomic analyses may offer an attractive method for understanding preservation processes. In this study, in-depth proteomic (LC-Orbitrap-MS/MS) analyses were carried out on 69 archaeological bovine bone and dentine samples from multiple European archaeological sites and compared with mitochondrial aDNA and amino acid racemisation (AAR) data. Comparisons of these data, including estimations of the relative abundances for seven selected non-collagenous proteins, indicate that the survival of aDNA in bone or dentine may correlate with the survival of some proteins, and that proteome complexity is a more useful predictor of aDNA survival than protein abundance or AAR. The lack of a strong correlation between the recovery of aDNA and the proteome abundance may indicate that the survival of aDNA is more closely linked to its ability to associate with bone hydroxyapatite crystals rather than to associate with proteins. Ancient biomolecule survival remains poorly understood, even with great advancements in 'omics' technologies, both in genomics and proteomics. This study investigates the survival of ancient DNA in relation to that of proteins, taking into account proteome complexity and the relative protein abundances to improve our understanding of survival mechanisms. The results show that although protein abundance is not necessarily directly related to aDNA survival, proteome complexity appears to be. Copyright © 2017. Published by Elsevier B.V.

  7. Analysis of Biostimulated Microbial Communities from Two Field Experiments Reveals Temporal and Spatial Differences in Proteome Profiles

    SciTech Connect

    Callister, Stephen J.; Wilkins, Michael J.; Nicora, Carrie D.; Williams, Kenneth H.; Banfield, Jillian F.; VerBerkmoes, Nathan; Hettich, Robert L.; N'Guessan, A. Lucie; Mouser, Paula; Elifantz, H.; Smith, Richard D.; Lovley, Derek R.; Lipton, Mary S.; Long, Philip E.

    2010-12-01

    Stimulated by acetate-amendment field experiments conducted in 2007 and 2008, anaerobic microbial populations in the aquifer at the Rifle Integrated Field Research Challenge site in Colorado reduced mobile U(VI) to insoluble U(IV). During this period, planktonic biomass was sampled at various time points and used to quantitatively evaluate proteomes, both spatially and temporally to study the dynamics of the microbial community proteome dynamics in relationship to geochemical measurements. As there were no comprehensive genome sequence data available at the time, we systematically evaluated different organisms to generate a "pseudo-metagenome" for proteomics analyses. Proteomics results supported the dominance of Geobacteraceae during biostimulation and revealed a shift from iron reduction to sulfate reduction, evidenced by changes in community membership. Because U(VI) is reduced at a lower rate during sulfate reduction, detecting this shift is important to maintaining the maximum rate of U(VI) reduction. In addition, the comparison of proteome measurements made at the end of the 2007 field experiment to the 2008 field experiment revealed a modified community structure. Importantly, the failure of a community to rebound following the cessation of biostimulation needs to be included in long-term remediation strategies.

  8. Mars Sample Return: The Value of Depth Profiles

    NASA Technical Reports Server (NTRS)

    Hausrath, E. M.; Navarre-Sitchler, A. K.; Moore, J.; Sak, P. B.; Brantley, S. L.; Golden, D. C.; Sutter, B.; Schroeder, C.; Socki, R.; Morris, R. V.; hide

    2008-01-01

    Sample return from Mars offers the promise of data from Martian materials that have previously only been available from meteorites. Return of carefully selected samples may yield more information about the history of water and possible habitability through Martian history. Here we propose that samples collected from Mars should include depth profiles of material across the interface between weathered material on the surface of Mars into unweathered parent rock material. Such profiles have the potential to yield chemical kinetic data that can be used to estimate the duration of water and information about potential habitats on Mars.

  9. Proteomic Profiling and Differential Messenger RNA Expression Correlate HSP27 and Serpin Family B Member 1 to Apical Periodontitis Outcomes.

    PubMed

    Cavalla, Franco; Biguetti, Claudia; Jain, Sameer; Johnson, Cleverick; Letra, Ariadne; Garlet, Gustavo Pompermaier; Silva, Renato Menezes

    2017-09-01

    Understanding protein expression profiles of apical periodontitis may contribute to the discovery of novel diagnostic or therapeutic molecular targets. Periapical tissue samples (n = 5) of patients with lesions characterized as nonhealing were submitted for proteomic analysis. Two differentially expressed proteins (heat shock protein 27 [HSP27] and serpin family B member 1 [SERPINB1]) were selected for characterization, localization by immunofluorescence, and association with known biomarkers of acute inflammatory response in human apical periodontitis (n = 110) and healthy periodontal ligaments (n = 26). Apical periodontitis samples were categorized as stable/inactive (n = 70) or progressive/active (n = 40) based on the ratio of expression of receptor activator of nuclear factor kappa-B ligand (RANKL)/osteoprotegerin (OPG). Next, the expression of HSP27, SERPINB1, C-X-C motif Chemokine Receptor 1 (CXCR1), matrix metalloproteinase 8 (MMP8), myeloperoxidase (MPO), and cathepsin G (CTSG) messenger RNA was evaluated using real-time polymerase chain reaction. Data analysis was performed using the Shapiro-Wilk test, analysis of variance, and the Pearson test. P values <.05 were considered statistically significant. Proteomic analysis revealed 48 proteins as differentially expressed in apical periodontitis compared with a healthy periodontium, with 30 of these proteins found to be expressed in all 4 lesions. The expression of HSP27 and SERPINB1 was ∼2-fold higher in apical periodontitis. Next, an increased expression of HSP27 was detected in epithelial cells, whereas SERPINB1 expression was noted in neutrophils and epithelial cells. HSP27 and SERPINB1 transcripts were highly expressed in stable/inactive lesions (P < .05). Significant negative correlations were found between the expression of HSP27 and SERPINB1 with biomarkers of acute inflammation including CXCR1, MPO, and CTSG. Our data suggest HSP27 and SERPINB1 as potential regulators of the inflammatory

  10. Temporal regulation of proteome profile in the fruit fly, Drosophila melanogaster

    PubMed Central

    Jayapalan, Jaime J.; Abdul-Rahman, Puteri S.; Arumugam, Manjula; Hashim, Onn H.

    2016-01-01

    Background. Diurnal rhythms of protein synthesis controlled by the biological clock underlie the rhythmic physiology in the fruit fly, Drosophila melanogaster. In this study, we conducted a proteome-wide investigation of rhythmic protein accumulation in D. melanogaster. Materials and Methods. Total protein collected from fly samples harvested at 4 h intervals over the 24 h period were subjected to two-dimensional gel electrophoresis, trypsin digestion and MS/MS analysis. Protein spots/clusters were identified with MASCOT search engine and Swiss-Prot database. Expression of proteins was documented as percentage of volume contribution using the Image Master 2D Platinum software. Results. A total of 124 protein spots/clusters were identified using MS/MS analysis. Significant variation in the expression of 88 proteins over the 24-h period was observed. A relatively higher number of proteins was upregulated during the night compared to the daytime. The complexity of temporal regulation of the D. melanogaster proteome was further reflected from functional annotations of the differently expressed proteins, with those that were upregulated at night being restricted to the heat shock proteins and proteins involved in metabolism, muscle activity, protein synthesis/folding/degradation and apoptosis, whilst those that were overexpressed in the daytime were apparently involved in metabolism, muscle activity, ion-channel/cellular transport, protein synthesis/folding/degradation, redox homeostasis, development and transcription. Conclusion. Our data suggests that a wide range of proteins synthesized by the fruit fly, D. melanogaster, is under the regulation of the biological clock. PMID:27257555

  11. Honeybee venom proteome profile of queens and winter bees as determined by a mass spectrometric approach.

    PubMed

    Danneels, Ellen L; Van Vaerenbergh, Matthias; Debyser, Griet; Devreese, Bart; de Graaf, Dirk C

