Yssouf, Amina; Socolovschi, Cristina; Leulmi, Hamza; Kernif, Tahar; Bitam, Idir; Audoly, Gilles; Almeras, Lionel; Raoult, Didier; Parola, Philippe
In the present study, a molecular proteomics (MALDI-TOF/MS) approach was used as a tool for identifying flea vectors. We measured the MS spectra from 38 flea specimens of 5 species including Ctenocephalides felis, Ctenocephalides canis, Archaeopsylla erinacei, Xenopsylla cheopis and Stenoponia tripectinata. A blind test performed with 24 specimens from species included in a library spectral database confirmed that MALDI-TOF/MS is an effective tool for discriminating flea species. Although fresh and 70% ethanol-conserved samples subjected to MALDI-TOF/MS in blind tests were correctly classified, only MS spectra of quality from fresh specimens were sufficient for accurate and significant identification. A cluster analysis highlighted that the MALDI Biotyper can be used for studying the phylogeny of fleas.
Monagas, María; Quintanilla-López, Jesús Eduardo; Gómez-Cordovés, Carmen; Bartolomé, Begoña; Lebrón-Aguilar, Rosa
Proanthocyanidins or condensed tannins are among the most abundant polyphenols compounds in our diet and may play a key role in the prevention of cardiovascular and neurodegenerative diseases and cancer. These antioxidants are widely distributed in the plant kingdom both in food plants and in non-food plants. The biological activity of plant proanthocyanidins depends on their chemical structure and concentration. However, due to their structural diversity and complexity, the qualitative and quantitative analysis of proanthocyanidins is a difficult task. Mass spectrometry has enabled great advances in the characterization of plant proanthocyanidins. Among these techniques, MALDI-TOF MS has proved to be highly suited for the analysis of highly polydisperse and heterogeneous proanthocyanidins. The objective of the present paper was to assess the potential, limitations and future challenges of the analysis of plant proanthocyanidins by MALDI-TOF MS techniques. Firstly, the fundamental of this technique, including modes of operation, advantages and limitations, as well as quantitative and qualitative operations, have been summarized. Applications of MALDI-TOF analysis to plant proanthocyanidins reported in the last decade (1997-2008) have been extensively covered, including the sample preparation protocols and conditions used for proanthocyanidin analysis, as well as the main findings regarding the determination of the structural features of different plant proanthocyanidin types (procyanidins, propelargonidins, prodelphinidins, profisetinidins and prorobinetinidins). Finally, attempts in the assessment of the molecular weight distribution of proanthocyanidins by MALDI-TOF are described.
This investigation aimed to assess whether MALDI-TOF MS analysis of proteomics could be applied to the study of Trichoderma, a fungal genus selected because it includes many species and is phylogenetically well defined. We also investigated whether MALDI-TOF MS analysis of proteomics would reveal ap...
Kafka, Alexandra P; Kleffmann, Torsten; Rades, Thomas; McDowell, Arlene
The development and quality assessment of modern biopharmaceuticals, particularly protein and peptide drugs, requires an array of analytical techniques to assess the integrity of the bioactive molecule during formulation and administration. Mass spectrometry is one of these methods and is particularly suitable for determining chemical modifications of protein and peptide drugs. The emphasis of this review is the identification of covalent interactions between protein and peptide bioactives with polymeric pharmaceutical formulations using mass spectrometry with the main focus on matrix-assisted laser desorption/ionization (MALDI) coupled tandem time-of-flight (TOF/TOF) mass spectrometry (MS). The basics of MALDI TOF MS and collision-induced dissociation (CID)-based ion fragmentation will be explained and applications for qualitative characterization of protein and peptide drugs and their interactions with pharmaceutical polymers will be discussed using three case studies.
Schlebusch, S; Price, G R; Gallagher, R L; Horton-Szar, V; Elbourne, L D H; Griffin, P; Venter, D J; Jensen, S O; Van Hal, S J
The use of MALDI-TOF MS (matrix-assisted laser desorption/ ionization-time of flight mass spectrometry) and WGS (whole genome sequencing) has been described for identification and strain relatedness determination. We describe the complementary use of MALDI-TOF MS and WGS in a VRE (vancomycin-resistant enterococci) outbreak investigation, and discuss some of the challenges with defining strain similarity across these two platforms. Although both assays indicated multiple clusters involved in the outbreak of vancomycin resistant Enterococcus faecium isolates from positive blood cultures of four haematology-oncology patients, the small cohort and discrepancies between findings indicate the limitations of MALDI-TOF MS and the cautious interpretation of MALDI-TOF MS dendrograms during outbreaks. For definitive determination of the evolutionary distance between isolates, WGS can be used.
Lüthje, Petra; Pranada, Arthur B; Carruthers-Lay, Duncan; Desjardins, Marc; Gaillot, Olivier; Wareham, David; Ciesielczuk, Holly; Özenci, Volkan
Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and chromogenic media are widely used in clinical microbiology laboratories to facilitate the rapid selection and identification of pathogens. The aim of this study was to evaluate whether usage of chromogenic media limits the diagnostic performance of MALDI-TOF MS for microbial identification. A total of 386 microorganisms collected and analyzed at five laboratories were included. Isolates were cultured on relevant chromogenic media and non-selective agar plates in parallel and identified using the Bruker MALDI-TOF MS. Among the tested isolates, no misidentification was recorded and there was no medium-related difference in the identification level. However, score values were overall slightly but significantly lower for isolates grown on chromogenic media. In conclusion, the use of chromogenic culture media tested here had no relevant impact on MALDI-TOF MS performance for diagnostic purposes.
van Belkum, Alex; Chatellier, Sonia; Girard, Victoria; Pincus, David; Deol, Parampal; Dunne, Wm Michael
Although classical proteomic approaches are still used regularly in routine clinical diagnostic procedures, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) MS has recently moved into diagnostic microbiology laboratories. MALDI-TOF MS is currently replacing phenotypic microbial identification. Many laboratories now use MALDI-TOF MS for its high efficiency, both from a diagnostic and a cost-per-analysis point of view. The US FDA has now cleared two of the commercially available systems for in vitro diagnostics. This will further spark development of MS applications in antimicrobial susceptibility testing and epidemiology. This review summarizes the state of affairs of MALDI-TOF MS in clinical microbiology; however, this is an active field of research subject to rapid evolution. We emphasize assessment of the clinical relevance and studies focusing on data obtained through comparative analyses of different MALDI-TOF MS instrumentation and multicenter validation studies. The future of MALDI-TOF MS, including antimicrobial susceptibility testing and epidemiological typing, is also highlighted.
Kodana, Masahiro; Tarumoto, Norihito; Kawamura, Tohru; Saito, Taeko; Ohno, Hideaki; Maesaki, Shigefumi; Ikebuchi, Kenji
In comparison to the conventional real-time polymerase chain reaction method (PCR method) or the DNA-DNA hybridization method (DDH method), the utility of NTM identification by the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) method has seldom been reported. In this study, 75 clinical NTM isolates from our hospital between April 2013 and July 2014 were identified and analyzed using PCR, DDH, and MALDI-TOF MS methods, and the results for the MALDI-TOF MS method were compared with the others. Identification at the species level was in agreement for 71 (94.5%) of the 75 isolates. For further details, identification was possible for 23 (95.8%) of 24 Mycobacterium avium, 11 (100%) of 11 Mycobacterium intracellulare, and 1 (50%) of 2 isolates mixed with M. avium and M. intracellulare. Mycobacterium ksansasii, Mycobacterium abscessus, Mycobacterium fortuitum, Mycobacterium gordonae, and Mycobacterium chelonae identified by DDH method were same result by MALDI-TOF MS. Additionally, Mycobacterium mucogenicum, which could not be identified by the DDH method, was identified by the MALDI-TOF MS method. However, two isolates identified as Mycobacterium terrae by DDH method could not be identified by the MALDI-TOF MS method and were determined to be Mycobacterium arupense by 16S ribosomal RNA (rRNA) sequence analysis. The present findings show that, for rare bacterial species, identification is sometimes not possible, but, in most cases, the results of identification by the MALDI-TOF MS method have a high concordance rate with the results of the PCR and DDH methods.
Zárate, Mariela S; Romano, Vanesa; Nievas, Jimena; Smayevsky, Jorgelina
The analysis by MALDI-TOF MS (Matrix-assited laser desorption/ionization time-of-flight mass spectrometry) has become a reference method for the identification of microorganisms in Clinical Microbiology. However, data on some groups of microorganisms are still controversial. The aim of this study is to determine the utility of MALDI-TOF MS for the identification of clinical isolates of anaerobic bacteria. One-hundred and six anaerobic bacteria isolates were analyzed by MALDI-TOF MS and by conventional biochemical tests. In those cases where identification by conventional methodology was not applicable or in the face of discordance between sequencing methodologies, 16 S rRNA gene sequence analysis was performed. The conventional method and MALDI-TOF MS agreed at genus and species level by 95.3 %. Concordance in gram-negative bacilli was 91.4% and 100% among gram-positive bacilli; there was also concordance both in the 8 isolates studied in gram-positive cocci and in the single gram-negative cocci included. The data obtained in this study demonstrate that MALDI-TOF MS offers the possibility of adequate identification of anaerobic bacteria.
Singhal, Neelja; Kumar, Manish; Virdi, Jugsharan Singh
Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is currently being used for rapid and reproducible identification of bacteria, viruses and fungi in clinical microbiological laboratories. However, some studies have also reported the use of MALDI-TOF MS for identification of parasites, like Leishmania, Giardia, Cryptosporidium, Entamoeba, ticks and fleas. The present review collates all the information available on the use of this technique for parasites, in an effort to assess its applicability and the constraints for identification/diagnosis of parasites and diseases caused by them. Though MALDI-TOF MS-based identification of parasites is currently done by reference laboratories only, in future, this promising technology might surely replace/augment molecular methods in clinical parasitology laboratories.
Relloso, María S; Nievas, Jimena; Fares Taie, Santiago; Farquharson, Victoria; Mujica, María T; Romano, Vanesa; Zarate, Mariela S; Smayevsky, Jorgelina
The matrix-assisted laser desorption/ionization time-of-flight mass spectrometry technique known as MALDI-TOF MS is a tool used for the identification of clinical pathogens by generating a protein spectrum that is unique for a given species. In this study we assessed the identification of clinical yeast isolates by MALDI-TOF MS in a university hospital from Argentina and compared two procedures for protein extraction: a rapid method and a procedure based on the manufacturer's recommendations. A short protein extraction procedure was applied in 100 isolates and the rate of correct identification at genus and species level was 98.0%. In addition, we analyzed 201 isolates, previously identified by conventional methods, using the methodology recommended by the manufacturer and there was 95.38% coincidence in the identification at species level. MALDI TOF MS showed to be a fast, simple and reliable tool for yeast identification.
Kostrzewa, Markus; Sparbier, Katrin; Maier, Thomas; Schubert, Sören
MALDI-TOF MS profiling for microorganism detection has already been demonstrated in the 1990s, but has evolved to the first-line identification method in many laboratories just during the past five years. While this application of MALDI-TOF MS has proven its broad applicability, accuracy, robustness, and cost-effectiveness it is of particular interest to expand the capabilities of the mass spectrometric platform. Resistance detection is the most desirable further application of MALDI-TOF MS in microbiology, but maybe also the most challenging. Different approaches have been published regarding diverse antibiotic drugs and distinct microorganism classes. The current review shall give an overview about the developments of the recent years and their potential to get transformed in clinical useful assays in the future.
Kilic, Abdullah; Baysallar, Mehmet
Bloodstream infections are substantial causes of morbidity and mortality worldwide. Staphylococcus species are the most commonly isolated microorganisms from blood cultures in clinical microbiology laboratories. MALDI-TOF MS (Matrix-Assisted Laser Desorption/Ionization Time- of- Flight Mass Spectrometry) system allows the identification of microorganisms directly from positive blood culture bottles. The aim of this study was to evaluate the MALDI-TOF MS system for the identification of staphylococci directly from the positive blood culture bottles which revealed the presence of gram-positive cocci by staining. A total of 96 positive blood culture bottles that yielded gram-positive cocci by Gram stain were evaluated. These blood cultures were obtained from 69 patients between December 2013-February 2014. Conventional methods and BD Phoenix™ automated bacterial identification system (Becton Dickinson, USA) were used for routine identification. The strains were also identified by real-time Taqman PCR (qPCR) which was considered as the reference method. In MALDI-TOF MS method, MALDI Sepsityper™ Kit was used for the bacterial identification and all measurements were carried out by using Microflex LT instrument and FlexControl 3.0 software (Bruker Daltonics, USA). Of 96 culture bottles positive for gram-positive cocci, 90 were correctly identified as staphylococci at genus level with all the three study methods (qPCR, BD Phoenix, Bruker MALDI-TOF MS). The other six samples were identified as Enterococcus faecium (n= 4) and Streptococcus pyogenes (n= 2) by both Phoenix and the MALDI-TOF systems. Of the 90 samples, 87 were identified at the species level (15 S.aureus, 33 S.epidermidis, 29 S.hominis, 10 S.haemolyticus) and three at the genus level by the reference qPCR method. When comparing the results obtained by qPCR and Bruker MALDI-TOF MS, incompatibility was detected for three isolates. Those isolates were identified as S.hominis by qPCR, however two of them were
Pavlovic, Melanie; Wudy, Corinna; Zeller-Peronnet, Veronique; Maggipinto, Marzena; Zimmermann, Pia; Straubinger, Alix; Iwobi, Azuka; Märtlbauer, Erwin; Busch, Ulrich; Huber, Ingrid
Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has recently emerged as a rapid and accurate identification method for bacterial species. Although it has been successfully applied for the identification of human pathogens, it has so far not been well evaluated for routine identification of veterinary bacterial isolates. This study was performed to compare and evaluate the performance of MALDI-TOF MS based identification of veterinary bacterial isolates with commercially available conventional test systems. Discrepancies of both methods were resolved by sequencing 16S rDNA and, if necessary, the infB gene for Actinobacillus isolates. A total of 375 consecutively isolated veterinary samples were collected. Among the 357 isolates (95.2%) correctly identified at the genus level by MALDI-TOF MS, 338 of them (90.1% of the total isolates) were also correctly identified at the species level. Conventional methods offered correct species identification for 319 isolates (85.1%). MALDI-TOF identification therefore offered more accurate identification of veterinary bacterial isolates. An update of the in-house mass spectra database with additional reference spectra clearly improved the identification results. In conclusion, the presented data suggest that MALDI-TOF MS is an appropriate platform for classification and identification of veterinary bacterial isolates.