    2015-10-30

    Venoms of invertebrates contain an enormous diversity of proteins, peptides, and other classes of substances. Insect venoms are characterized by a large interspecific variation resulting in extended lists of venom compounds. The venom composition of several hymenopterans also shows different intraspecific variation. For instance, venom from different honeybee castes, more specifically queens and workers, shows quantitative and qualitative variation, while the environment, like seasonal changes, also proves to be an important factor. The present study aimed at an in-depth analysis of the intraspecific variation in the honeybee venom proteome. In summer workers, the recent list of venom proteins resulted from merging combinatorial peptide ligand library sample pretreatment and targeted tandem mass spectrometry realized with a Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR MS/MS). Now, the same technique was used to determine the venom proteome of queens and winter bees, enabling us to compare it with that of summer bees. In total, 34 putative venom toxins were found, of which two were never described in honeybee venoms before. Venom from winter workers did not contain toxins that were not present in queens or summer workers, while winter worker venom lacked the allergen Api m 12, also known as vitellogenin. Venom from queen bees, on the other hand, was lacking six of the 34 venom toxins compared to worker bees, while it contained two new venom toxins, in particularly serine proteinase stubble and antithrombin-III. Although people are hardly stung by honeybees during winter or by queen bees, these newly identified toxins should be taken into account in the characterization of a putative allergic response against Apis mellifera stings.

  12. Honeybee Venom Proteome Profile of Queens and Winter Bees as Determined by a Mass Spectrometric Approach

    PubMed Central

    Danneels, Ellen L.; Van Vaerenbergh, Matthias; Debyser, Griet; Devreese, Bart; de Graaf, Dirk C.

    2015-01-01

    Venoms of invertebrates contain an enormous diversity of proteins, peptides, and other classes of substances. Insect venoms are characterized by a large interspecific variation resulting in extended lists of venom compounds. The venom composition of several hymenopterans also shows different intraspecific variation. For instance, venom from different honeybee castes, more specifically queens and workers, shows quantitative and qualitative variation, while the environment, like seasonal changes, also proves to be an important factor. The present study aimed at an in-depth analysis of the intraspecific variation in the honeybee venom proteome. In summer workers, the recent list of venom proteins resulted from merging combinatorial peptide ligand library sample pretreatment and targeted tandem mass spectrometry realized with a Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR MS/MS). Now, the same technique was used to determine the venom proteome of queens and winter bees, enabling us to compare it with that of summer bees. In total, 34 putative venom toxins were found, of which two were never described in honeybee venoms before. Venom from winter workers did not contain toxins that were not present in queens or summer workers, while winter worker venom lacked the allergen Api m 12, also known as vitellogenin. Venom from queen bees, on the other hand, was lacking six of the 34 venom toxins compared to worker bees, while it contained two new venom toxins, in particularly serine proteinase stubble and antithrombin-III. Although people are hardly stung by honeybees during winter or by queen bees, these newly identified toxins should be taken into account in the characterization of a putative allergic response against Apis mellifera stings. PMID:26529016

  13. Proteome-wide quantitative multiplexed profiling of protein expression: carbon-source dependency in Saccharomyces cerevisiae.

    PubMed

    Paulo, Joao A; O'Connell, Jeremy D; Gaun, Aleksandr; Gygi, Steven P

    2015-11-05

    The global proteomic alterations in the budding yeast Saccharomyces cerevisiae due to differences in carbon sources can be comprehensively examined using mass spectrometry-based multiplexing strategies. In this study, we investigate changes in the S. cerevisiae proteome resulting from cultures grown in minimal media using galactose, glucose, or raffinose as the carbon source. We used a tandem mass tag 9-plex strategy to determine alterations in relative protein abundance due to a particular carbon source, in triplicate, thereby permitting subsequent statistical analyses. We quantified more than 4700 proteins across all nine samples; 1003 proteins demonstrated statistically significant differences in abundance in at least one condition. The majority of altered proteins were classified as functioning in metabolic processes and as having cellular origins of plasma membrane and mitochondria. In contrast, proteins remaining relatively unchanged in abundance included those having nucleic acid-related processes, such as transcription and RNA processing. In addition, the comprehensiveness of the data set enabled the analysis of subsets of functionally related proteins, such as phosphatases, kinases, and transcription factors. As a resource, these data can be mined further in efforts to understand better the roles of carbon source fermentation in yeast metabolic pathways and the alterations observed therein, potentially for industrial applications, such as biofuel feedstock production. © 2015 Paulo et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  14. Optimized Sample Handling Strategy for Metabolic Profiling of Human Feces.

    PubMed

    Gratton, Jasmine; Phetcharaburanin, Jutarop; Mullish, Benjamin H; Williams, Horace R T; Thursz, Mark; Nicholson, Jeremy K; Holmes, Elaine; Marchesi, Julian R; Li, Jia V

    2016-05-03

    Fecal metabolites are being increasingly studied to unravel the host-gut microbial metabolic interactions. However, there are currently no guidelines for fecal sample collection and storage based on a systematic evaluation of the effect of time, storage temperature, storage duration, and sampling strategy. Here we derive an optimized protocol for fecal sample handling with the aim of maximizing metabolic stability and minimizing sample degradation. Samples obtained from five healthy individuals were analyzed to assess topographical homogeneity of feces and to evaluate storage duration-, temperature-, and freeze-thaw cycle-induced metabolic changes in crude stool and fecal water using a (1)H NMR spectroscopy-based metabolic profiling approach. Interindividual variation was much greater than that attributable to storage conditions. Individual stool samples were found to be heterogeneous and spot sampling resulted in a high degree of metabolic variation. Crude fecal samples were remarkably unstable over time and exhibited distinct metabolic profiles at different storage temperatures. Microbial fermentation was the dominant driver in time-related changes observed in fecal samples stored at room temperature and this fermentative process was reduced when stored at 4 °C. Crude fecal samples frozen at -20 °C manifested elevated amino acids and nicotinate and depleted short chain fatty acids compared to crude fecal control samples. The relative concentrations of branched-chain and aromatic amino acids significantly increased in the freeze-thawed crude fecal samples, suggesting a release of microbial intracellular contents. The metabolic profiles of fecal water samples were more stable compared to crude samples. Our recommendation is that intact fecal samples should be collected, kept at 4 °C or on ice during transportation, and extracted ideally within 1 h of collection, or a maximum of 24 h. Fecal water samples should be extracted from a representative amount (∼15 g

  15. The human adrenal gland proteome defined by transcriptomics and antibody-based profiling.

    PubMed

    Bergman, Julia; Botling, Johan; Fagerberg, Linn; Hallström, Björn M; Djureinovic, Dijana; Uhlén, Mathias; Pontén, Fredrik

    2016-11-30

    The adrenal gland is a composite endocrine organ with vital functions that include the synthesis and release of glucocorticoids and catecholamines. To define the molecular landscape that underlies the specific functions of the adrenal gland, we combined a genome-wide transcriptomics approach based on mRNA sequencing of human tissues with immunohistochemistry-based protein profiling on tissue microarrays. Approximately two-thirds of all putative protein coding genes were expressed in the adrenal gland and the analysis identified 253 genes with an elevated pattern of expression in the adrenal gland, with only 37 genes showing a markedly higher expression level (>5-fold) in the adrenal gland compared to 31 other normal human tissue types analyzed. The analyses allowed for an assessment of the relative expression levels for well-known proteins involved in adrenal gland function, but also identified previously poorly characterized proteins in the adrenal cortex, such as FERM domain containing 5 (FRMD5) and protein NOV homolog (NOV). In summary, we provide a global analysis of the adrenal gland transcriptome and proteome, with a comprehensive list of genes with elevated expression in the adrenal gland and spatial information with examples of protein expression patterns for corresponding proteins. These genes and proteins constitute important starting points for an improved understanding of the normal function and pathophysiology of the adrenal glands.

  16. Proteome profile of peritoneal effluents in children on glucose- or icodextrin-based peritoneal dialysis.

    PubMed

    Bruschi, Maurizio; Candiano, Giovanni; Santucci, Laura; Petretto, Andrea; Mangraviti, Salvatore; Canepa, Alberto; Perri, Katia; Ghiggeri, Gian Marco; Verrina, Enrico

    2011-01-01

    We compared the proteome profile of peritoneal effluents obtained with icodextrin (Ico) or glucose (Glu) in paediatric patients and defined the oxido-redox status of proteins. Sixteen patients underwent two 14-h daytime dwells performed on subsequent days with 7.5% Ico and 3.86% Glu solutions. Protein composition was analysed by two-dimensional electrophoresis and mass spectrometry; oxidized products were evaluated by cyanine labelling. Peritoneal transport kinetics of β2-microglobulin and cystatin C was linear for both solutions, but was significantly higher with Ico than with Glu, suggesting a better efficiency for these molecules. There was a linear correlation between total protein removal during Ico and Glu dialysis in the same patient, suggesting that it is a function of peritoneal membrane characteristics. The ratio between proteins removed by Ico and by Glu solutions was higher at low removal rate. Image gel analysis revealed 1064 and 774 spots, respectively, in Ico and Glu solutions; 524 were common, and 314 were higher in Ico than Glu effluents. Analysis of protein oxido-redox status showed a greater amount of oxidized albumin in Ico dialysate that was correlated with lower serum levels. Our results indicate a better efficiency of Ico in removing small proteins. Removal of big proteins and their oxidized isoforms reflects potentially opposite effects. The long-term clinical consequences of removing also potentially important molecules are to be defined.

  17. Alterations of protein profile in zebrafish liver cells exposed to methyl parathion: a membrane proteomics approach.

    PubMed

    Huang, Qingyu; Huang, He-Qing

    2012-03-01

    Methyl parathion (MP) is an extensively used organophosphorus pesticide, which has been associated with a wide spectrum of toxic effects on environmental organisms. The aim of this study is to investigate the alterations of membrane protein profiles in zebrafish liver (ZFL) cell line exposed to MP for 24 h using proteomic approaches. Two-dimensional gel electrophoresis revealed a total of 13 protein spots, whose expression levels were significantly altered by MP. These differential proteins were subjected to matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry analysis, and nine proteins were identified to be membrane proteins, among which seven were up-regulated, while two were down-regulated. In addition, the mRNA levels corresponding to these differential membrane proteins were further analyzed by quantitative real-time PCR. And the differential expression of arginase-2 was specially validated via Western blotting. Regarding the physiological functions, these proteins are involved in molecular chaperon, cytoskeleton system, cell metabolism, signal transduction, transport and hormone receptor respectively, suggesting the complexity of MP-mediated toxicity to ZFL cell. These data could provide useful insights for better understanding the hepatotoxic mechanisms of MP and develop novel protein biomarkers for effectively monitoring MP contamination level in aquatic environment.

  18. Altered protein profile in chronic myeloid leukemia chronic phase identified by a comparative proteomic study.

    PubMed

    Pizzatti, Luciana; Sá, Lílian Ayres; de Souza, Jamison Menezes; Bisch, Paulo Mascarello; Abdelhay, Eliana

    2006-05-01

    Chronic myeloid leukemia is a hematological disorder in which the Ph chromosome is a marker of the disease, detected virtually in all cases. The chimeric transcripts encode a 210-kDa chimeric protein with altered tyrosine kinase activity, responsible for the disease phenotype. In this work, we tried to identify which are the molecular changes common to chronic phase patients, those that represent the chronic phase molecular phenotype. To address this problem we analyzed through a comparative proteomic approach, several CML bone marrow cells protein profile from patients in chronic phase and healthy bone marrow donors. From these results, we identified 31 differentially expressed proteins. Among these proteins, we pointed out c-Myc binding protein 1, 53BP1, Mdm4, OSBP-related protein 3 and Mortalin as putative candidates to BCR-ABL targets in chronic phase. Moreover, we describe for the first time the cytoplasmic protein map from bone marrow cells that helped in the elucidation of the changes we were looking for.

  19. Deciphering the proteomic profile of rice (Oryza sativa) bran: a pilot study.

    PubMed

    Ferrari, Fabio; Fumagalli, Marco; Profumo, Antonella; Viglio, Simona; Sala, Alberto; Dolcini, Lorenzo; Temporini, Caterina; Nicolis, Stefania; Merli, Daniele; Corana, Federica; Casado, Begona; Iadarola, Paolo

    2009-12-01

    The exact knowledge of the qualitative and quantitative protein components of rice bran is an essential aspect to be considered for a better understanding of the functional properties of this resource. Aim of the present investigation was to extract the largest number of rice bran proteins and to obtain their qualitative characterization. For this purpose, three different extraction protocols have been applied either on full-fat or on defatted rice bran. Likewise, to identify the highest number of proteins, MS data collected from 1-DE, 2-DE and gel-free procedures have been combined. These approaches allowed to unambiguously identify 43 proteins that were classified as signalling/regulation proteins (30%), proteins with enzymatic activity (30%), storage proteins (30%), transfer (5%) and structural (5%) proteins. The fact that all extraction and identification procedures have been performed in triplicate with an excellent reproducibility provides a rationale for considering the platform of proteins shown in this study as the potential proteome profile of rice bran. It also represents a source of information to evaluate better the qualities of rice bran as food resource.

  20. Proteomic profiling of small-molecule inhibitors reveals dispensability of MTH1 for cancer cell survival

    PubMed Central

    Kawamura, Tatsuro; Kawatani, Makoto; Muroi, Makoto; Kondoh, Yasumitsu; Futamura, Yushi; Aono, Harumi; Tanaka, Miho; Honda, Kaori; Osada, Hiroyuki

    2016-01-01

    Since recent publications suggested that the survival of cancer cells depends on MTH1 to avoid incorporation of oxidized nucleotides into the cellular DNA, MTH1 has attracted attention as a potential cancer therapeutic target. In this study, we identified new purine-based MTH1 inhibitors by chemical array screening. However, although the MTH1 inhibitors identified in this study targeted cellular MTH1, they exhibited only weak cytotoxicity against cancer cells compared to recently reported first-in-class inhibitors. We performed proteomic profiling to investigate the modes of action by which chemically distinct MTH1 inhibitors induce cancer cell death, and found mechanistic differences among the first-in-class MTH1 inhibitors. In particular, we identified tubulin as the primary target of TH287 and TH588 responsible for the antitumor effects despite the nanomolar MTH1-inhibitory activity in vitro. Furthermore, overexpression of MTH1 did not rescue cells from MTH1 inhibitor–induced cell death, and siRNA-mediated knockdown of MTH1 did not suppress cancer cell growth. Taken together, we conclude that the cytotoxicity of MTH1 inhibitors is attributable to off-target effects and that MTH1 is not essential for cancer cell survival. PMID:27210421

  1. Pancreatic islet proteome profile in Zucker fatty rats chronically treated with a grape seed procyanidin extract.

    PubMed

    Cedó, Lídia; Castell-Auví, Anna; Pallarès, Victor; Ubaida Mohien, Ceereena; Baiges, Isabel; Blay, Mayte; Ardévol, Anna; Pinent, Montserrat

    2012-12-01

    Grape seed procyanidin extract (GSPE) has been reported to modify glucose metabolism and β-cell functionality through its lipid-lowering effects in a diet-induced obesity model. The objective of the present study was to evaluate the effects of chronically administrated GSPE on the proteomic profile of pancreatic islets from Zucker fatty (ZF) rats. An isobaric tag for relative and absolute quantitation (iTRAQ) experiment was conducted and 31 proteins were found to be differentially expressed in ZF rats treated with GSPE compared to untreated ZF rats. Of these proteins, five subcategories of biological processes emerged: hexose metabolic processes, response to hormone stimulus, apoptosis and cell death, translation and protein folding, and macromolecular complex assembly. Gene expression analysis supported the role of the first three biological processes, concluding that GSPE limits insulin synthesis and secretion and modulates factors involved in apoptosis, but these molecular changes are not sufficient to counteract the genetic background of the Zucker model at a physiological level. Copyright © 2012 Elsevier Ltd. All rights reserved.