Zhu, Bingqing; Xiao, Di; Zhang, Huifang; Zhang, Yongchan; Gao, Yuan; Xu, Li; Lv, Jing; Wang, Yingtong; Zhang, Jianzhong; Shao, Zhujun
Nontypable Haemophilus influenzae (NTHi) and Haemophilus haemolyticus exhibit different pathogenicities, but to date, there remains no definitive and reliable strategy for differentiating these strains. In this study, we evaluated matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) as a potential method for differentiating NTHi and H. haemolyticus. The phylogenetic analysis of concatenated 16S rRNA and recombinase A (recA) gene sequences, outer membrane protein P6 gene sequencing and single-gene PCR were used as reference methods. The original reference database (ORD, provided with the Biotyper software) and new reference database (NRD, extended with Chinese strains) were compared for the evaluation of MALDI-TOF MS. Through a search of the ORD, 76.9% of the NTHi (40/52) and none of the H. haemolyticus (0/20) strains were identified at the species level. However, all NTHi and H. haemolyticus strains used for identification were accurately recognized at the species level when searching the NRD. From the dendrogram clustering of the main spectra projections, the Chinese and foreign H. influenzae reference strains were categorized into two distinct groups, and H. influenzae and H. haemolyticus were also separated into two categories. Compared to the existing methods, MALDI-TOF MS has the advantage of integrating high throughput, accuracy and speed. In conclusion, MALDI-TOF MS is an excellent method for differentiating NTHi and H. haemolyticus. This method can be recommended for use in appropriately equipped laboratories.
Rotova, Veronika; Papagiannitsis, Costas C; Skalova, Anna; Chudejova, Katerina; Hrabak, Jaroslav
A comparison of carbapenem molecules for the detection of carbapenemase-producing bacteria by MALDI-TOF MS showed that imipenem exhibited higher sensitivity (97%) and specificity (100%) scores for Pseudomonas aeruginosa than meropenem. However, meropenem was more efficient (98% sensitivity and 100% specificity) against Enterobacteriaceae.
Yssouf, Amina; Almeras, Lionel; Terras, Jérôme; Socolovschi, Cristina; Raoult, Didier; Parola, Philippe
Background Matrix Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) has been shown to be an effective tool for the rapid identification of arthropods, including tick vectors of human diseases. Methodology/Principal Findings The objective of the present study was to evaluate the use of MALDI-TOF MS to identify tick species, and to determine the presence of rickettsia pathogens in the infected Ticks. Rhipicephalus sanguineus and Dermacentor marginatus Ticks infected or not by R. conorii conorii or R. slovaca, respectively, were used as experimental models. The MS profiles generated from protein extracts prepared from tick legs exhibited mass peaks that distinguished the infected and uninfected Ticks, and successfully discriminated the Rickettsia spp. A blind test was performed using Ticks that were laboratory-reared, collected in the field or removed from patients and infected or not by Rickettsia spp. A query against our in-lab arthropod MS reference database revealed that the species and infection status of all Ticks were correctly identified at the species and infection status levels. Conclusions/Significance Taken together, the present work demonstrates the utility of MALDI-TOF MS for a dual identification of tick species and intracellular bacteria. Therefore, MALDI-TOF MS is a relevant tool for the accurate detection of Rickettsia spp in Ticks for both field monitoring and entomological diagnosis. The present work offers new perspectives for the monitoring of other vector borne diseases that present public health concerns. PMID:25659152
Background The genus Brucella contains highly infectious species that are classified as biological threat agents. The timely detection and identification of the microorganism involved is essential for an effective response not only to biological warfare attacks but also to natural outbreaks. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) is a rapid method for the analysis of biological samples. The advantages of this method, compared to conventional techniques, are rapidity, cost-effectiveness, accuracy and suitability for the high-throughput identification of bacteria. Discrepancies between taxonomy and genetic relatedness on the species and biovar level complicate the development of detection and identification assays. Results In this study, the accurate identification of Brucella species using MALDI-TOF-MS was achieved by constructing a Brucella reference library based on multilocus variable-number tandem repeat analysis (MLVA) data. By comparing MS-spectra from Brucella species against a custom-made MALDI-TOF-MS reference library, MALDI-TOF-MS could be used as a rapid identification method for Brucella species. In this way, 99.3% of the 152 isolates tested were identified at the species level, and B. suis biovar 1 and 2 were identified at the level of their biovar. This result demonstrates that for Brucella, even minimal genomic differences between these serovars translate to specific proteomic differences. Conclusions MALDI-TOF-MS can be developed into a fast and reliable identification method for genetically highly related species when potential taxonomic and genetic inconsistencies are taken into consideration during the generation of the reference library. PMID:22192890
Lafri, Ismail; Almeras, Lionel; Bitam, Idir; Caputo, Aurelia; Yssouf, Amina; Forestier, Claire-Lise; Izri, Arezki; Raoult, Didier; Parola, Philippe
Background Phlebotomine sand flies are known to transmit Leishmania parasites, bacteria and viruses that affect humans and animals in many countries worldwide. Precise sand fly identification is essential to prevent phlebotomine-borne diseases. Over the past two decades, progress in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has emerged as an accurate tool for arthropod identification. The objective of the present study was to investigate the usefulness of MALDI-TOF MS as a tool for identifying field-caught phlebotomine. Methodology/Principal Findings Sand flies were captured in four sites in north Algeria. A subset was morphologically and genetically identified. Six species were found in these areas and a total of 28 stored frozen specimens were used for the creation of the reference spectrum database. The relevance of this original method for sand fly identification was validated by two successive blind tests including the morphological identification of 80 new specimens which were stored at -80°C, and 292 unknown specimens, including engorged specimens, which were preserved under different conditions. Intra-species reproducibility and inter-species specificity of the protein profiles were obtained, allowing us to distinguish specimens at the gender level. Querying of the sand fly database using the MS spectra from the blind test groups revealed concordant results between morphological and MALDI-TOF MS identification. However, MS identification results were less efficient for specimens which were engorged or stored in alcohol. Identification of 362 phlebotomine sand flies, captured at four Algerian sites, by MALDI-TOF MS, revealed that the subgenus Larroussius was predominant at all the study sites, except for in M’sila where P. (Phlebotomus) papatasi was the only sand fly species detected. Conclusion The present study highlights the application of MALDI-TOF MS for monitoring sand fly fauna captured in the field
Wang, Sheng; Wei, Bangguo; Yang, Pengyuan; Chen, Gang
In this report, alternating current-assisted on-plate proteolysis has been developed for rapid peptide mapping. Protein solutions containing trypsin were allowed to digest directly on the spots of a stainless steel MALDI plate with the assistance of low-voltage alternating current electricity. Alternating current (AC) was allowed to pass through the protein solutions via the MALDI plate and a platinum disc electrode. The feasibility and performance of the novel proteolysis approach were investigated by the digestion of BSA and cytochrome c (Cyt-c). It was demonstrated that AC substantially enhanced the efficiency of proteolysis and the digestion time was significantly reduced to 5 min. The digests were identified by MALDI-TOF MS with sequence coverages of 42% (BSA) and 77% (Cyt-c) that were comparable to those obtained by using conventional in-solution tryptic digestion. The present proteolysis strategy is simple and efficient, offering great promise for MALDI-TOF MS peptide mapping.
Knox, James; Palombo, Enzo
A MALDI-TOF MS(1)-based imipenem hydrolysis assay was modified by adding ZnSO4. This improved detection of metallo-β-lactamase producing strains without compromising detection of other carbapenemase types. Using 129 genetically characterized Gram-negative bacilli, the sensitivity and specificity were 98.5% (95% confidence interval [CI]: 91.9-99.7%) and 100% (95% CI: 94.3-100%), respectively.
Burcar, Bradley T.; Cassidy, Lauren M.; Moriarty, Elizabeth M.; Joshi, Prakash C.; Coari, Kristin M.; McGown, Linda B.
Demonstration of the abiotic polymerization of ribonucleotides under conditions consistent with conditions that may have existed on the prebiotic Earth is an important goal in "RNA world" research. Recent reports of abiotic RNA polymerization with and without catalysis rely on techniques such as HPLC, gel electrophoresis, and MALDI-TOF MS to analyze the reaction products. It is essential to understand the limitations of these techniques in order to accurately interpret the results of these analyses. In particular, techniques that rely on mass for peak identification may not be able to distinguish between a single, linear RNA oligomer and stable aggregates of smaller linear and/or cyclic RNA molecules. In the case of MALDI-TOF MS, additional complications may arise from formation of salt adducts and MALDI matrix complexes. This is especially true for abiotic RNA polymerization reactions because the concentration of longer RNA chains can be quite low and RNA, as a polyelectrolyte, is highly susceptible to adduct formation and aggregation. Here we focus on MALDI-TOF MS analysis of abiotic polymerization products of imidazole-activated AMP in the presence and absence of montmorillonite clay as a catalyst. A low molecular weight oligonucleotide standard designed for use in MALDI-TOF MS and a 3'-5' polyadenosine monophosphate reference standard were also run for comparison and calibration. Clay-catalyzed reaction products of activated GMP and UMP were also examined. The results illustrate the ambiguities associated with assignment of m/z values in MALDI mass spectra and the need for accurate calibration of mass spectra and careful sample preparation to minimize the formation of adducts and other complications arising from the MALDI process.
Wolters, Manuel; Rohde, Holger; Maier, Thomas; Belmar-Campos, Cristina; Franke, Gefion; Scherpe, Stefanie; Aepfelbacher, Martin; Christner, Martin
Early detection of outbreaks of methicillin-resistant Staphylococcus aureus (MRSA) and initiation of adequate infection control measures are important objectives in hospital hygiene. To reach these goals, prompt determination of epidemiologic relatedness of clinical MRSA isolates is essential. Genetic typing methods like pulsed-field gel electrophoresis (PFGE), spa typing, or multilocus sequence typing (MLST) have a high discriminatory power, however, these methods are time consuming and cost intensive. The aim of this study was to investigate the potential of matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) for discrimination of major MRSA lineages. By analysis of mass spectra from 25 representative MRSA isolates belonging to the 5 major hospital-acquired (HA) MRSA clonal complexes (CC5, CC8, CC22, CC30, CC45; deduced from spa typing), reproducible spectrum differences were observed at 13 characteristic m/z values allowing robust discrimination of the clonal complexes. When 60 independent clinical MRSA isolates were tested for the presence or absence of the 13 characteristic MALDI-TOF MS peaks, 15 different profiles (MALDI types) could be detected. Hierarchical clustering of the MALDI types showed high concordance with the clonal complexes. Our results suggest that MALDI-TOF MS has the potential to become a valuable first-line tool for inexpensive and rapid typing of MRSA in infection control.
The microbiological management of patients with suspected bacterial infection includes the identification of the pathogen and the determination of the antibiotic susceptibility. These traditional approaches, based on the pure culture of the microorganism, require at least 36-48h. A new method, Matrix Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (MALDI-TOF MS), has been recently developed to profile bacterial proteins from whole cell extracts and obtain a bacterial fingerprint able to discriminate microorganisms from different genera and species. By whole cell-mass spectrometry, microbial identification can be achieved within minutes from cultured isolate, rather than traditional phenotypic or genotypic characterizations. From the year 2009 an explosion of applications of this technology has been observed with promising results. Several studies have been performed and showed that MALDI-TOF represents a reliable alternative method for rapid bacteria and fungi identification in clinical setting. A future area of expansion is represented by the application of MALDI-TOF technology to the antibiotic susceptibility test. In conclusion, the revision of the literature available up to date demonstrated that MALDI-TOF MS represents an innovative technology for the rapid and accurate identification of bacterial and fungal isolates in clinical settings. By an earlier microbiological diagnosis, MALDI-TOF MS contributes to a reduced mortality and hospitalization time of the patients and consequently has a significant impact on cost savings and public health.
Pinto, Tatiana C. A.; Costa, Natalia S.; Castro, Luciana F. S.; Ribeiro, Rachel L.; Botelho, Ana Caroline N.; Neves, Felipe P. G.; Peralta, Jose Mauro; Teixeira, Lucia M.