  2. Proteomic profiling of halloysite clay nanotube exposure in intestinal cell co-culture

    PubMed Central

    Lai, Xianyin; Agarwal, Mangilal; Lvov, Yuri M.; Pachpande, Chetan; Varahramyan, Kody; Witzmann, Frank A.

    2013-01-01

    Halloysite is aluminosilicate clay with a hollow tubular structure with nanoscale internal and external diameters. Assessment of halloysite biocompatibility has gained importance in view of its potential application in oral drug delivery. To investigate the effect of halloysite nanotubes on an in vitro model of the large intestine, Caco-2/HT29-MTX cells in monolayer co-culture were exposed to nanotubes for toxicity tests and proteomic analysis. Results indicate that halloysite exhibits a high degree of biocompatibility characterized by an absence of cytotoxicity, in spite of elevated pro-inflammatory cytokine release. Exposure-specific changes in expression were observed among 4081 proteins analyzed. Bioinformatic analysis of differentially expressed protein profiles suggest that halloysite stimulates processes related to cell growth and proliferation, subtle responses to cell infection, irritation and injury, enhanced antioxidant capability, and an overall adaptive response to exposure. These potentially relevant functional effects warrant further investigation in in vivo models and suggest that chronic or bolus occupational exposure to halloysite nanotubes may have unintended outcomes. PMID:23606564

  3. Comparative proteomic profiles reveal characteristic Mycobacterium tuberculosis proteins induced by cholesterol during dormancy conditions.

    PubMed

    Garcia-Morales, Lazaro; Leon-Solis, Lizbel; Monroy-Muñoz, Irma E; Talavera-Paulin, Moises; Serafin-López, Jeanet; Estrada-Garcia, Iris; Rivera-Gutierrez, Sandra; Cerna-Cortes, Jorge F; Helguera-Repetto, Addy C; Gonzalez-Y-Merchand, Jorge A

    2017-08-01

    Cholesterol has been reported to play an important role during Mycobacterium tuberculosis infection and during its dormant state inside the host. We present the determination of proteomic profiles of M. tuberculosis H37Rv in the presence of cholesterol as the sole carbon source under exponential growth and in two in vitro dormancy phases (NRP1 and NRP2). Using 2D-PAGE, we detected that M. tuberculosis expressed a high diversity of proteins in both exponential and non-replicative phases. We also found that cholesterol was involved in the overexpression of some proteins related to sulfur metabolism (CysA2), electron transport (FixB), cell wall synthesis (Ald), iron storage (BfrB), protein synthesis (Tig and EF-Tu) and dormancy maintenance (HspX and TB 31.7). According to our results we propose that proteins Ald, BfrB, FadA5 and TB31.7 are likely to play a fundamental role during in vitro dormancy of M. tuberculosis in the presence of cholesterol, helping to counteract its intracellular hostile microenvironment.

  4. [Changes in proteome profiles of rat liver microsomes induced by silicon dioxide nanoparticles].

    PubMed

    Tananova, O N; Arianova, E A; Gmoshinskii, I V; Toropygin, I Yu; Khryapova, E V; Trusov, N V; Khotimchenko, S A; Tutel'yan, V A

    2015-01-01

    The effect of daily intragastric administration of an aqueous dispersion of silicon nanoparticles (NPs) (the dose range from 1.0 mg/kg to 100 mg/kg body weight for 28 days) to rats on the proteomic profile of liver microsomes has been investigated by 2D-electrophoresis followed by subsequent mass spectrometry identification. The liver microsomal fraction was isolated by differential centrifugation and its protein composition was analyzed by 2D-polyacrylamide gel electrophoresis. Identification of protein spots was carried out using MALDI-TOF mass spectrometric analysis. The mass spectrometry analysis revealed the protein GRP78 (78 kD glucose-regulated protein precursor), belonging to the family of heat shock proteins. This protein present in animals of the control group was not detected in NP-treated rats of group 2 (1 mg/kg body weight/day) and group 3 (10 mg/kg body weight/day). This protein predominantly localized in the liver cell endoplasmic reticulum and plasma membrane has the chaperone biological activity. Possible mechanisms of the effects of engineered nanoparticles on biosynthetic processes in the body are discussed.

  5. Proteomic profiling of mitochondria: what does it tell us about the ageing brain?

    PubMed

    Ingram, Thomas; Chakrabarti, Lisa

    2016-12-13

    Mitochondrial dysfunction is evident in numerous neurodegenerative and age-related disorders. It has also been linked to cellular ageing, however our current understanding of the mitochondrial changes that occur are unclear. Functional studies have made some progress reporting reduced respiration, dynamic structural modifications and loss of membrane potential, though there are conflicts within these findings. Proteomic analyses, together with functional studies, are required in order to profile the mitochondrial changes that occur with age and can contribute to unravelling the complexity of the ageing phenotype. The emergence of improved protein separation techniques, combined with mass spectrometry analyses has allowed the identification of age and cell-type specific mitochondrial changes in energy metabolism, antioxidants, fusion and fission machinery, chaperones, membrane proteins and biosynthesis pathways. Here, we identify and review recent data from the analyses of mitochondria from rodent brains. It is expected that knowledge gained from understanding age-related mitochondrial changes of the brain should lead to improved biomarkers of normal ageing and also age-related disease progression.

  6. Proteomic profiling of mitochondria: what does it tell us about the ageing brain?

    PubMed Central

    Ingram, Thomas; Chakrabarti, Lisa

    2016-01-01

    Mitochondrial dysfunction is evident in numerous neurodegenerative and age-related disorders. It has also been linked to cellular ageing, however our current understanding of the mitochondrial changes that occur are unclear. Functional studies have made some progress reporting reduced respiration, dynamic structural modifications and loss of membrane potential, though there are conflicts within these findings. Proteomic analyses, together with functional studies, are required in order to profile the mitochondrial changes that occur with age and can contribute to unravelling the complexity of the ageing phenotype. The emergence of improved protein separation techniques, combined with mass spectrometry analyses has allowed the identification of age and cell-type specific mitochondrial changes in energy metabolism, antioxidants, fusion and fission machinery, chaperones, membrane proteins and biosynthesis pathways. Here, we identify and review recent data from the analyses of mitochondria from rodent brains. It is expected that knowledge gained from understanding age-related mitochondrial changes of the brain should lead to improved biomarkers of normal ageing and also age-related disease progression. PMID:27992860

  7. A review on symptoms, treatments protocols, and proteomic profile in sulfur mustard-exposed victims.

    PubMed

    Panahi, Yunes; Abdolghaffari, Amir H; Sahebkar, Amirhossein

    2017-06-28

    Sulfur mustard (SM) as an alkylating and vesicating agent was used for 100 years as a chemical weapon. SM as bi-functional mustard can attacks and alkylates lots of biomolecules. Different cellular mechanism and molecular pathways are responsible for damages to body tissues. Such as DNA damages, oxidative stress, Apoptosis, and inflammation. Sulfur mustard penetrated body organs and induces long term eye, skin, lung, gastrointestinal, urogenital damages and can cause carcinogenic and mutagenic consequences. Currently there is no definitive treatment protocol for SM exposed patients. The goal of treatment is relieving the symptoms with fast healing rate and retrieval of damaged tissues to normal function and appearance in short period of time. Evaluation of proteomics profile in SM-exposed victims has been performed in animal model and human patients. These studies revealed that different protein were involved in the patients with SM damages to skin and lungs. Apolipoprotein A1, type I cytokeratins K14, K16 and K17, S100 calcium-binding protein A8, α1 haptoglobin isoforms, Amyloid A1, albumin, haptoglobin, and keratin isoforms, immunoglobulin kappa chain are defined expressed proteins in the damaged tissues. © 2017 Wiley Periodicals, Inc.

  8. Serum proteomic profiling and haptoglobin polymorphisms in patients with GVHD after allogeneic hematopoietic cell transplantation

    PubMed Central

    McGuirk, Joseph; Hao, Gang; Hou, Weijian; Abhyankar, Sunil; Williams, Casey; Yan, Weisi; Yuan, Jianda; Guan, Xiuqin; Belt, Robert; Dejarnette, Shaun; Wieman, Jeffery; Yan, Ying

    2009-01-01

    We studied serum proteomic profiling in patients with graft versus host disease (GVHD) after allogeneic hematopoietic cell transplantation (allo-HCT) by two-dimensional gel electrophoresis (2-DE) and mass spectrometry analysis. The expression of a group of proteins, haptoglobin (Hp), alpha-1-antitrypsin, apolipoprotein A-IV, serum paraoxonase and Zn-alpha-glycoprotein were increased and the proteins, clusterin precursor, alpha-2-macroglobulin, serum amyloid protein precursor, sex hormone-binding globulin, serotransferrin and complement C4 were decreased in patients with extensive chronic GVHD (cGVHD). Serum haptoglobin (Hp) levels in patients with cGVHD were demonstrated to be statistically higher than in patients without cGVHD and normal controls (p < 0.01). We used immunoblotting and PCR in combination with 2-DE gel image analysis to determine Hp polymorphisms in 25 allo-HCT patients and 16 normal donors. The results demonstrate that patients with cGVHD had a higher incidence of HP 2-2 phenotype (43.8%), in comparison to the patients without cGVHD (0%) and normal donors (18.7%), suggesting the possibility that specific Hp polymorphism may play a role in the development of cGVHD after allo-HCT. In this study, quantitative serum Hp levels were shown to be related to cGVHD development. Further, the data suggest the possibility that specific Hp polymorphisms may be associated with cGVHD development and warrant further investigation. PMID:19379511

  9. Discovery metabolite profiling--forging functional connections between the proteome and metabolome.

    PubMed

    Saghatelian, Alan; Cravatt, Benjamin F

    2005-08-19

    Of primary interest for every enzyme is the identification of its physiological substrates. However, the vast structural diversity of endogenous metabolites, coupled with the overlapping activities of numerous enzymes, makes it difficult to deduce the identity of natural substrates for a given enzyme based on in vitro experiments. To address this challenge, we recently introduced an LC-MS based analytical method termed discovery metabolite profiling (DMP) to evaluate the global metabolic effects of enzyme inactivation in vivo. We have applied DMP to study mice lacking the enzyme fatty acid amide hydrolase (FAAH), which degrades the endocannabinoid family of signaling lipids. DMP identified several previously uncharacterized FAAH substrates, including a structurally novel class of brain lipids that represent conjugates of very long chain fatty acids with the amino acid derivative taurine [N-acyl taurines (NATs)]. These findings show that DMP can establish direct connections between the proteome and metabolome and thus offers a powerful strategy to assign physiological functions to enzymes in the post-genomic era.

  10. MS/MS-based strategies for proteomic profiling of invasive cell structures.

    PubMed

    Havrylov, Serhiy; Park, Morag

    2015-01-01

    Acquired capacity of cancer cells to penetrate through the extracellular matrix of surrounding tissues is a prerequisite for tumour metastatic spread - the main source of cancer-associated mortality. Through combined efforts of many research groups, we are beginning to understand that the ability of cells to invade through the extracellular matrix is a multi-faceted phenomenon supported by variety of specialised protrusive cellular structures, primarily pseudopodia, invadopodia and podosomes. Additionally, secreted extracellular vesicles are being increasingly recognised as important mediators of invasive cell phenotypes and therefore may be considered bona fide invasive cell structures. Dissection of the molecular makings underlying biogenesis and function of all of these structures is crucial to identify novel targets for specific anti-metastatic therapies. Rapid advances and growing accessibility of MS/MS-based protein identification made this family of techniques a suitable and appropriate choice for proteomic profiling of invasive cell structures. In this review, we provide a summary of current progress in the characterisation of protein composition and topology of protein interaction networks of pseudopodia, invadopodia, podosomes and extracellular vesicles, as well as outline challenges and perspectives of the field.

  11. Proteomic profiling of birch (Betula verrucosa) pollen extracts from different origins.

    PubMed

    Erler, Anja; Hawranek, Thomas; Krückemeier, Leif; Asam, Claudia; Egger, Matthias; Ferreira, Fátima; Briza, Peter

    2011-04-01

    Pollen of the European white birch is a major source of spring pollinosis in Europe. Pollen-allergy diagnosis and treatment by specific immunotherapy commonly rely on extracts of natural origin. To gain insight into the protein content and its variability, we evaluated the profile of allergenic and non-allergenic proteins in extracts of pollen from different origins by MS-based proteomics. Aqueous extracts prepared from commercially available Swedish birch pollen, pollen collected from Austrian trees and a commercial skin prick extract were analyzed by 1-DE, 2-DE, immunoblotting and mass spectrometry, resulting in a complete inventory of extractable, disease-relevant pollen proteins. A main focus of this study was on the isoform distribution of Bet v 1, the major allergen of birch pollen. Using a combination of intact mass determination and peptide sequencing, five isoforms (a, b, d, f and j) were unequivocally identified in Swedish and Austrian birch pollen extracts, while the skin prick extract contained only isoforms a, b and d. Using the same methods as for Bet v 1, divergencies in the sequence of birch profilin (Bet v 2), a plant panallergen, were solved. The molecular characterization of pollen extracts is relevant for standardization and development of new reagents for specific immunotherapy.

  12. Proteomic profiling of neuromas reveals alterations in protein composition and local protein synthesis in hyper-excitable nerves

    PubMed Central

    Huang, Hong-Lei; Cendan, Cruz-Miguel; Roza, Carolina; Okuse, Kenji; Cramer, Rainer; Timms, John F; Wood, John N

    2008-01-01

    Neuropathic pain may arise following peripheral nerve injury though the molecular mechanisms associated with this are unclear. We used proteomic profiling to examine changes in protein expression associated with the formation of hyper-excitable neuromas derived from rodent saphenous nerves. A two-dimensional difference gel electrophoresis (2D-DIGE) profiling strategy was employed to examine protein expression changes between developing neuromas and normal nerves in whole tissue lysates. We found around 200 proteins which displayed a >1.75-fold change in expression between neuroma and normal nerve and identified 55 of these proteins using mass spectrometry. We also used immunoblotting to examine the expression of low-abundance ion channels Nav1.3, Nav1.8 and calcium channel α2δ-1 subunit in this model, since they have previously been implicated in neuronal hyperexcitability associated with neuropathic pain. Finally, S35methionine in vitro labelling of neuroma and control samples was used to demonstrate local protein synthesis of neuron-specific genes. A number of cytoskeletal proteins, enzymes and proteins associated with oxidative stress were up-regulated in neuromas, whilst overall levels of voltage-gated ion channel proteins were unaffected. We conclude that altered mRNA levels reported in the somata of damaged DRG neurons do not necessarily reflect levels of altered proteins in hyper-excitable damaged nerve endings. An altered repertoire of protein expression, local protein synthesis and topological re-arrangements of ion channels may all play important roles in neuroma hyper-excitability. PMID:18700027

  13. Gas chromatographic column for the storage of sample profiles

    NASA Technical Reports Server (NTRS)

    Dimandja, J. M.; Valentin, J. R.; Phillips, J. B.