Streptococcus pneumoniae can be classified in more than 90 capsular types, as traditionally determined by serological methods and more recently by PCR-based techniques. Such methods, however, can be expensive, laborious or unable to accurately discriminate among certain serotypes. Therefore, determination of capsular types, although extremely important for epidemiological purposes and for estimating the impact of pneumococcal conjugate vaccines, is mainly restricted to research laboratories, being rarely performed in the clinical setting. In the present study, MALDI-TOF MS was evaluated as an alternative tool to characterize 416 pneumococcal isolates belonging to serotypes 6A, 6B, 6C, 9N, 9V or 14. For MALDI-TOF MS analysis, each isolate was submitted to an extraction protocol using formic acid and acetonitrile. Measurements were performed with a Bruker Microflex LT mass spectrometer using default parameters and generating spectra in the range of 2,000–20,000 m/z. Spectra were analyzed with the BioNumerics software v7.6. Isolates were mainly distributed according to the capsular type in a Neighbor Joining tree and serotypes investigated were successfully discriminated by the presence/absence of 14 selected biomarkers. The results suggest that MALDI-TOF MS is a promising alternative for typing pneumococcal strains, highlighting its usefulness for rapid and cost-effective routine application in clinical laboratories. PMID:28349999
Šalplachta, Jiří; Kubesová, Anna; Horký, Jaroslav; Matoušková, Hana; Tesařová, Marie; Horká, Marie
Dickeya and Pectobacterium species represent an important group of broad-host-range phytopathogens responsible for blackleg and soft rot diseases on numerous plants including many economically important plants. Although these species are commonly detected using cultural, serological, and molecular methods, these methods are sometimes insufficient to classify the bacteria correctly. On that account, this study was undertaken to investigate the feasibility of three individual analytical techniques, capillary zone electrophoresis (CZE), capillary isoelectric focusing (CIEF), and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), for reliable classification of Dickeya and Pectobacterium species. Forty-three strains, representing different Dickeya and Pectobacterium species, namely Dickeya dianthicola, Dickeya dadantii, Dickeya dieffenbachiae, Dickeya chrysanthemi, Dickeya zeae, Dickeya paradisiaca, Dickeya solani, Pectobacterium carotovorum, and Pectobacterium atrosepticum, were selected for this purpose. Furthermore, the selected bacteria included one strain which could not be classified using traditional microbiological methods. Characterization of the bacteria was based on different pI values (CIEF), migration velocities (CZE), or specific mass fingerprints (MALDI-TOF MS) of intact cells. All the examined strains, including the undetermined bacterium, were characterized and classified correctly into respective species. MALDI-TOF MS provided the most reliable results in this respect.
Pérez-Sancho, Marta; Vela, Ana Isabel; García-Seco, Teresa; Gottschalk, Marcelo; Domínguez, Lucas; Fernández-Garayzábal, José Francisco
The accuracy of matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) for identifying Streptococcus suis isolates obtained from pigs, wild animals, and humans was evaluated using a PCR-based identification assay as the gold standard. In addition, MALDI-TOF MS was compared with the commercial multi-tests Rapid ID 32 STREP system. From the 129 S. suis isolates included in the study and identified by the molecular method, only 31 isolates (24.03%) had score values ≥2.300 and 79 isolates (61.24%) gave score values between 2.299 and 2.000. After updating the currently available S. suis MALDI Biotyper database with the spectra of three additional clinical isolates of serotypes 2, 7, and 9, most isolates had statistically significant higher score values (mean score: 2.65) than those obtained using the original database (mean score: 2.182). Considering the results of the present study, we suggest using a less restrictive threshold score of ≥2.000 for reliable species identification of S. suis. According to this cut-off value, a total of 125 S. suis isolates (96.9%) were correctly identified using the updated database. These data indicate an excellent performance of MALDI-TOF MS for the identification of S. suis. PMID:26347858
Meltretter, Jasmin; Birlouez-Aragon, Inès; Becker, Cord-Michael; Pischetsrieder, Monika
The formation of the Amadori product from lactose (protein lactosylation) is a major parameter to evaluate the quality of processed milk. Here, MALDI-TOF-MS was used for the relative quantification of lactose-adducts in heated milk. Milk was heated at a temperature of 70, 80, and 100 degrees C between 0 and 300 min, diluted, and subjected directly to MALDI-TOF-MS. The lactosylation rate of alpha-lactalbumin increased with increasing reaction temperature and time. The results correlated well with established markers for heat treatment of milk (concentration of total soluble protein, soluble alpha-lactalbumin and beta-lactoglobulin at pH 4.6, and fluorescence of advanced Maillard products and soluble tryptophan index; r=0.969-0.997). The method was also applied to examine commercially available dairy products. In severely heated products, protein pre-purification by immobilized metal affinity chromatography improved spectra quality. Relative quantification of protein lactosylation by MALDI-TOF-MS proved to be a very fast and reliable method to monitor early Maillard reaction during milk processing.
Soler, Laura; Labas, Valérie; Thélie, Aurore; Grasseau, Isabelle; Teixeira-Gomes, Ana-Paula; Blesbois, Elisabeth
Currently, evaluation of sperm quality is primarily based on in vitro measures of sperm function such as motility, viability and/or acrosome reaction. However, results are often poorly correlated with fertility, and alternative diagnostic tools are therefore needed both in veterinary and human medicine. In a recent pilot study, we demonstrated that MS profiles from intact chicken sperm using MALDI-TOF profiles could detect significant differences between fertile/subfertile spermatozoa showing that such profiles could be useful for in vitro male fertility testing. In the present study, we performed larger standardized experimental procedures designed for the development of fertility- predictive mathematical models based on sperm cell MALDI-TOF MS profiles acquired through a fast, automated method. This intact cell MALDI-TOF MS-based method showed high diagnostic accuracy in identifying fertile/subfertile males in a large male population of known fertility from two distinct genetic lineages (meat and egg laying lines). We additionally identified 40% of the m/z peaks observed in sperm MS profiles through a top-down high-resolution protein identification analysis. This revealed that the MALDI-TOF MS spectra obtained from intact sperm cells contained a large proportion of protein degradation products, many implicated in important functional pathways in sperm such as energy metabolism, structure and movement. Proteins identified by our predictive model included diverse and important functional classes providing new insights into sperm function as it relates to fertility differences in this experimental system. Thus, in addition to the chicken model system developed here, with the use of appropriate models these methods should effectively translate to other animal taxa where similar tests for fertility are warranted.
Kemptner, Jasmin; Marchetti-Deschmann, Martina; Siekmann, Juergen; Turecek, Peter L; Schwarz, Hans Peter; Allmaier, Günter
One of the most prominent polymer group applied for drug conjugation is poly(ethylene) glycol (PEG). Since drug production is subjected to strict restrictions on the part of the FDA and EMEA, also PEG has to be characterized accurately. Particularly its molecular mass distribution (MMD) and polydispersity can result in unrequested inhomogeneous final products. Therefore evaluation of PEG before applying it to drug conjugation is essential. In this study a new analytical method for size and molecular mass determination based on electrophoretic mobility called GEMMA is used to characterize linear PEGs with two differing terminating functional groups. To confirm the data acquired by GEMMA a second, well-established method for molecular weight determination, MALDI-TOF MS (matrix-assisted laser desorption ionization time-of-flight mass spectrometry), was applied. Utilizing these two analytical approaches four monomethoxylated PEG-succinimidyl succinate (mPEG-SS) derivatives were investigated in terms of polydispersity and MMD. Although based on differing principles, both analytical methods yield comparable results. All obtained MMD maxima for the mPEG-SS batches lie within the company stated specifications, MMD+/-10% (based on MALDI-TOF MS data). For mPEG-SS 2K a polydispersity of 1.02 and for mPEG-SS 5K, 10K and 20K a polydispersity of 1.01 were determined from GEMMA as well as from MALDI-TOF MS data and are in agreement with the company's data (based on GPC data), namely 1.05-1.10.
Senneby, E; Göransson, L; Weiber, S; Rasmussen, M
The purpose of this study was to determine the incidence of aerococcal bacteraemia in the MALDI-TOF MS-era, to describe the clinical presentation and to determine the MIC values of aerococci for ten antibiotics. Aerococci in blood cultures were identified through searches in the laboratory database for the years 2012-2014. MALDI-TOF MS, sequencing of the 16S rRNA gene and a PYR test were used for species identification. Patients' medical charts were systematically reviewed. Etests were used to determine MIC values. Seventy-seven patients were identified (Aerococcus urinae n = 49, Aerococcus viridans n = 14, Aerococcus sanguinicola n = 13 and Aerococcus christensenii n = 1) corresponding to incidences of 14 cases per 1,000,000 inhabitants per year (A. urinae) and 3.5 cases per 1,000,000 inhabitants per year (A. sanguinicola and A.viridans). A. urinae was in pure culture in 61 %, A. sanguinicola in 46 % and A. viridans in 36 % of the cases. The A. urinae and A. sanguinicola patients were old and many had urinary tract disorders, and a majority had a suspected urinary tract focus of the bacteraemia. Eighty percent of the A. urinae patients were men. Five A. urinae patients were diagnosed with infective endocarditis. Six patients died within 30 days. Most isolates had low MICs to penicillins and carbapenems. MALDI-TOF MS has led to an increased identification of aerococcal bacteremia. A. urinae remains the most common Aerococcus in blood cultures and in aerococcal IE.
Santos, Cledir; Ventura, José Aires; Lima, Nelson
Fusarium is one of the most economically important fungal genus, since it includes many pathogenic species which cause a wide range of plant diseases. Morphological or molecular biology identification of Fusarium species is a limiting step in the fast diagnosis and treatment of plant disease caused by these fungi. Mass spectrometry by matrix-assisted laser/desorption ionisation-time-of-flight (MALDI-TOF)-based fingerprinting approach was applied to the fungal growth monitoring and direct detection of strain Fusarium guttiforme E-480 inoculated in both pineapple cultivars Pérola and Imperial side shoots, that are susceptible and resistant, respectively, to this fungal strain. MALDI-TOF MS technique was capable to detect fungal molecular mass peaks in the susceptible pineapple stem side shoot tissue. It is assumed that these molecular masses are mainly constituted by ribosomal proteins. MALDI-TOF-based fingerprinting approach has herein been demonstrated to be sensitive and accurate for the direct detection of F. guttiforme E-480 molecular masses on both susceptible and resistant pineapple side stem free of any pre-treatment. According to the results obtained, the changing on molecular mass peaks of infected susceptible pineapple tissue together with the possibility of fungal molecular masses analysis into this pineapple tissue can be a good indication for an early diagnosis by MALDI-TOF MS of pineapple fusariosis.
Toh, Benjamin E W; Paterson, David L; Kamolvit, Witchuda; Zowawi, Hosam; Kvaskoff, David; Sidjabat, Hanna; Wailan, Alexander; Peleg, Anton Y; Huber, Charlotte A
Acinetobacter baumannii, one of the more clinically relevant species in the Acinetobacter genus is well known to be multi-drug resistant and associated with bacteremia, urinary tract infection, pneumonia, wound infection and meningitis. However, it cannot be differentiated from closely related species such as Acinetobacter calcoaceticus, Acinetobacter pittii and Acinetobacter nosocomialis by most phenotypic tests and can only be differentiated by specific, time consuming genotypic tests with very limited use in clinical microbiological laboratories. As a result, these species are grouped into the A. calcoaceticus-A. baumannii (Acb) complex. Herein we investigated the mass spectra of 73 Acinetobacter spp., representing ten different species, using an AB SCIEX 5800 MALDI-TOF MS to differentiate members of the Acinetobacter genus, including the species of the Acb complex. RpoB gene sequencing, 16S rRNA sequencing, and gyrB multiplex PCR were also evaluated as orthogonal methods to identify the organisms used in this study. We found that whilst 16S rRNA and rpoB gene sequencing could not differentiate A. pittii or A. calcoaceticus, they can be differentiated using gyrB multiplex PCR and MALDI-TOF MS. All ten Acinetobacter species investigated could be differentiated by their MALDI-TOF mass spectra.
Fuchs, Beate; Schiller, Jürgen
Many diseases as atherosclerosis and metabolic dysfunctions are known to correlate with changes of the lipid profile of tissues and body fluids. Therefore, the importance of reliable methods of lipid analysis is obvious. Although matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS) was so far primarily used for protein analysis, this method has itself proven to be very useful in lipid analysis, too. This review provides an overview of applications of MALDI-TOF MS in lipid analysis and summarizes the specific advantages and drawbacks of this modern soft-ionization method. The focus will be on the analysis of body fluids and cells as well as the diagnostic potential of the method in the lipid field. It will be shown that MALDI-TOF mass spectra can be recorded in a very short time and provide important information on the lipid as well as the fatty acyl composition of the lipids of an unknown sample. However, it will also be shown that only selected lipid classes (in particular those with quaternary ammonia groups as phosphatidylcholine) are detected if crude mixtures are analyzed as they are more sensitively detectable than other ones. This review ends with a short outlook emphasizing current methodological developments.
Del Chierico, Federica; Di Cave, David; Accardi, Cristel; Santoro, Maristella; Masotti, Andrea; D'Alfonso, Rossella; Berrilli, Federica; Urbani, Andrea; Putignani, Lorenza
Over the years, the potential pathogenicity of Acanthamoeba for humans and animals has gained increasing attention from the scientific community. More than 24 species belong to this genus, however only some of them are causative agents of keratitis and encephalitis in humans. Due to technical difficulties in diagnosis, these infections are likely to be under-detected. The introduction of 18S rDNA amplification for the identification of Acanthamoeba has dramatically enhanced diagnosis performances, but the attestation of genotyping requires supplementary sequencing-based procedures. In this study, 15 Acanthamoeba strains were collected and grown on nutrient agar media. Each strain was genotyped by end-point PCR assay for the amplification of the 18S rDNA gene and the genotype was assigned by sequencing analysis through neighbor joining phylogenetic tree. In order to optimize standardization of the MALDI-TOF MS assay, we established the collection time point at the cystic phase. Two strains of each genotype were randomly chosen to customize the biotyper database. For all strains, 24 spectral measurements were acquired and submitted to identification and cluster analysis of spectra. The obtained results highlighted the correct identification of Acanthamoeba strains and the overlapping of spectra dendrogram clusters to the 18S genotype assignations. In conclusion, the MALDI-TOF MS Biotyper revealed the capability to identify and genotype the Acanthamoeba strains, providing a new frontier in the diagnostic identification of amaebae and in taxonomic and phylogenetic studies.