    1994-01-01

    The concept of a sample retention column that preserves the true time profile of an analyte of interest is studied. This storage system allows for the detection to be done at convenient times, as opposed to the nearly continuous monitoring that is required by other systems to preserve a sample time profile. The sample storage column is essentially a gas chromatography column, although its use is not the separation of sample components. The functions of the storage column are the selective isolation of the component of interest from the rest of the components present in the sample and the storage of this component as a function of time. Using octane as a test substance, the sample storage system was optimized with respect to such parameters as storage and readout temperature, flow rate through the storage column, column efficiency and storage time. A 3-h sample profile was collected and stored at 30 degrees C for 20 h. The profile was then retrieved, essentially intact, in 5 min at 130 degrees C.

  14. Proteomic analysis of complex protein samples by MALDI-TOF mass spectrometry.

    PubMed

    Calvano, Cosima Damiana; De Ceglie, Cristina; Zambonin, Carlo G

    2014-01-01

    MALDI MS has become a technique of considerable impact on many fields, from proteomics to lipidomics, including polymer analysis and, more recently, even low molecular weight analytes due to the introduction of matrix-less ionization techniques (e.g., DIOS) or new matrices such as ionic liquids, proton sponges, and metal nanoparticles. However, protein identification by peptide mass fingerprint (PMF) still remains the main routine application. In the last few years, MALDI MS has played an emerging role in food chemistry especially in detection of food adulterations, characterization of food allergens, and investigation of protein structural modifications, induced by various industrial processes that could be detrimental for food quality and safety. Sample handling and pretreatment can be very different depending on the physical state, liquid or solid, of the analyzed matrices. Here, we describe simple protocols for protein extraction and MALDI MS analysis of liquid (milk) and solid (hazelnuts) samples taken as model. A classic approach based on a preliminary SDS gel electrophoresis separation followed by in-gel digestion and a faster approach based on in-solution digestion of whole samples are described and compared.

  15. Method validation for preparing urine samples for downstream proteomic and metabolomic applications.

    PubMed

    Ammerlaan, Wim; Trezzi, Jean-Pierre; Mathay, Conny; Hiller, Karsten; Betsou, Fay

    2014-10-01

    Formal validation of methods for biospecimen processing in the context of accreditation in laboratories and biobanks is lacking. A protocol for processing of a biospecimen (urine) was validated for fitness-for-purpose in terms of key downstream endpoints. Urine processing was optimized for centrifugation conditions on the basis of microparticle counts at room temperature (RT) and at 4°C. The optimal protocol was validated for performance (microparticle counts), and for reproducibility and robustness for centrifugation temperature (4°C vs. RT) and brake speed (soft, medium, hard). Acceptance criteria were based on microparticle counts, cystatin C and creatinine concentrations, and the metabolomic profile. The optimal protocol was a 20-min, 12,000 g centrifugation at 4°C, and was validated for urine collection in terms of microparticle counts. All reproducibility acceptance criteria were met. The protocol was robust for centrifugation at 4°C versus RT for all parameters. The protocol was considered robust overall in terms of brake speeds, although a hard brake gave significantly fewer microparticles than a soft brake. We validated a urine processing method suitable for downstream proteomic and metabolomic applications. Temperature and brake speed can influence analytic results, with 4°C and high brake speed considered optimal. Laboratories and biobanks should ensure these conditions are systematically recorded in the scope of accreditation.

  16. Plasma proteomic profiles from disease-discordant monozygotic twins suggest that molecular pathways are shared in multiple systemic autoimmune diseases*

    PubMed Central

    2011-01-01

    Introduction Although systemic autoimmune diseases (SAID) share many clinical and laboratory features, whether they also share some common features of pathogenesis remains unclear. We assessed plasma proteomic profiles among different SAID for evidence of common molecular pathways that could provide insights into pathogenic mechanisms shared by these diseases. Methods Differential quantitative proteomic analyses (one-dimensional reverse-phase liquid chromatography-mass spectrometry) were performed to assess patterns of plasma protein expression. Monozygotic twins (four pairs discordant for systemic lupus erythematosus, four pairs discordant for juvenile idiopathic arthritis and two pairs discordant for juvenile dermatomyositis) were studied to minimize polymorphic gene effects. Comparisons were also made to 10 unrelated, matched controls. Results Multiple plasma proteins, including acute phase reactants, structural proteins, immune response proteins, coagulation and transcriptional factors, were differentially expressed similarly among the different SAID studied. Multivariate Random Forest modeling identified seven proteins whose combined altered expression levels effectively segregated affected vs. unaffected twins. Among these seven proteins, four were also identified in univariate analyses of proteomic data (syntaxin 17, α-glucosidase, paraoxonase 1, and the sixth component of complement). Molecular pathway modeling indicated that these factors may be integrated through interactions with a candidate plasma biomarker, PON1 and the pro-inflammatory cytokine IL-6. Conclusions Together, these data suggest that different SAID may share common alterations of plasma protein expression and molecular pathways. An understanding of the mechanisms leading to the altered plasma proteomes common among these SAID may provide useful insights into their pathogeneses. PMID:22044644

  17. Proteomic and Glycoproteomic Profilings Reveal That Post-translational Modifications of Toxins Contribute to Venom Phenotype in Snakes.

    PubMed

    Andrade-Silva, Débora; Zelanis, André; Kitano, Eduardo S; Junqueira-de-Azevedo, Inácio L M; Reis, Marcelo S; Lopes, Aline S; Serrano, Solange M T

    2016-08-05

    Snake venoms are biological weapon systems composed of secreted proteins and peptides that are used for immobilizing or killing prey. Although post-translational modifications are widely investigated because of their importance in many biological phenomena, we currently still have little understanding of how protein glycosylation impacts the variation and stability of venom proteomes. To address these issues, here we characterized the venom proteomes of seven Bothrops snakes using a shotgun proteomics strategy. Moreover, we compared the electrophoretic profiles of native and deglycosylated venoms and, in order to assess their subproteomes of glycoproteins, we identified the proteins with affinity for three lectins with different saccharide specificities and their putative glycosylation sites. As proteinases are abundant glycosylated toxins, we examined the effect of N-deglycosylation on their catalytic activities and show that the proteinases of the seven venoms were similarly affected by removal of N-glycans. Moreover, we prospected putative glycosylation sites of transcripts of a B. jararaca venom gland data set and detected toxin family related patterns of glycosylation. Based on our global analysis, we report that Bothrops venom proteomes and glycoproteomes contain a core of components that markedly define their composition, which is conserved upon evolution in parallel to other molecular markers that determine their phylogenetic classification.