Calderaro, Adriana; Gorrini, Chiara; Piccolo, Giovanna; Montecchini, Sara; Buttrini, Mirko; Rossi, Sabina; Piergianni, Maddalena; Arcangeletti, Maria Cristina; De Conto, Flora; Chezzi, Carlo; Medici, Maria Cristina
Lyme borreliosis (LB) is a multisystemic disease caused by Borrelia burgdorferi sensu lato (sl) complex transmitted to humans by Ixodes ticks. B. burgdorferi sl complex, currently comprising at least 19 genospecies, includes the main pathogenic species responsible for human disease in Europe: B. burgdorferi sensu stricto (ss), B. afzelii, and B. garinii. In this study, for the first time, MALDI-TOF MS was applied to Borrelia spp., supplementing the existing database, limited to the species B. burgdorferi ss, B . spielmanii and B. garinii, with the species B. afzelii, in order to enable the identification of all the species potentially implicated in LB in Europe. Moreover, we supplemented the database also with B. hermsii, which is the primary cause of tick-borne relapsing fever in western North America, B. japonica, circulating in Asia, and another reference strain of B. burgdorferi ss (B31 strain). The dendrogram obtained by analyzing the protein profiles of the different Borrelia species reflected Borrelia taxonomy, showing that all the species included in the Borrelia sl complex clustered in a unique branch, while Borrelia hermsii clustered separately. In conclusion, in this study MALDI-TOF MS proved a useful tool suitable for identification of Borrelia spp. both for diagnostic purpose and epidemiological surveillance.
Fuchs, Beate; Schiller, Jürgen; Süss, Rosmarie; Zscharnack, Matthias; Bader, Augustinus; Müller, Peter; Schürenberg, Martin; Becker, Michael; Suckau, Detlev
MALDI-TOF MS is traditionally used for "proteomics", but is also a useful tool for lipid analysis. Depending on the applied matrix, however, some lipid classes are more sensitively detected than other ones and this may even lead to suppression effects if complex mixtures are analyzed. Therefore, a previous separation into the individual lipid classes is necessary. Using artificial lipid mixtures or easily available tissue extracts, it has been already shown that HPTLC-(High Performance Thin-Layer Chromatography)-separated lipids can be conveniently analyzed by MALDI-TOF MS directly on the TLC plate. Here we present an initial TLC-MALDI study of the lipid composition of ovine mesenchymal stem cells. Due to the complex composition of these cells, data are also compared to lipids extracted from human erythrocytes. It will be shown that even very minor lipid classes can be easily detected and with much higher sensitivity than by common staining protocols. Additionally, MS images of the developed TLC plates will be shown and potential applications, new methods of data analysis as well as problems discussed.
Olate, Verónica R; Nachtigall, Fabiane M; Santos, Leonardo S; Soto, Alex; Araya, Macarena; Oyanedel, Sandra; Díaz, Verónica; Marchant, Vanessa; Rios-Momberg, Mauricio
Piscirickettsia salmonis is a pathogenic bacteria known as the aetiological agent of the salmonid rickettsial syndrome and causes a high mortality in farmed salmonid fishes. Detection of P. salmonis in farmed fishes is based mainly on molecular biology and immunohistochemistry techniques. These techniques are in most of the cases expensive and time consuming. In the search of new alternatives to detect the presence of P. salmonis in salmonid fishes, this work proposed the use of MALDI-TOF-MS to compare serum protein profiles from Salmo salar fish, including experimentally infected and non-infected fishes using principal component analysis (PCA). Samples were obtained from a controlled bioassay where S. salar was challenged with P. salmonis in a cohabitation model and classified according to the presence or absence of the bacteria by real time PCR analysis. MALDI spectra of the fish serum samples showed differences in its serum protein composition. These differences were corroborated with PCA analysis. The results demonstrated that the use of both MALDI-TOF-MS and PCA represents a useful tool to discriminate the fish status through the analysis of salmonid serum samples.
Weiz, Stefan; Kamalakumar, Aryaline; Biskup, Karina; Blanchard, Véronique
Many biologically relevant glycoproteins need to be separated on 1D- or 2D-gels prior to analysis and are available in picomole amounts. Therefore, it is important to have optimized methods to unravel the glycome that combine in-gel digestions with MALDI-TOF-MS. In this technical report, we investigated how the detection of in-gel released N-glycans could be improved by MALDI-TOF-MS. First, an AnchorChip target was tested and compared to ground steel target using several reference oligosaccharides. The highest signals were obtained with an AnchorChip target and D-arabinosazone as the matrix; a LOD of 1.3 to 10 fmol was attained. Then, the effect of octyl-β-glucopyranoside, a nonionic detergent, was studied during in-gel peptide-N(4) -(acetyl-ß-glucosaminyl) asparagine amidase F digestion of standard glycoproteins and during glycan extraction. Octyl-β-glucopyranoside increased the intensity and the amount of detected neutral as well as acidic N-glycans. A LOD of under 7 pmol glycoprotein could be achieved.
Mlynáriková, Katarína; Šedo, Ondrej; Růžička, Filip; Zdráhal, Zbyněk; Holá, Veronika; Mahelová, Martina
Matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is, currently, used as a rapid and reliable tool in microbial diagnostics. The discriminatory power of the method extends its applicability also beyond species level. This study examined the possibility to use MALDI-TOF MS to differentiate between Candida parapsilosis sensu stricto biofilm-positive (n = 12) and biofilm-negative (n = 9) strains. The results indicated a grouping trend within MALDI-TOF mass spectra belonging to each of the tested groups. However, these trends were eclipsed by mass spectral variations resulting from limited repeatability of the method, making its application for the selected purpose impossible. Improvement in the discriminatory power of the method was not obtained neither by using different matrices (α-cyano-4-hydroxycinnamic acid, ferulic acid, 5-chloro-2-mercaptobenzothionazole) for MALDI-TOF MS analysis nor by testing different culture conditions (cultivation length, culture media).
Matilde da Silva-Costa, Sueli; Balieiro, Juliane Cristina; Fernandes, Marcela Scabello Amaral; Alves, Rogério Marins; Guerra, Andrea Trevas Maciel; Marcondes, Ana Maria; Sartorato, Edi Lúcia
Purpose Leber hereditary optic neuropathy (LHON) is a mitochondrial disease characterized by bilateral vision loss. More than 95% of LHON cases are associated with one of the three main mtDNA mutations: G11778A, T14484C, and G3460A. The other 5% of cases are due to other rare mutations related to the disease. The aim of this study was to identify the prevalence and spectrum of LHON mtDNA mutations, including the haplogroup, in a cohort of Brazilian patients with optic neuropathy and to evaluate the usefulness of iPLEX Gold/matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) technology in detecting LHON mutations. Methods We analyzed a total of 101 patients; 67 had a clinical diagnosis of LHON and 34 had optic neuropathy of unknown etiology. Direct sequencing and iPLEX Gold/MALDI-TOF MS were used to screen for the most common pathogenic point mutations in LHON, together with the rare mutations G3733A, C4171A, T10663C, G14459A, C14482G, A14495G, C14568T, and C14482A. Results We identified mutations in 36 patients, of whom 83.3% carried the G11778A mutation and 16.7% carried the T14484C mutation. In individuals with mutations, the haplogroups found were L1/L2, L3, C, R, U, D, and H. Rare mutations were not detected in any of the patients analyzed. Conclusions The frequencies of the main LHON mutations were similar to those previously reported for Latin America. A different frequency was found only for the A3460G mutation. The most frequent haplogroups identified were of African origin. The iPLEX Gold/MALDI-TOF MS technology proved to be highly accurate and efficient for screening mutations and identifying the haplogroups related to LHON. The MassArray platform, combined with other techniques, enabled definitive diagnosis of LHON in 36% (36/101) of the cases studied. PMID:27582625
MALDI-TOF MS has been utilized as a reliable and rapid tool for microbial fingerprinting at the genus and species levels. Recently, there has been keen interest in using MALDI-TOF MS beyond the genus and species levels to rapidly identify antibiotic resistant strains of bacteria. The purpose of this...
Lauer, Franziska; Seifert, Stephan; Kneipp, Janina; Weidner, Steffen M
Matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) is a well-implemented analytical technique for the investigation of complex biological samples. In MS, the sample preparation strategy is decisive for the success of the measurements. Here, sample preparation processes and target materials for the investigation of different pollen grains are compared. A reduced and optimized sample preparation process prior to MALDI-TOF measurement is presented using conductive carbon tape as target. The application of conductive tape yields in enhanced absolute signal intensities and mass spectral pattern information, which leads to a clear separation in subsequent pattern analysis. The results will be used to improve the taxonomic differentiation and identification, and might be useful for the development of a simple routine method to identify pollen based on mass spectrometry.
Lauer, Franziska; Seifert, Stephan; Kneipp, Janina; Weidner, Steffen M.
Matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) is a well-implemented analytical technique for the investigation of complex biological samples. In MS, the sample preparation strategy is decisive for the success of the measurements. Here, sample preparation processes and target materials for the investigation of different pollen grains are compared. A reduced and optimized sample preparation process prior to MALDI-TOF measurement is presented using conductive carbon tape as target. The application of conductive tape yields in enhanced absolute signal intensities and mass spectral pattern information, which leads to a clear separation in subsequent pattern analysis. The results will be used to improve the taxonomic differentiation and identification, and might be useful for the development of a simple routine method to identify pollen based on mass spectrometry. PMID:28273807
Rodriguez-Lozano, Jesús; Pérez-Llantada, Enrique; Agüero, Jesús; Rodríguez-Fernández, Ana; Ruiz de Alegria, Carlos; Martinez-Martinez, Luis
Introduction: Gordonia spp. infections are uncommon. However, a few clinical cases have been reported in the literature, particularly those involving immunocompromised hosts. Advanced microbiology diagnosis techniques, such as matrix-assisted laser desorption ionization-time of flight MS (MALDI-TOF MS), have been recently introduced in clinical microbiology laboratories in order to improve microbial identification, resulting in better patient management. Case presentation: Here, we present a new clinical case of persistent wound infection caused by Gordonia bronchialis in a 64-year-old woman after a mitral valve replacement, using two MALDI-TOF-based systems for identifying this micro-organism. Conclusion: Both MALDI-TOF systems were able to identify Gordonia spp.; thus, providing a useful tool that overcomes the current limitations of phenotypic identification associated with this micro-organism. Although the technique validation deserves additional verification, our study provides guidance about MALDI-TOF as a fast and easy method for Gordonia spp. identification. PMID:28348789
Popović, Natalija Topić; Kazazić, Snježana P; Strunjak-Perović, Ivančica; Čož-Rakovac, Rozelindra
Identification of bacteria in aquatic and environmental applications, for monitoring purposes and research, for health assessments and therapy considerations of farmed and free-living aquatic organisms, still relies on conventional phenotypic and biochemical protocols. Although molecular techniques based on DNA amplification and sequencing are finding ways into diagnostic laboratories, they are time-consuming, costly and difficult in the case of multiplex assays. Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) is a rapid and accurate proteomic method reliable for identification of unknown bacteria to the genus and species level. Upon extension of databases, it will certainly find its position in environmental sciences. The paper presents an overview of the principle of the method, its effectiveness in comparison with conventional and molecular identification procedures, and applicability on environmental and aquatic isolates, discussing its advantages and shortcomings, as well as possible future implementations.
Roth, Michael J; Maresh, Erica M; Plymire, Daniel A; Zhang, Junmei; Corbett, John R; Robbins, Roger; Patrie, Steven M
Immunoassays are employed in academia and the healthcare and biotech industries for high-throughput, quantitative screens of biomolecules. We have developed monolayer-based immunoassays for MALDI-TOF MS. To improve parallelization, we adapted the workflow to photolithography-generated arrays. Our work shows Parylene-C coatings provide excellent "solvent pinning" for reagents and biofluids, enabling sensitive MS detection of immobilized components. With a unique MALDI-matrix crystallization technique we show routine interassay RSD <10% at picomolar concentrations and highlight platform compatibility for relative and label-free quantitation applications. Parylene-arrays provide high sample densities and promise screening throughputs exceeding 1000 samples/h with modern liquid-handlers and MALDI-TOF systems.
Phillips, Nancy J; John, Constance M; Jarvis, Gary A
Lipooligosaccharides (LOS) are major microbial virulence factors displayed on the outer membrane of rough-type Gram-negative bacteria. These amphipathic glycolipids are comprised of two domains, a core oligosaccharide linked to a lipid A moiety. Isolated LOS samples are generally heterogeneous mixtures of glycoforms, with structural variability in both domains. Traditionally, the oligosaccharide and lipid A components of LOS have been analyzed separately following mild acid hydrolysis, although important acid-labile moieties can be cleaved. Recently, an improved method was introduced for analysis of intact LOS by MALDI-TOF MS using a thin layer matrix composed of 2,4,6-trihydroxyacetophenone (THAP) and nitrocellulose. In addition to molecular ions, the spectra show in-source "prompt" fragments arising from regiospecific cleavage between the lipid A and oligosaccharide domains. Here, we demonstrate the use of traveling wave ion mobility spectrometry (TWIMS) for IMS-MS and IMS-MS/MS analyses of intact LOS from Neisseria spp. ionized by MALDI. Using IMS, the singly charged prompt fragments for the oligosaccharide and lipid A domains of LOS were readily separated into resolved ion plumes, permitting the extraction of specific subspectra, which led to increased confidence in assigning compositions and improved detection of less abundant ions. Moreover, IMS separation of precursor ions prior to collision-induced dissociation (CID) generated time-aligned, clean MS/MS spectra devoid of fragments from interfering species. Incorporating IMS into the profiling of intact LOS by MALDI-TOF MS exploits the unique domain structure of the molecule and offers a new means of extracting more detailed information from the analysis. Graphical Abstract ᅟ.
Gottardo, Rossella; Chiarini, Anna; Dal Prà, Ilaria; Seri, Catia; Rimondo, Claudia; Serpelloni, Giovanni; Armato, Ubaldo; Tagliaro, Franco
Since 2004, a number of herbal blends containing different synthetic compounds mimicking the pharmacological activity of cannabinoids and displaying a high toxicological potential have appeared in the market. Their availability is mainly based on the so-called "e-commerce", being sold as legal alternatives to cannabis and cannabis derivatives. Although highly selective, sensitive, accurate, and quantitative methods based on GC-MS and LC-MS are available, they lack simplicity, rapidity, versatility and throughput, which are required for product monitoring. In this context, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) offers a simple and rapid operation with high throughput. Thus, the aim of the present work was to develop a MALDI-TOF MS method for the rapid qualitative direct analysis of herbal blend preparations for synthetic cannabinoids to be used as front screening of confiscated clandestine preparations. The sample preparation was limited to herbal blend leaves finely grinding in a mortar and loading onto the MALDI plate followed by addition of 2 µl of the matrix/surfactant mixture [α-cyano-4-hydroxy-cinnamic acid/cetyltrimethylammonium bromide (CTAB)]. After drying, the sample plate was introduced into the ion source for analysis. MALDI-TOF conditions were as follows: mass spectra were analyzed in the range m/z 150-550 by averaging the data from 50 laser shots and using an accelerating voltage of 20 kV. The described method was successfully applied to the screening of 31 commercial herbal blends, previously analyzed by GC-MS. Among the samples analyzed, 21 contained synthetic cannabinoids (namely JWH-018, JWH-073, JWH-081, JWH-250, JWH-210, JWH-019, and AM-694). All the results were in agreement with GC-MS, which was used as the reference technique.
Fiedoruk, Krzysztof; Daniluk, Tamara; Fiodor, Angelika; Drewicka, Ewa; Buczynska, Katarzyna; Leszczynska, Katarzyna; Bideshi, Dennis Ken; Swiecicka, Izabela
The number of foodborne intoxications caused by emetic Bacillus cereus isolates has increased significantly. As such, rapid and reliable methods to identify emetic strains appear to be clinically relevant. In this study, intact cell matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was used to differentiate emetic and non-emetic bacilli. The phyloproteomic clustering of 34 B. cereus emetic and 88 non-emetic isolates classified as B. cereus, Bacillus thuringiensis, Bacillus weihenstephanensis, and Bacillus mycoides, showed (i) a clear separation of both groups at a similarity level of 43%, and (ii) a high relatedness among the emetic isolates (similarity of 78%). Specifically, 83 mass peak classes were recognized in the spectral window range between m/z 4000 and 12 000 that were tentatively assigned to 41 protein variants based on a bioinformatic approach. Mass variation between the emetic and the non-emetic subsets was recorded for 27 of them, including ten ribosomal subunit proteins, for which inter-strain polymorphism was confirmed by gene sequencing. Additional peaks were assigned to other proteins such as small acid soluble proteins, cold shock proteins and hypothetical proteins, e.g., carbohydrate kinase. Moreover, the results were supported by in silico analysis of the biomarkers in 259 members of B. cereus group, including Bacillus anthracis, based on their whole-genome sequences. In conclusion, the proteomic profiling by MALDI-TOF MS is a promising and rapid method for pre-screening B. cereus to identify medically relevant isolates and for epidemiologic purposes.
Branquinho, Raquel; Sousa, Clara; Lopes, João; Pintado, Manuela E; Peixe, Luísa V; Osório, Hugo
Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) despite being increasingly used as a method for microbial identification, still present limitations in which concerns the differentiation of closely related species. Bacillus pumillus and Bacillus safensis, are species of biotechnological and pharmaceutical significance, difficult to differentiate by conventional methodologies. In this study, using a well-characterized collection of B. pumillus and B. safensis isolates, we demonstrated the suitability of MALDI-TOF-MS combined with chemometrics to accurately and rapidly identify them. Moreover, characteristic species-specific ion masses were tentatively assigned, using UniProtKB/Swiss-Prot and UniProtKB/TrEMBL databases and primary literature. Delineation of B. pumilus (ions at m/z 5271 and 6122) and B. safensis (ions at m/z 5288, 5568 and 6413) species were supported by a congruent characteristic protein pattern. Moreover, using a chemometric approach, the score plot created by partial least square discriminant analysis (PLSDA) of mass spectra demonstrated the presence of two individualized clusters, each one enclosing isolates belonging to a species-specific spectral group. The generated pool of species-specific proteins comprised mostly ribosomal and SASPs proteins. Therefore, in B. pumilus the specific ion at m/z 5271 was associated with a small acid-soluble spore protein (SASP O) or with 50S protein L35, whereas in B. safensis specific ions at m/z 5288 and 5568 were associated with SASP J and P, respectively, and an ion at m/z 6413 with 50S protein L32. Thus, the resulting unique protein profile combined with chemometric analysis, proved to be valuable tools for B. pumilus and B. safensis discrimination, allowing their reliable, reproducible and rapid identification.
Phillips, Nancy J.; John, Constance M.; Jarvis, Gary A.
Lipooligosaccharides (LOS) are major microbial virulence factors displayed on the outer membrane of rough-type Gram-negative bacteria. These amphipathic glycolipids are comprised of two domains, a core oligosaccharide linked to a lipid A moiety. Isolated LOS samples are generally heterogeneous mixtures of glycoforms, with structural variability in both domains. Traditionally, the oligosaccharide and lipid A components of LOS have been analyzed separately following mild acid hydrolysis, although important acid-labile moieties can be cleaved. Recently, an improved method was introduced for analysis of intact LOS by MALDI-TOF MS using a thin layer matrix composed of 2,4,6-trihydroxyacetophenone (THAP) and nitrocellulose. In addition to molecular ions, the spectra show in-source "prompt" fragments arising from regiospecific cleavage between the lipid A and oligosaccharide domains. Here, we demonstrate the use of traveling wave ion mobility spectrometry (TWIMS) for IMS-MS and IMS-MS/MS analyses of intact LOS from Neisseria spp. ionized by MALDI. Using IMS, the singly charged prompt fragments for the oligosaccharide and lipid A domains of LOS were readily separated into resolved ion plumes, permitting the extraction of specific subspectra, which led to increased confidence in assigning compositions and improved detection of less abundant ions. Moreover, IMS separation of precursor ions prior to collision-induced dissociation (CID) generated time-aligned, clean MS/MS spectra devoid of fragments from interfering species. Incorporating IMS into the profiling of intact LOS by MALDI-TOF MS exploits the unique domain structure of the molecule and offers a new means of extracting more detailed information from the analysis.
Miescher Schwenninger, S; Freimüller Leischtfeld, S; Gantenbein-Demarchi, C
Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a powerful biotyping tool increasingly used for high-throughput identification of clinical microbial isolates, however, in food fermentation research this approach is still not well established. This study examines the microbial biodiversity of cocoa bean fermentation based on the isolation of micro-organisms in cocoa-producing regions, followed by MALDI-TOF MS in Switzerland. A preceding 6-week storage test to mimic lengthy transport of microbial samples from cocoa-producing regions to Switzerland was performed with strains of Lactobacillus plantarum, Acetobacter pasteurianus and Saccharomyces cerevisiae. Weekly MALDI-TOF MS analysis was able to successfully identify microbiota to the species level after storing live cultures on slant agar at mild temperatures (7°C) and/or in 75% aqueous ethanol at differing temperatures (-20, 7 and 30°C). The efficacy of this method was confirmed by on-site recording of the microbial biodiversity in cocoa bean fermentation in Bolivia and Brazil, with a total of 1126 randomly selected isolates. MALDI-TOF MS analyses revealed known dominant cocoa bean fermentation species with Lact. plantarum and Lactobacillus fermentum in the lactic acid bacteria taxon, Hanseniaspora opuntiae and S. cerevisiae in the yeast taxon, and Acet. pasteurianus, Acetobacter fabarum, Acetobacter ghanensis and Acetobacter senegalensis in the acetic acid bacteria taxon.
Agergaard, Charlotte N; Knudsen, Elisa; Dargis, Rimtas; Nielsen, Xiaohui C; Christensen, Jens J; Justesen, Ulrik S
This study compared two MALDI-TOF MS systems (Biotyper and VITEK MS) on clinical Streptococcus bovis group isolates (n=66). The VITEK MS gave fewer misidentifications and a higher rate of correct identifications than the Biotyper. Only the identification of S. lutetiensis by the VITEK MS was reliable. Additional optimization of the available system databases is needed.
Hsu, Yen-Michael S; Burnham, Carey-Ann D
Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has emerged as a tool for identifying clinically relevant anaerobes. We evaluated the analytical performance characteristics of the Bruker Microflex with Biotyper 3.0 software system for identification of anaerobes and examined the impact of direct formic acid (FA) treatment and other pre-analytical factors on MALDI-TOF MS performance. A collection of 101 anaerobic bacteria were evaluated, including Clostridium spp., Propionibacterium spp., Fusobacterium spp., Bacteroides spp., and other anaerobic bacterial of clinical relevance. The results of our study indicate that an on-target extraction with 100% FA improves the rate of accurate identification without introducing misidentification (P<0.05). In addition, we modify the reporting cutoffs for the Biotyper "score" yielding acceptable identification. We found that a score of ≥1.700 can maximize the rate of identification. Of interest, MALDI-TOF MS can correctly identify anaerobes grown in suboptimal conditions, such as on selective culture media and following oxygen exposure. In conclusion, we report on a number of simple and cost-effective pre- and post-analytical modifications could enhance MALDI-TOF MS identification for anaerobic bacteria.
Nagy, Elisabeth; Urbán, Edit; Becker, Simone; Kostrzewa, Markus; Vörös, Andrea; Hunyadkürti, Judit; Nagy, István
Matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is widely used today for species determination of bacteria and fungi in routine microbiological laboratories, and can also be used for subtyping of bacteria, such as Bacteroides fragilis. Propionibacterium acnes is frequently referred to as an anaerobic skin commensal of relatively low pathogenicity. In addition to its accepted pathogenic role in acne, P. acnes is now emerging as an important opportunistic pathogen in many other clinical situations, including late-stage prosthetic joint infections, osteomyelitis, endocarditis, endophthalmitis, post-neurosurgical infections and possibly prostate cancer. At the population genetic level, P. acnes can be differentiated into a number of distinct phylogroups, known as types IA1, IA2, IB, IC, II and III, which may be associated with different types of infections and clinical conditions. The aim of the present study was to evaluate MS-based typing for resolution of these genetic groups after routine identification by MALDI-TOF MS (Bruker MALDI Biotyper). The software package ClinProTools 2.2 was used to analyze the protein based mass spectra of reference strains belonging to types IA, IB, IC, II and III. Phylogroup-specific peaks and peak shifts were then identified visually. In addition, peak variations between the different types of P. acnes were investigated by using FlexAnalysis 3.3 software (Bruker). A differentiating library was created, which was used to type further 48 clinical isolates of P. acnes. Typing data obtained by MALDI-TOF MS were then compared with the results from Multilocus Sequence Typing (MLST). Most of the clinical isolates (n = 19) belonged to the type IA grouping according to MALDI-TOF MS. By MLST, all isolates were identified as type IA1. Twenty-one clinical isolates belonged to the type IB cluster based on both MALDI-TOF MS and MLST typing. Eight clinical isolates were identified as type II strains
Bille, E; Dauphin, B; Leto, J; Bougnoux, M-E; Beretti, J-L; Lotz, A; Suarez, S; Meyer, J; Join-Lambert, O; Descamps, P; Grall, N; Mory, F; Dubreuil, L; Berche, P; Nassif, X; Ferroni, A
All organisms usually isolated in our laboratory are now routinely identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) using the Andromas software. The aim of this study was to describe the use of this strategy in a routine clinical microbiology laboratory. The microorganisms identified included bacteria, mycobacteria, yeasts and Aspergillus spp. isolated on solid media or extracted directly from blood cultures. MALDI-TOF MS was performed on 2665 bacteria isolated on solid media, corresponding to all bacteria isolated during this period except Escherichia coli grown on chromogenic media. All acquisitions were performed without extraction. After a single acquisition, 93.1% of bacteria grown on solid media were correctly identified. When the first acquisition was not contributory, a second acquisition was performed either the same day or the next day. After two acquisitions, the rate of bacteria identified increased to 99.2%. The failures reported on 21 strains were due to an unknown profile attributed to new species (9) or an insufficient quality of the spectrum (12). MALDI-TOF MS has been applied to 162 positive blood cultures. The identification rate was 91.4%. All mycobacteria isolated during this period (22) were correctly identified by MALDI-TOF MS without any extraction. For 96.3% and 92.2% of yeasts and Aspergillus spp., respectively, the identification was obtained with a single acquisition. After a second acquisition, the overall identification rate was 98.8% for yeasts (160/162) and 98.4% (63/64) for Aspergillus spp. In conclusion, the MALDI-TOF MS strategy used in this work allows a rapid and efficient identification of all microorganisms isolated routinely.
Rahi, Praveen; Prakash, Om; Shouche, Yogesh S
Matrix-assisted laser desorption/ionization time-of-flight mass-spectrometry (MALDI-TOF MS) based biotyping is an emerging technique for high-throughput and rapid microbial identification. Due to its relatively higher accuracy, comprehensive database of clinically important microorganisms and low-cost compared to other microbial identification methods, MALDI-TOF MS has started replacing existing practices prevalent in clinical diagnosis. However, applicability of MALDI-TOF MS in the area of microbial ecology research is still limited mainly due to the lack of data on non-clinical microorganisms. Intense research activities on cultivation of microbial diversity by conventional as well as by innovative and high-throughput methods has substantially increased the number of microbial species known today. This important area of research is in urgent need of rapid and reliable method(s) for characterization and de-replication of microorganisms from various ecosystems. MALDI-TOF MS based characterization, in our opinion, appears to be the most suitable technique for such studies. Reliability of MALDI-TOF MS based identification method depends mainly on accuracy and width of reference databases, which need continuous expansion and improvement. In this review, we propose a common strategy to generate MALDI-TOF MS spectral database and advocated its sharing, and also discuss the role of MALDI-TOF MS based high-throughput microbial identification in microbial ecology studies.