  18. The Human Pancreas Proteome Defined by Transcriptomics and Antibody-Based Profiling

    PubMed Central

    Fagerberg, Linn; Hallström, Björn M.; Schwenk, Jochen M.; Uhlén, Mathias; Korsgren, Olle; Lindskog, Cecilia

    2014-01-01

    The pancreas is composed of both exocrine glands and intermingled endocrine cells to execute its diverse functions, including enzyme production for digestion of nutrients and hormone secretion for regulation of blood glucose levels. To define the molecular constituents with elevated expression in the human pancreas, we employed a genome-wide RNA sequencing analysis of the human transcriptome to identify genes with elevated expression in the human pancreas. This quantitative transcriptomics data was combined with immunohistochemistry-based protein profiling to allow mapping of the corresponding proteins to different compartments and specific cell types within the pancreas down to the single cell level. Analysis of whole pancreas identified 146 genes with elevated expression levels, of which 47 revealed a particular higher expression as compared to the other analyzed tissue types, thus termed pancreas enriched. Extended analysis of in vitro isolated endocrine islets identified an additional set of 42 genes with elevated expression in these specialized cells. Although only 0.7% of all genes showed an elevated expression level in the pancreas, this fraction of transcripts, in most cases encoding secreted proteins, constituted 68% of the total mRNA in pancreas. This demonstrates the extreme specialization of the pancreas for production of secreted proteins. Among the elevated expression profiles, several previously not described proteins were identified, both in endocrine cells (CFC1, FAM159B, RBPJL and RGS9) and exocrine glandular cells (AQP12A, DPEP1, GATM and ERP27). In summary, we provide a global analysis of the pancreas transcriptome and proteome with a comprehensive list of genes and proteins with elevated expression in pancreas. This list represents an important starting point for further studies of the molecular repertoire of pancreatic cells and their relation to disease states or treatment effects. PMID:25546435

  19. Protein Profiling in Hepatocellular Carcinoma by Label-Free Quantitative Proteomics in Two West African Populations

    PubMed Central

    Fye, Haddy K. S.; Wright-Drakesmith, Cynthia; Kramer, Holger B.; Camey, Suzi; da Costa, Andre Nogueira; Jeng, Adam; Bah, Alasana; Kirk, Gregory D.; Sharif, Mohamed I. F.; Ladep, Nimzing G.; Okeke, Edith; Hainaut, Pierre; Taylor-Robinson, Simon D.; Kessler, Benedikt M.; Mendy, Maimuna E.

    2013-01-01

    Background Hepatocellular Carcinoma is the third most common cause of cancer related death worldwide, often diagnosed by measuring serum AFP; a poor performance stand-alone biomarker. With the aim of improving on this, our study focuses on plasma proteins identified by Mass Spectrometry in order to investigate and validate differences seen in the respective proteomes of controls and subjects with LC and HCC. Methods Mass Spectrometry analysis using liquid chromatography electro spray ionization quadrupole time-of-flight was conducted on 339 subjects using a pooled expression profiling approach. ELISA assays were performed on four significantly differentially expressed proteins to validate their expression profiles in subjects from the Gambia and a pilot group from Nigeria. Results from this were collated for statistical multiplexing using logistic regression analysis. Results Twenty-six proteins were identified as differentially expressed between the three subject groups. Direct measurements of four; hemopexin, alpha-1-antitrypsin, apolipoprotein A1 and complement component 3 confirmed their change in abundance in LC and HCC versus control patients. These trends were independently replicated in the pilot validation subjects from Nigeria. The statistical multiplexing of these proteins demonstrated performance comparable to or greater than ALT in identifying liver cirrhosis or carcinogenesis. This exercise also proposed preliminary cut offs with achievable sensitivity, specificity and AUC statistics greater than reported AFP averages. Conclusions The validated changes of expression in these proteins have the potential for development into high-performance tests usable in the diagnosis and or monitoring of HCC and LC patients. The identification of sustained expression trends strengthens the suggestion of these four proteins as worthy candidates for further investigation in the context of liver disease. The statistical combinations also provide a novel inroad of

  20. Proteomic profiling of proteins associated with methamphetamine-induced neurotoxicity in different regions of rat brain.

    PubMed

    Li, Xuefeng; Wang, Huijun; Qiu, Pingming; Luo, Hong

    2008-01-01

    It is well documented that methamphetamine (MA) can cause obvious damage to the brain, but the exact mechanism is still unknown. In the present study, proteomic methods of two-dimensional gel electrophoresis in combination with mass spectrometry analysis were used to identify global protein profiles associated with MA-induced neurotoxicity. For the first time, 30 protein spots have been found differentially expressed in different regions of rat brain, including 14 in striatum, 12 in hippocampus and 4 in frontal cortex. The proteins identified by tandem mass spectrometry were Cu, Zn superoxide dismutase, dimethylarginine dimethylaminohydrolase 1, alpha synuclein, ubiquitin-conjugating enzyme E2N, stathmin 1, calcineurin B, cystatin B, subunit of mitochondrial H-ATP synthase, ATP synthase D chain, mitochondrial, NADH dehydrogenase(ubiquinone) Fe-S protein 8, glia maturation factor, beta, Ash-m, neurocalcin delta, myotrophin, profiling IIa, D-dopachrome tautomerase, and brain lipid binding protein. The known functions of these proteins were related to the pathogenesis of MA-induced neurotoxicity, including oxidative stress, degeneration/apoptosis, mitochontrial/energy metabolism and others. Of these proteins, alpha-synuclein was up-regulated, and ATP synthase D chain, mitochondrial was down-regulated in all brain regions. Two proteins, Cu, Zn superoxide dismutase, subunit of mitochondrial H-ATPsynthase were down-regulated and Ubiquitin-conjugating enzyme E2N, NADH dehydrogenase (ubiquinone) Fe-S protein 8 were up-regulated simultaneously in striatum and hippocaltum. The expression of dimethylarginine dimethylaminohydrolase 1 (DDAH 1) increased both in striatum and frontal cortex. The parallel expression patterns of these proteins suggest that the pathogenesis of MA neurotoxicity in different brain regions may share some same pathways.

  1. The human pancreas proteome defined by transcriptomics and antibody-based profiling.

    PubMed

    Danielsson, Angelika; Pontén, Fredrik; Fagerberg, Linn; Hallström, Björn M; Schwenk, Jochen M; Uhlén, Mathias; Korsgren, Olle; Lindskog, Cecilia

    2014-01-01

    The pancreas is composed of both exocrine glands and intermingled endocrine cells to execute its diverse functions, including enzyme production for digestion of nutrients and hormone secretion for regulation of blood glucose levels. To define the molecular constituents with elevated expression in the human pancreas, we employed a genome-wide RNA sequencing analysis of the human transcriptome to identify genes with elevated expression in the human pancreas. This quantitative transcriptomics data was combined with immunohistochemistry-based protein profiling to allow mapping of the corresponding proteins to different compartments and specific cell types within the pancreas down to the single cell level. Analysis of whole pancreas identified 146 genes with elevated expression levels, of which 47 revealed a particular higher expression as compared to the other analyzed tissue types, thus termed pancreas enriched. Extended analysis of in vitro isolated endocrine islets identified an additional set of 42 genes with elevated expression in these specialized cells. Although only 0.7% of all genes showed an elevated expression level in the pancreas, this fraction of transcripts, in most cases encoding secreted proteins, constituted 68% of the total mRNA in pancreas. This demonstrates the extreme specialization of the pancreas for production of secreted proteins. Among the elevated expression profiles, several previously not described proteins were identified, both in endocrine cells (CFC1, FAM159B, RBPJL and RGS9) and exocrine glandular cells (AQP12A, DPEP1, GATM and ERP27). In summary, we provide a global analysis of the pancreas transcriptome and proteome with a comprehensive list of genes and proteins with elevated expression in pancreas. This list represents an important starting point for further studies of the molecular repertoire of pancreatic cells and their relation to disease states or treatment effects.