Rahi, Praveen; Prakash, Om; Shouche, Yogesh S.
Matrix-assisted laser desorption/ionization time-of-flight mass-spectrometry (MALDI-TOF MS) based biotyping is an emerging technique for high-throughput and rapid microbial identification. Due to its relatively higher accuracy, comprehensive database of clinically important microorganisms and low-cost compared to other microbial identification methods, MALDI-TOF MS has started replacing existing practices prevalent in clinical diagnosis. However, applicability of MALDI-TOF MS in the area of microbial ecology research is still limited mainly due to the lack of data on non-clinical microorganisms. Intense research activities on cultivation of microbial diversity by conventional as well as by innovative and high-throughput methods has substantially increased the number of microbial species known today. This important area of research is in urgent need of rapid and reliable method(s) for characterization and de-replication of microorganisms from various ecosystems. MALDI-TOF MS based characterization, in our opinion, appears to be the most suitable technique for such studies. Reliability of MALDI-TOF MS based identification method depends mainly on accuracy and width of reference databases, which need continuous expansion and improvement. In this review, we propose a common strategy to generate MALDI-TOF MS spectral database and advocated its sharing, and also discuss the role of MALDI-TOF MS based high-throughput microbial identification in microbial ecology studies. PMID:27625644
Ziegler, Dominik; Pothier, Joël F; Ardley, Julie; Fossou, Romain Kouakou; Pflüger, Valentin; de Meyer, Sofie; Vogel, Guido; Tonolla, Mauro; Howieson, John; Reeve, Wayne; Perret, Xavier
Accurate identification of soil bacteria that form nitrogen-fixing associations with legume crops is challenging given the phylogenetic diversity of root nodule bacteria (RNB). The labor-intensive and time-consuming 16S ribosomal RNA (rRNA) sequencing and/or multilocus sequence analysis (MLSA) of conserved genes so far remain the favored molecular tools to characterize symbiotic bacteria. With the development of mass spectrometry (MS) as an alternative method to rapidly identify bacterial isolates, we recently showed that matrix-assisted laser desorption ionization (MALDI) time-of-flight (TOF) can accurately characterize RNB found inside plant nodules or grown in cultures. Here, we report on the development of a MALDI-TOF RNB-specific spectral database built on whole cell MS fingerprints of 116 strains representing the major rhizobial genera. In addition to this RNB-specific module, which was successfully tested on unknown field isolates, a subset of 13 ribosomal proteins extracted from genome data was found to be sufficient for the reliable identification of nodule isolates to rhizobial species as shown in the putatively ascribed ribosomal protein masses (PARPM) database. These results reveal that data gathered from genome sequences can be used to expand spectral libraries to aid the accurate identification of bacterial species by MALDI-TOF MS.
Lin, Jian-feng; Chen, Qing-xi; Tian, Hong-yu; Gao, Xia; Yu, Mei-lan; Xu, Gen-jun; Zhao, Fu-kun
Visible stain is still the most popular protein staining method used in proteomic approaches. However, most published data have been derived from comparisons between visible dyes and fluorescent dyes. In this work, we have focused on seven widely used visible staining procedures--Neuhoff CCB, blue silver, and five silver stains (LKB SN, He SN, Yan SN, Vorum SN, and Blum SN)--and studied their stain efficiencies and MALDI-TOF MS compatibilities on 1-D and 2-D PAGE. It was concluded that blue silver is slightly better in terms of stain efficiency than Neuhoff CCB, but it presented worse MS compatibility. Neuhoff CCB presented better MS compatibility and superior linearity but worse sensitivity than silver stains. Among the five silvering procedures, He SN showed the best MS compatibility and a reasonable staining efficiency; Yan SN lowered the chances of obtaining the protein identity by PMF but gave the best stain efficiency; Vorum SN gave a very clear background and a great contrast, while Blum SN was the worst in this respect. The implications of these results for the selection of a convenient stain are discussed according to specific objectives as well as practical aspects.
Lou, Xianwen; de Waal, Bas F M; Milroy, Lech-Gustav; van Dongen, Joost L J
In matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS), analyte signals can be substantially suppressed by other compounds in the sample. In this technical note, we describe a modified thin-layer sample preparation method that significantly reduces the analyte suppression effect (ASE). In our method, analytes are deposited on top of the surface of matrix preloaded on the MALDI plate. To prevent embedding of analyte into the matrix crystals, the sample solution were prepared without matrix and efforts were taken not to re-dissolve the preloaded matrix. The results with model mixtures of peptides, synthetic polymers and lipids show that detection of analyte ions, which were completely suppressed using the conventional dried-droplet method, could be effectively recovered by using our method. Our findings suggest that the incorporation of analytes in the matrix crystals has an important contributory effect on ASE. By reducing ASE, our method should be useful for the direct MALDI MS analysis of multicomponent mixtures.
Zhu, Wenming; Sieradzki, Krzysztof; Albrecht, Valerie; McAllister, Sigrid; Lin, Wen; Stuchlik, Olga; Limbago, Brandi; Pohl, Jan; Kamile Rasheed, J
The Bruker Biotyper MALDI-TOF MS (Biotyper) system, with a modified 30 minute formic acid extraction method, was evaluated by its ability to identify 216 clinical Staphylococcus isolates from the CDC reference collection comprising 23 species previously identified by conventional biochemical tests. 16S rDNA sequence analysis was used to resolve discrepancies. Of these, 209 (96.8%) isolates were correctly identified: 177 (84.7%) isolates had scores ≥2.0, while 32 (15.3%) had scores between 1.70 and 1.99. The Biotyper identification was inconsistent with the biochemical identification for seven (3.2%) isolates, but the Biotyper identifications were confirmed by 16S rDNA analysis. The distribution of low scores was strongly species-dependent, e.g. only 5% of Staphylococcus epidermidis and 4.8% of Staphylococcus aureus isolates scored below 2.0, while 100% of Staphylococcus cohnii, 75% of Staphylococcus sciuri, and 60% of Staphylococcus caprae produced low but accurate Biotyper scores. Our results demonstrate that the Biotyper can reliably identify Staphylococcus species with greater accuracy than conventional biochemicals. Broadening of the reference database by inclusion of additional examples of under-represented species could further optimize Biotyper results.
Alharbi, Fahad J.; Geberhiwot, Tarekegn; Hughes, Derralynn A.; Ward, Douglas G.
Fabry disease is an X-linked lysosomal storage disorder caused by deficiency of α-galactosidase A, resulting in the accumulation of glycosphingolipids in various organs. Globotriaosylceramide (Gb3) and its isoforms and analogues have been identified and quantified as biomarkers of disease severity and treatment efficacy. The current study aimed to establish rapid methods for urinary Gb3 extraction and quantitation. Urine samples from 15 Fabry patients and 21 healthy control subjects were processed to extract Gb3 by mixing equal volumes of urine, methanol containing an internal standard, and chloroform followed by sonication and centrifugation. Thereafter, the lower phase was analyzed by MALDI-TOF MS and the relative peak areas of the internal standard and four major species of Gb3 determined. The results showed high reproducibility with intra- and inter-assay coefficients variation of 9.9% and 13.7%, respectively. The limit of detection was 0.15 ng/μL and the limit of quantitation was 0.30 ng/μL. Total urinary Gb3 levels in both genders of classic Fabry patients were significantly higher than in healthy controls (p < 0.0001). Gb3 levels in Fabry males were higher than in Fabry females (p = 0.08). We have established a novel assay for urinary total Gb3 that takes less than 15 min from start to finish.
Alharbi, Fahad J; Geberhiwot, Tarekegn; Hughes, Derralynn A; Ward, Douglas G
Fabry disease is an X-linked lysosomal storage disorder caused by deficiency of α-galactosidase A, resulting in the accumulation of glycosphingolipids in various organs. Globotriaosylceramide (Gb3) and its isoforms and analogues have been identified and quantified as biomarkers of disease severity and treatment efficacy. The current study aimed to establish rapid methods for urinary Gb3 extraction and quantitation. Urine samples from 15 Fabry patients and 21 healthy control subjects were processed to extract Gb3 by mixing equal volumes of urine, methanol containing an internal standard, and chloroform followed by sonication and centrifugation. Thereafter, the lower phase was analyzed by MALDI-TOF MS and the relative peak areas of the internal standard and four major species of Gb3 determined. The results showed high reproducibility with intra- and inter-assay coefficients variation of 9.9% and 13.7%, respectively. The limit of detection was 0.15 ng/μL and the limit of quantitation was 0.30 ng/μL. Total urinary Gb3 levels in both genders of classic Fabry patients were significantly higher than in healthy controls (p < 0.0001). Gb3 levels in Fabry males were higher than in Fabry females (p = 0.08). We have established a novel assay for urinary total Gb3 that takes less than 15 min from start to finish. Graphical Abstract ᅟ.
Hammarström, Björn; Nilson, Bo; Laurell, Thomas; Nilsson, Johan; Ekström, Simon
Matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is currently changing the clinical routine for identification of microbial pathogens. One important application is the rapid identification of bacteria for the diagnosis of bloodstream infections (BSI). A novel approach based on acoustic trapping and an integrated selective enrichment target (ISET) microchip that improves the sample preparation step for this type of analysis is presented. The method is evaluated on clinically relevant samples in the form of Escherichia coli infected blood cultures. It is shown that noncontact acoustic trapping enables capture, enrichment, and washing of bacteria directly from the complex background of crude blood cultures. The technology replaces centrifugation-based separation with a faster and highly automated sample preparation method that minimizes manual handling of hazardous pathogens. The presented method includes a solid phase extraction step that was optimized for enrichment of the bacterial proteins and peptides that are used for bacterial identification. The acoustic trapping-based assay provided correct identification in 12 out 12 cases of E. coli positive blood cultures with an average score of 2.19 ± 0.09 compared to 1.98 ± 0.08 when using the standard assay. This new technology opens up the possibility to automate and speed up an important and widely used diagnostic assay for bloodstream infections.
Roman, Eric; Lunde, Mai Lill Suhr; Miron, Talia; Warnakulasauriya, Saman; Johannessen, Anne Christine; Vasstrand, Endre Normann; Ibrahim, Salah Osman
In this study, two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionisation-time of flight mass spectrometry (MALDI-TOF-MS) technology was used to examine differentially expressed proteins in oral squamous cell carcinoma (OSCC) tissues from Norway (n=15) and the UK (n=45). Twenty-nine proteins were found to be significantly overexpressed in the OSCCs examined compared to the normal controls. Identified proteins included, family of annexin proteins that play important roles in signal transduction pathways and regulation of cellular growth, keratin-1, heat-shock proteins (HSP), squamous cell carcinoma antigen (SCC-Ag), cytoskeleton proteins, and proteins involved in mitochondrial and intracellular signalling pathways. The expression of four selected proteins (annexin II and V, HSP-27, and SCC-Ag) was verified using western blot analysis of 76 fresh tissue biopsy specimens in total, from Norway (n=53) and the UK (n=23). Proteomic analysis of OSCCs examined here demonstrated involvement of several proteins that might function as potential biomarkers and molecular targets for early cancer diagnostics, and may contribute to a novel approach to therapeutics and for predicting prognosis of OSCC.
Dong, Xiaoli; Cheng, Jinsheng; Li, Jinghong; Wang, Yinsheng
Graphene was utilized for the first time as matrix for the analysis of low-molecular weight compounds using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Polar compounds including amino acids, polyamines, anticancer drugs and nucleosides could be successfully analyzed. Additionally, nonpolar compounds including steroids could be detected with high resolution and sensitivity. Compared with conventional matrix, graphene exhibited high desorption/ionization efficiency for nonpolar compounds. The graphene matrix functions as substrate to trap analytes, and it transfers energy to the analytes upon laser irradiation, which allowed for the analytes to be readily desorbed/ionized and interference of intrinsic matrix ions to be eliminated. The use of graphene as matrix avoided the fragmentation of analytes and provided good reproducibility and high salt tolerance, underscoring the potential application of graphene as matrix for MALDI-MS analysis of practical samples in complex sample matrices. We also demonstrated that the use of graphene as adsorbent for the solid-phase extraction of squalene could improve greatly the detection limit. This work not only opens a new field for applications of graphene, but also offers a new technique for high-speed analysis of low-molecular weight compounds in areas such as metabolism research and natural products characterization. PMID:20565059
Driedger, D R; Sporns, P
A sample purification technique was developed for the detection of potato glycoalkaloids (GAs) in blood serum by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). GAs were extracted from spiked serum (5 mL) using a C(18) solid-phase extraction cartridge. The GAs were then selectively captured on antibody-coated agarose beads. The agarose beads were washed with water and the GAs eluted with 25 microL of methanol. MALDI-TOF MS was used to detect the GAs in the methanol eluent. Immunoaffinity sample purification of the GAs effectively reduced the signal suppression observed during the analysis of unpurified samples. alpha-Chaconine and alpha-solanine were detected in serum spiked with 1 ng/mL of each GA.
Terracciano, Rosa; Pasqua, Luigi; Casadonte, Francesca; Frascà, Stella; Preianò, Mariaimmacolata; Falcone, Daniela; Savino, Rocco
Matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) is a promising tool for large-scale screening of body fluids for the early detection of human diseases. Proteins, peptides, and metabolites present in cells, tissues, or in body fluids constitute the molecular signatures of individuals. The design and generation of material-based platforms for capturing molecular signatures from body fluids has gained increasing interest in recent years. Highly selective materials are attractive candidates for a wide range of applications in biofluid proteomics. We have therefore developed a procedure based on mesoporous silica particles for the selective binding and enrichment of low molecular weight plasma/serum proteins by MALDI MS analysis ( Terracciano, R., Gaspari, M., Testa, F., Pasqua, L., Cuda G., Tagliaferri, P., Cheng, M. C., Nijdam, A. J., Petricoin, E. F., Liotta, L. A., Ferrari, M., and Venuta, S. ( 2006 ) Selective binding and enrichment for low-molecular weight biomarker molecules in human plasma after exposure to nanoporous silica particles . Proteomics 6, 3243-3250 ). Mesoporous silica beads (MSB) are able to harvest peptides from plasma and serum by means of nanosized porous channels with high surface area, while excluding large size proteins. Moreover, the absorption properties can be modified since the pore walls can be functionalized with different chemical species due to the high concentration of silanol groups at the surface. In this study, we performed derivatization of MSB with different functionalities, and we evaluated the derivatized materials for plasma and urine peptidomic profiling. Aminopropyl, N-(2-aminoethyl)-3-aminopropyl, and N,N,N' tris-carboxymethyl ethylene diamine, have been introduced onto the mesoporous silica surfaces in order to modulate selective peptide enrichment. We also explored various experimental conditions in order to optimize the performance of chemically modified MSB in the peptide
Sandrin, Todd R; Goldstein, Jason E; Schumaker, Stephanie
Since the advent of the use of matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry (TOF MS) as a tool for microbial characterization, efforts to increase the taxonomic resolution of the approach have been made. The rapidity and efficacy of the approach have suggested applications in counter-bioterrorism, prevention of food contamination, and monitoring the spread of antibiotic-resistant bacteria. Strain-level resolution has been reported with diverse bacteria, using library-based and bioinformatics-enabled approaches. Three types of characterization at the strain level have been reported: strain categorization, strain differentiation, and strain identification. Efforts to enhance the library-based approach have involved sample pre-treatment and data reduction strategies. Bioinformatics approaches have leveraged the ever-increasing amount of publicly available genomic and proteomic data to attain strain-level characterization. Bioinformatics-enabled strategies have facilitated strain characterization via intact biomarker identification, bottom-up, and top-down approaches. Rigorous quantitative and advanced statistical analyses have fostered success at the strain level with both approaches. Library-based approaches can be limited by effects of sample preparation and culture conditions on reproducibility, whereas bioinformatics-enabled approaches are typically limited to bacteria, for which genetic and/or proteomic data are available. Biological molecules other than proteins produced in strain-specific manners, including lipids and lipopeptides, might represent other avenues by which strain-level resolution might be attained. Immunological and lectin-based chemistries have shown promise to enhance sensitivity and specificity. Whereas the limits of the taxonomic resolution of MALDI TOF MS profiling of bacteria appears bacterium-specific, recent data suggest that these limits might not yet have been reached.
Ren, Shi-fang; Zhang, Li; Cheng, Zhi-hong; Guo, Yin-long
In this work, we reported on the advantages of immobilized carbon nanotubes as a novel MALDI-matrix. Recently, carbon nanotubes have been reported to be an effective MALDI matrix for small molecules (Anal. Chem.2003, 75, 6191), as it can eliminate the interfering matrix peaks as well as form a web morphology to fully disperse the analyte and allow strong ultraviolet absorption for enhanced pulsed laser desorption and ionization. In our study, to overcome the problem that the carbon nanotube matrix may fly off from the target, a type of polyurethane adhesive, NIPPOLAN-DC-205, is introduced to immobilize carbon nanotubes on the target, which enables widespread application of carbon nanotubes as matrix for MALDI-MS analysis. At the same time, the properties of the carbon nanotubes as an efficient matrix remained after immobilization. The presence of NIPPOLAN-DC-205 increases the time for analysis at a particular desorption spot by minimizing the time-consuming search for "hot spots" and facilitating experiments such as post source decay (PSD) which need longer-lasting signals. Moreover, NIPPOLAN-DC-205 produces no interference peaks and can easily be cleaned with acetone. Fast evaporation technology may be used to enhance signal reproducibility in MALDI analysis using carbon nanotubes as matrix. Consequently, the applicability of the carbon nanotube as matrix for matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analysis of low molecular mass analytes is highly improved. The feasibility of the method employing polyurethane is demonstrated by comparison of the results produced from the carbon nanotube matrix with and without immobilization. In addition, neutral small carbohydrates, which are difficult to be ionized normally, can be cationized with high efficiency by MALDI-TOF-MS using the immobilized carbon nanotube matrix. The method was further applied to analyze peptides and detect urine glucose successfully.
Verroken, A; Defourny, L; Lechgar, L; Magnette, A; Delmée, M; Glupczynski, Y
Speeding up the turn-around time of positive blood culture identifications is essential in order to optimize the treatment of septic patients. Several sample preparation techniques have been developed allowing direct matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) identification of positive blood cultures. Yet, the hands-on time restrains their routine workflow. In this study, we evaluated an approach whereby MALDI-TOF MS identification without any additional steps was carried out on short subcultured colonies from positive blood bottles with the objective of allowing results reporting on the day of positivity detection. Over a 7-month period in 2012, positive blood cultures detected by 9 am with an automated system were inoculated onto a Columbia blood agar and processed after a 5-h incubation on a MALDI-TOF MicroFlex platform (Bruker Daltonik GmbH). Single-spotted colonies were covered with 1 μl formic acid and 1 μl matrix solution. The results were compared to the validated identification techniques. A total of 925 positive blood culture bottles (representing 470 bacteremic episodes) were included. Concordant identification was obtained in 727 (81.1 %) of the 896 monomicrobial blood cultures, with failure being mostly observed with anaerobes and yeasts. In 17 episodes of polymicrobic bacteremia, the identification of one of the two isolates was achieved in 24/29 (82.7 %) positive cultures. Routine implementation of MALDI-TOF MS identification on young positive blood subcultures provides correct results to the clinician in more than 80 % of the bacteremic episodes and allows access to identification results on the day of blood culture positivity detection, potentially accelerating the implementation of targeted clinical treatments.
Brauer, Heather Ann; Lampe, Paul D.; Yasui, Yutaka Y.; Hamajima, Nobuyuki; Stolowitz, Mark L.
A novel approach to immunoaffinity MS is described wherein antibodies are appended to a patterned gold Biochip surface. The Biochip surface is patterned with an array of concentric immunocapture zones comprised of highly hydrophilic central zones surrounded by moderately hydrophilic zones that reside on a non-wetting background, with protein attachment via electrochemically cleavable linkers. After linker cleavage, matrix application forms a discrete spot suitable for MALDI-TOF-MS. Use of the Biochip to purify Transthyretin from human serum allowed distinct resolution of four disulfide conjugates and one truncated form isoforms with good mass resolution and sensitivity. PMID:20957758
Lobasso, Simona; Lopalco, Patrizia; Angelini, Roberto; Vitale, Rita; Huber, Harald; Müller, Volker; Corcelli, Angela
The lipidome of the marine hyperthermophilic archaeon Pyrococcus furiosus was studied by means of combined thin-layer chromatography and MALDI-TOF/MS analyses of the total lipid extract. 80–90% of the major polar lipids were represented by archaeol lipids (diethers) and the remaining part by caldarchaeol lipids (tetraethers). The direct analysis of lipids on chromatography plate showed the presence of the diphytanylglycerol analogues of phosphatidylinositol and phosphatidylglycerol, the N-acetylglucosamine-diphytanylglycerol phosphate plus some caldarchaeol lipids different from those previously described. In addition, evidence for the presence of the dimeric ether lipid cardiolipin is reported, suggesting that cardiolipins are ubiquitous in archaea. PMID:23193375
Lacroix, C; Gicquel, A; Sendid, B; Meyer, J; Accoceberry, I; François, N; Morio, F; Desoubeaux, G; Chandenier, J; Kauffmann-Lacroix, C; Hennequin, C; Guitard, J; Nassif, X; Bougnoux, M-E
Candida spp. are responsible for severe infections in immunocompromised patients and those undergoing invasive procedures. The accurate identification of Candida species is important because emerging species can be associated with various antifungal susceptibility spectra. Conventional methods have been developed to identify the most common pathogens, but have often failed to identify uncommon species. Several studies have reported the efficiency of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for the identification of clinically relevant Candida species. In this study, we evaluated two commercially available MALDI-TOF systems, Andromas™ and Bruker Biotyper™, for Candida identification in routine diagnosis. For this purpose, we investigated 1383 Candida isolates prospectively collected in eight hospital laboratories during routine practice. MALDI-TOF MS results were compared with those obtained using conventional phenotypic methods. Analysis of rDNA gene sequences with internal transcribed regions or D1-D2 regions is considered the reference standard for identification. Both MALDI-TOF MS systems could accurately identify 98.3% of the isolates at the species level (1359/1383 for Andromas™; 1360/1383 for Bruker Biotyper™) vs. 96.5% for conventional techniques. Furthermore, whereas conventional methods failed to identify rare or emerging species, these were correctly identified by MALDI-TOF MS. Both MALDI-TOF MS systems are accurate and cost-effective alternatives to conventional methods for mycological identification of clinically relevant Candida species and should improve the diagnosis of fungal infections as well as patient management.
Rodríguez-Sánchez, Belén; Alcalá, Luis; Marín, Mercedes; Ruiz, Adrián; Alonso, Elena; Bouza, Emilio
Information regarding the use of MALDI-TOF MS as an alternative to conventional laboratory methods for the rapid and reliable identification of bacterial isolates is still limited. In this study, MALDI-TOF MS was evaluated on 295 anaerobic isolates previously identified by 16S rRNA gene sequencing and with biochemical tests (Rapid ID 32A system, BioMérieux). In total, 85.8% of the isolates were identified by MALDI-TOF MS at the species level vs 49.8% using the Rapid ID 32A system (p < 0.0001). None of the isolates was discordantly identified at the genus level using MALDI-TOF MS and only 9 of them could not be identified using the method. Thus, our results show that MALDI-TOF MS is a robust and reliable tool for the identification of anaerobic isolates in the microbiology laboratory. Its implementation will reduce the turnaround time for a final identification and the number of isolates that require 16S rRNA sequencing.
Avila, C C; Almeida, F G; Palmisano, G
Accurate and rapid determination of trypanosomatids is essential in epidemiological surveillance and therapeutic studies. Matrix-assisted laser desorption ionization/time of flight mass spectrometry (MALDI-TOF MS) has been shown to be a useful and powerful technique to identify bacteria, fungi, metazoa and human intact cells with applications in clinical settings. Here, we developed and optimized a MALDI-TOF MS method to profile trypanosomatids. trypanosomatid cells were deposited on a MALDI target plate followed by addition of matrix solution. The plate was then subjected to MALDI-TOF MS measurement to create reference mass spectra library and unknown samples were identified by pattern matching using the BioTyper software tool. Several m/z peaks reproducibly and uniquely identified trypanosomatids species showing the potentials of direct identification of trypanosomatids by MALDI-TOF MS. Moreover, this method discriminated different life stages of Trypanosoma cruzi, epimastigote and bloodstream trypomastigote and Trypanosoma brucei, procyclic and bloodstream. T. cruzi Discrete Typing Units (DTUs) were also discriminated in three clades. However, it was not possible to achieve enough resolution and software-assisted identification at the strain level. Overall, this study shows the importance of MALDI-TOF MS for the direct identification of trypanosomatids and opens new avenues for mass spectrometry-based detection of parasites in biofluids. Copyright © 2016 John Wiley & Sons, Ltd.
Brand, Henk; Imangaliyev, Sultan; Tsivtsivadze, Evgeni; van der Weijden, Fridus; de Jong, Ad; Paauw, Armand; Crielaard, Wim; Keijser, Bart; Veerman, Enno
A cross-sectional observational study was conducted to evaluate the inter-individual variation in the MALDI-TOF MS peptide profiles of unstimulated whole saliva in a population of 268 systemically healthy adults aged 18–30 yr (150 males and 118 females) with no apparent caries lesions or periodontal disease. Using Spectral Clustering, four subgroups of individuals were identified within the study population. These subgroups were delimited by the pattern of variation in 9 peaks detected in the 2–15 kDa m/z range. An Unsupervised Feature Selection algorithm showed that P-C peptide, a 44 residue-long salivary acidic proline-rich protein, and three of its fragments (Fr. 1–25, Fr. 15–35 and Fr. 15–44) play a central role in delimiting the subgroups. Significant differences were found in the salivary biochemistry of the subgroups with regard to lysozyme and chitinase, two enzymes that are part of the salivary innate defense system (p < 0.001). These results suggest that MALDI-TOF MS salivary peptide profiles may relate information on the underlying state of the oral ecosystem and may provide a useful reference for salivary disease biomarker discovery studies. PMID:27258023
Schurr, Benjamin C; Behr, Jürgen; Vogel, Rudi F
Due to the harsh environment, microorganisms encounter in beer, spoilage bacteria must be able to customise their metabolism and physiology in an order to master various kinds of perturbations. Proteomic approaches have been used to examine differences between various beer spoilage bacteria and between different stress conditions, such as acid and hop (Humulus lupulus) stress. However, these investigations cannot detect changes in low molecular weight (lmw) proteins (<150 amino acids). Therefore, for the first time, we herein present data from a proteomic study of lmw proteins for two Lactobacillus (L.) brevis strains exposed to acid stress or, respectively, two different qualities of hop induced stress. We used MALDI-TOF MS as analytical tool for the detection of lmw stress response proteins due to its high sensitivity and low throughput times. Comparing a hop-sensitive and a hop-tolerant strain, detection of the fatty acid biosynthesis-associated acyl carrier protein varied between different stress conditions and incubation times. The findings coincide with previous studies of our group regarding the fatty acid cell membrane composition of beer spoiling L. brevis. It is demonstrated that MALDI-TOF MS is a fast tool to detect and characterise stress situations in beer spoiling bacteria along the lmw sub-proteome.