  2. Global proteome profiling of dental cementum under experimentally-induced apposition.

    PubMed

    Salmon, Cristiane R; Giorgetti, Ana Paula O; Paes Leme, Adriana Franco; Domingues, Romênia R; Sallum, Enilson Antonio; Alves, Marcelo C; Kolli, Tamara N; Foster, Brian L; Nociti, Francisco H

    2016-06-01

    Dental cementum (DC) covers the tooth root and has important functions in tooth attachment and position. DC can be lost to disease, and regeneration is currently unpredictable due to limited understanding of DC formation. This study used a model of experimentally-induced apposition (EIA) in mice to identify proteins associated with new DC formation. Mandibular first molars were induced to super-erupt for 6 and 21days after extracting opposing maxillary molars. Decalcified and formalin-fixed paraffin-embedded mandible sections were prepared for laser capture microdissection. Microdissected protein extracts were analyzed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), and the data submitted to repeated measure ANOVA test (RM-ANOVA, alpha=5%). A total of 519 proteins were identified, with 97 (18.6%) proteins found exclusively in EIA sites and 50 (9.6%) proteins exclusively expressed in control sites. Fifty six (10.7%) proteins were differentially regulated by RM-ANOVA (p<0.05), with 24 regulated by the exclusive effect of EIA (12 proteins) or the interaction between EIA and time (12 proteins), including serpin 1a, procollagen C-endopeptidase enhancer, tenascin X (TNX), and asporin (ASPN). In conclusion, proteomic analysis demonstrated significantly altered protein profile in DC under EIA, providing new insights on DC biology and potential candidates for tissue engineering applications. Dental cementum (DC) is a mineralized tissue that covers the tooth root surface and has important functions in tooth attachment and position. DC and other periodontal tissues can be lost to disease, and regeneration is currently unpredictable due to lack of understanding of DC formation. This study used a model of experimentally-induced apposition (EIA) in mice to promote new cementum formation, followed by laser capture microdissection (LCM) and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) proteomic analysis. This approach identified

  3. Proteomic characterization of intermediate and advanced glycation end-products in commercial milk samples.

    PubMed

    Renzone, Giovanni; Arena, Simona; Scaloni, Andrea

    2015-03-18

    The Maillard reaction consists of a number of chemical processes affecting the structure of the proteins present in foods. We previously accomplished the proteomic characterization of the lactosylation targets in commercial milk samples. Although characterizing the early modification derivatives, this analysis did not describe the corresponding advanced glycation end-products (AGEs), which may be formed from the further oxidation of former ones or by reaction of oxidized sugars with proteins, when high temperatures are exploited. To fill this gap, we have used combined proteomic procedures for the systematic characterization of the lactosylated and AGE-containing proteins from the soluble and milk fat globule membrane fraction of various milk products. Besides to confirm all lactulosyl-lysines described previously, 40 novel lactosylation sites were identified. More importantly, 308 additional intermediate and advanced glyco-oxidation derivatives (including cross-linking adducts) were characterized in 31 proteins, providing the widest qualitative inventory of modified species ascertained in commercial milk samples so far. Amadori adducts with glucose/galactose, their dehydration products, carboxymethyllysine and glyoxal-, 3-deoxyglucosone/3-deoxygalactosone- and 3-deoxylactosone-derived dihydroxyimidazolines and/or hemiaminals were the most frequent derivatives observed. Depending on thermal treatment, a variable number of modification sites was identified within each protein; their number increased with harder food processing conditions. Among the modified proteins, species involved in assisting the delivery of nutrients, defense response against pathogens and cellular proliferation/differentiation were highly affected by AGE formation. This may lead to a progressive decrease of the milk nutritional value, as it reduces the protein functional properties, abates the bioavailability of the essential amino acids and eventually affects food digestibility. These aspects

  4. MStern Blotting–High Throughput Polyvinylidene Fluoride (PVDF) Membrane-Based Proteomic Sample Preparation for 96-Well Plates*

    PubMed Central

    Berger, Sebastian T.; Ahmed, Saima; Muntel, Jan; Cuevas Polo, Nerea; Bachur, Richard; Kentsis, Alex; Steen, Judith; Steen, Hanno

    2015-01-01

    We describe a 96-well plate compatible membrane-based proteomic sample processing method, which enables the complete processing of 96 samples (or multiples thereof) within a single workday. This method uses a large-pore hydrophobic PVDF membrane that efficiently adsorbs proteins, resulting in fast liquid transfer through the membrane and significantly reduced sample processing times. Low liquid transfer speeds have prevented the useful 96-well plate implementation of FASP as a widely used membrane-based proteomic sample processing method. We validated our approach on whole-cell lysate and urine and cerebrospinal fluid as clinically relevant body fluids. Without compromising peptide and protein identification, our method uses a vacuum manifold and circumvents the need for digest desalting, making our processing method compatible with standard liquid handling robots. In summary, our new method maintains the strengths of FASP and simultaneously overcomes one of the major limitations of FASP without compromising protein identification and quantification. PMID:26223766

  5. Serum proteomic analysis identifies sex-specific differences in lipid metabolism and inflammation profiles in adults diagnosed with Asperger syndrome

    PubMed Central

    2014-01-01

    Background The higher prevalence of Asperger Syndrome (AS) and other autism spectrum conditions in males has been known for many years. However, recent multiplex immunoassay profiling studies have shown that males and females with AS have distinct proteomic changes in serum. Methods Here, we analysed sera from adults diagnosed with AS (males = 14, females = 16) and controls (males = 13, females = 16) not on medication at the time of sample collection, using a combination of multiplex immunoassay and shotgun label-free liquid chromatography mass spectrometry (LC-MSE). The main objective was to identify sex-specific serum protein changes associated with AS. Results Multiplex immunoassay profiling led to identification of 16 proteins that were significantly altered in AS individuals in a sex-specific manner. Three of these proteins were altered in females (ADIPO, IgA, APOA1), seven were changed in males (BMP6, CTGF, ICAM1, IL-12p70, IL-16, TF, TNF-alpha) and six were changed in both sexes but in opposite directions (CHGA, EPO, IL-3, TENA, PAP, SHBG). Shotgun LC-MSE profiling led to identification of 13 serum proteins which had significant sex-specific changes in the AS group and, of these, 12 were altered in females (APOC2, APOE, ARMC3, CLC4K, FETUB, GLCE, MRRP1, PTPA, RN149, TLE1, TRIPB, ZC3HE) and one protein was altered in males (RGPD4). The free androgen index in females with AS showed an increased ratio of 1.63 compared to controls. Conclusion Taken together, the serum multiplex immunoassay and shotgun LC-MSE profiling results indicate that adult females with AS had alterations in proteins involved mostly in lipid transport and metabolism pathways, while adult males with AS showed changes predominantly in inflammation signalling. These results provide further evidence that the search for biomarkers or novel drug targets in AS may require stratification into male and female subgroups, and could lead to the development of novel targeted treatment

  6. Proteomic and oxidative stress analysis in human brain samples of Huntington disease.

    PubMed

    Sorolla, Ma Alba; Reverter-Branchat, Gemma; Tamarit, Jordi; Ferrer, Isidre; Ros, Joaquim; Cabiscol, Elisa

    2008-09-01

    Huntington disease (HD) is a neurodegenerative disorder caused by expansion of CAG repeats in exon 1 of the huntingtin gene, affecting initially the striatum and progressively the cortex. This work reports a proteomic analysis of human brain postmortem samples obtained from striatum and cortex of patients with HD compared to samples of age- and sex-matched controls. Antioxidant defense proteins that were strongly induced in striatum, but also detectable in cortex, were identified as peroxiredoxins 1, 2, and 6, as well as glutathione peroxidases 1 and 6. The activities of other antioxidant enzymes such as mitochondrial superoxide dismutase and catalase were also increased in HD. Aconitase, a protein involved in energy metabolism, showed decreased activities in striatum of HD patients. Protein carbonyls, used as markers of oxidative stress, were increased in HD, and glial fibrillary acidic protein, aconitase, gamma-enolase, and creatine kinase B were identified as the main targets. Taken together, these results indicate that oxidative stress and damage to specific macromolecules would participate in the disease progression. Also, these data support the rationale for therapeutic strategies that either potentiate antioxidant defenses or avoid oxidative stress generation to delay disease progression.

  7. Plasma Proteomic</