Ma, Yu-rong; Zhang, Xiao-le; Zeng, Tao; Cao, Dong; Zhou, Zhen; Li, Wen-hui; Niu, Hongyun; Cai, Ya-qi
Polydopamine-coated Fe(3)O(4) nanoparticles (Fe(3)O(4)@PDA NPs) were synthesized and applied as matrix for the detection of pollutants by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The synthesis of Fe(3)O(4)@PDA NPs was accomplished in 30 min by in situ polymerization of dopamine without any toxic reagent. Using Fe(3)O(4)@PDA NPs as matrix of MALDI-TOF, eleven small molecule pollutants (molecular weight from 251.6 to 499.3), including Benzo(a)pyrene (BaP), three perfluorinated compounds (PFCs), and seven antibiotics, were successfully detected in either positive or negative reflection mode without background interference. Furthermore, the Fe(3)O(4)@PDA NPs can also enrich trace amounts of hydrophobic target, such as BaP, from solution to nanoparticles surface. Then the Fe(3)O(4)@PDA-BaP can be isolated through magnetic sedimentation step and directly spotted on the stainless steel plate for MALDI measurement. With Fe(3)O(4)@PDA NPs as adsorbent and matrix, we also realized the analysis of BaP in tap water and lake water samples. Thus, a magnetic solid-phase extraction (MSPE)-MALDI-TOF-MS method was established for the measurement of BaP.
Wang, Baojie; Liu, Mei; Jiang, Keyong; Zhang, Guofan; Wang, Lei
Peptides in shrimp hemolymph play an important role in the innate immune response. Analysis of hemolymph will help to detect and identify potential novel biomarkers of microbial infection. We used magnetic bead-based purification (ClinProt system) and matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) to characterize shrimp hemolymph peptides. Shrimp serum and plasma were used as the source of samples for comparative analysis, and it was found that serum was more suitable for shrimp hemolymph peptidomic analysis. To screen potential specific biomarkers in serum of immune-challenged shrimps, we applied magnetic bead-based MALDI-TOF MS to serum samples from 10 immune-challenged and 10 healthy shrimps. The spectra were analyzed using FlexAnalysis 3.0 and ClinProTools 2.1 software. Thirteen peptide peaks significantly different between the two groups were selected as candidate biomarkers of lipopolysaccharide (LPS)-infection. The diagnostic model established by genetic algorithm using five of these peaks was able to discriminate LPS-challenged shrimps from healthy control shrimps with a sensitivity of 90% and a specificity of 100%. Our approach in MALDITOF MS-based peptidomics is a powerful tool for screening bioactive peptides or biomarkers derived from hemolymph, and will help to enable a better understanding of the innate immune response of shrimps.
Calvano, Cosima Damiana; De Ceglie, Cristina; Monopoli, Antonio; Zambonin, Carlo Giorgio
In dairy field, one of the most common frauds is the adulteration of higher value types of milk (sheep's and goat's) with milk of lower value (cow's milk). This illegal practice has an economic advantage for milk producers and poses a threat for consumers' health because of the presence of hidden allergens as, for example, cow milk proteins, in particular, α(s1)-casein and β-lactoglobulin. The urgent need of sensitive techniques to detect this kind of fraud brought to the development of chromatographic, immunoenzymatic, electrophoretic and mass spectrometric assays. In the current work, we present a fast, reproducible and sensitive method based on the direct matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) MS analysis of milk tryptic digests for the detection of milk adulteration by evaluating specie-specific markers in the peptide profiles. Several pure raw and commercial milk samples and binary mixtures containing cows' and goats', cows' and sheep's and goats' and sheep's milk (concentrations of each milk varied from 0% to 100%) were prepared, and tryptic digests were analyzed by MALDI-TOF MS. The use of the new MALDI matrix α-cyano-4-chlorocinnamic acid allowed to detect cow and goat milk peptide markers up to 5% level of adulteration. Finally, from preliminary data, it seems that the strategy could be successfully applied also to detect similar adulterations in cheese samples.
Liu, Junyan; Liu, Yang; Gao, Mingxia; Zhang, Xiangmin
In this work, a new pre-analysis method for tetracyclines (TCs) detection from the milk samples was established. As a good accomplishment for the existing accurate quantification strategies for TCs detection, the new pre-analysis method was demonstrated to be simple, sensitive, fast, cost effective, and high throughput, which would do a great favor to the routine quality pre-analysis of TCs from milk samples. Graphene or graphene oxide was utilized, for the first time, as a duel-platform to enrich and detect the TCs by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). All together, four TCs were chosen as models: tetracycline, oxytetracycline, demeclocycline, and chlortetracycline. Due to the excellent electronic, thermal, and mechanical properties, graphene and graphene oxide were successfully applied as matrices for MALDI-TOF MS with free background inference in low mass range. Meanwhile, graphene or graphene oxide has a large surface area and strong interaction force with the analytes. By taking the advantage of these features, TCs were effectively enriched with the limit of detection (LOD) as low as 2 nM.
Shimazaki, Youji; Nishimura, Yuri; Saito, Masaki
A combination of methods is required to achieve separation of intact proteins and subsequently perform structure analysis to examine their unstable or external structures. The aim of this study was to develop a method of structure analysis in intact proteins after purification. Transferrin from human plasma was trapped by membrane-immobilized anti-transferrin antibody, which was produced by non-denaturing two-dimensional electrophoresis (2-DE), and transferred to a polyvinylidene fluoride (PVDF) membrane and stained with Ponceau S. The antigen transferrin was eluted by rinsing the membrane with trifluoroacetic acid (TFA) or aspartic acid. In addition, a method was established by which the purified human transferrin was enzymatically digested on a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) target plate. Thus, after purification of the human transferrin antigen from tens of microlitres of human plasma using an immunoaffinity membrane, transferrin polypeptide fragments were obtained on the plate following digestion with pepsin in the presence of 0.1% TFA or endoproteinase Lys-C or Lys-C/trypsin with 0.001% sodium dodecyl sulphate (SDS). The results indicated that the combined methods of isolation using an immunoaffinity membrane and enzymatic digestion on a MALDI-TOF MS plate could be applied to the purification and microanalysis of antigens. This approach would be particularly applicable to the analysis of the primary structure and the less stable and highly accessible regions of antigens from limited sample volumes.
Veloo, A C M; de Vries, E D; Jean-Pierre, H; Justesen, U S; Morris, T; Urban, E; Wybo, I; van Winkelhoff, A J
Gram-positive anaerobic cocci (GPAC) account for 24%-31% of the anaerobic bacteria isolated from human clinical specimens. At present, GPAC are under-represented in the Biotyper MALDI-TOF MS database. Profiles of new species have yet to be added. We present the optimization of the matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS) database for the identification of GPAC. Main spectral profiles (MSPs) were created for 108 clinical GPAC isolates. Identity was confirmed using 16S rRNA gene sequencing. Species identification was considered to be reliable if the sequence similarity with its closest relative was ≥98.7%. The optimized database was validated using 140 clinical isolates. The 16S rRNA sequencing identity was compared with the MALDI-TOF MS result. MSPs were added from 17 species that were not yet represented in the MALDI-TOF MS database or were under-represented (fewer than five MSPs). This resulted in an increase from 53.6% (75/140) to 82.1% (115/140) of GPAC isolates that could be identified at the species level using MALDI-TOF MS. An improved log score was obtained for 51.4% (72/140) of the strains. For strains with a sequence similarity <98.7% with their closest relative (n = 5) or with an inconclusive sequence identity (n = 4), no identification was obtained by MALDI-TOF MS or in the latter case an identity with one of its relatives. For some species the MSP of the type strain was not part of the confined cluster of the corresponding clinical isolates. Also, not all species formed a homogeneous cluster. It emphasizes the necessity of adding sufficient MSPs of human clinical isolates.
Saracli, Mehmet A; Fothergill, Annette W; Sutton, Deanna A; Wiederhold, Nathan P
MALDI-TOF MS can rapidly identify microorganisms to the species level and may be able to detect antimicrobial resistance. We evaluated the ability of this technology to detect triazole resistance in Candida species.35 C. albicans, 35 C. glabrata, and 37 C. tropicalis strains were exposed to fluconazole, voriconazole, or posaconazole at two different concentrations plus a drug-free control: a midrange concentration (CLSI clinical breakpoint or epidemiologic cut-off value), and a high concentration (fluconazole 64 μg/ml, voriconazole & posaconazole 16 μg/ml). The MALDI-TOF MS spectra at these concentrations were used to create the individual composite correlation index (CCI) matrices for each isolate. When the CCI of the midrange/highest concentration was lower than that of the midrange/null concentration, the strain was classified as resistant. These results were then compared to the classifications for susceptible or resistant obtained by measuring the MICs according to the CLSI M27-A3 antifungal susceptibility testing (AFST) method.The MALDI-TOF MS assay was able to classify triazole susceptibility against all strains. Overall, essential agreement between MALDI-TOF MS and AFST varied between 54% and 97%, and was highest for posaconazole against C. glabrata. The reproducibility of the MALDI-TOF MS assay varied between 54.3 and 82.9% and was best for fluconazole against C. albicans and posaconazole against C. glabrata. Reproducibility was also higher for C. glabrata isolates compared to C. albicans and C. tropicalis.These results demonstrate that MALDI-TOF MS may be used to simultaneously determine the Candida species and classification as susceptible or resistant to triazole antifungals. Further studies are needed to refine the methodology and improve the reproducibility of this assay.
Shi, X L; Li, C W; Liang, B C; He, K H; Li, X Y
We investigated weak cation magnetic separation technology and matrix-assisted laser desorption ionization-time of flight-mass spectrometry (MALDI-TOF-MS) in screening serum protein markers of primary type I osteoporosis. We selected 16 postmenopausal women with osteoporosis and nine postmenopausal women as controls to find a new method for screening biomarkers and establishing a diagnostic model for primary type I osteoporosis. Serum samples were obtained from controls and patients. Serum protein was extracted with the WCX protein chip system; protein fingerprints were examined using MALDI-TOF-MS. The preprocessed and model construction data were handled by the ProteinChip system. The diagnostic models were established using a genetic arithmetic model combined with a support vector machine (SVM). The SVM model with the highest Youden index was selected. Combinations with the highest accuracy in distinguishing different groups of data were selected as potential biomarkers. From the two groups of serum proteins, 123 cumulative MS protein peaks were selected. Significant intensity differences in the protein peaks of 16 postmenopausal women with osteoporosis were screened. The difference in Youden index between the four groups of protein peaks showed that the highest peaks had mass-to-charge ratios of 8909.047, 8690.658, 13745.48, and 15114.52. A diagnosis model was established with these four markers as the candidates, and the model specificity and sensitivity were found to be 100%. Two groups of specimens in the SVM results on the scatterplot were distinguishable. We established a diagnosis model, and provided a new serological method for screening and diagnosis of osteoporosis with high sensitivity and specificity.
Xiao, Di; Ye, Changyun; Zhang, Huifang; Kan, Biao; Lu, Jingxing; Xu, Jianguo; Jiang, Xiugao; Zhao, Fei; You, Yuanhai; Yan, Xiaomei; Wang, Duochun; Hu, Yuan; Zhang, Maojun; Zhang, Jianzhong
Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is an emerging technique for the rapid and high-throughput identification of microorganisms. There remains a dearth of studies in which a large number of pathogenic microorganisms from a particular country or region are utilized for systematic analyses. In this study, peptide mass reference spectra (PMRS) were constructed and evaluated from numerous human pathogens (a total of 1019 strains from 94 species), including enteric (46 species), respiratory (21 species), zoonotic (17 species), and nosocomial pathogens (10 species), using a MALDI-TOF MS Biotyper system (MBS). The PMRS for 380 strains of 52 species were new contributions to the original reference database (ORD). Compared with the ORD, the new reference database (NRD) allowed for 28.2% (from 71.5% to 99.7%) and 42.3% (from 51.3% to 93.6%) improvements in identification at the genus and species levels, respectively. Misidentification rates were 91.7% and 57.1% lower with the NRD than with the ORD for genus and species identification, respectively. Eight genera and 25 species were misidentified. For genera and species that are challenging to accurately identify, identification results must be manually determined and adjusted in accordance with the database parameters. Through augmentation, the MBS demonstrated a high identification accuracy and specificity for human pathogenic microorganisms. This study sought to provide theoretical guidance for using PMRS databases in various fields, such as clinical diagnosis and treatment, disease control, quality assurance, and food safety inspection. PMID:25181391
Chang, Susane; Porto Carneiro-Leão, Mariele; Ferreira de Oliveira, Benny; Souza-Motta, Cristina; Lima, Nelson; Santos, Cledir; Tinti de Oliveira, Neiva
Fusarium verticillioides is considered one of the most important global sources of fumonisins contamination in food and feed. Corn is one of the main commodities produced in the Northeastern Region of Brazil. The present study investigated potential mycotoxigenic fungal strains belonging to the F. verticillioides species isolated from corn kernels in 3 different Regions of the Brazilian State of Pernambuco. A polyphasic approach including classical taxonomy, molecular biology, MALDI-TOF MS and MALDI-TOF MS/MS for the identification and characterisation of the F. verticillioides strains was used. Sixty F. verticillioides strains were isolated and successfully identified by classical morphology, proteomic profiles of MALDI-TOF MS, and by molecular biology using the species-specific primers VERT-1 and VERT-2. FUM1 gene was further detected for all the 60 F. verticillioides by using the primers VERTF-1 and VERTF-2 and through the amplification profiles of the ISSR regions using the primers (GTG)5 and (GACA)4. Results obtained from molecular analysis shown a low genetic variability among these isolates from the different geographical regions. All of the 60 F. verticillioides isolates assessed by MALDI-TOF MS/MS presented ion peaks with the molecular mass of the fumonisin B1 (721.83 g/mol) and B2 (705.83 g/mol). PMID:26927172
Gorton, Rebecca L; Ramnarain, P; Barker, K; Stone, N; Rattenbury, S; McHugh, T D; Kibbler, C C
Fungaemia diagnosis could be improved by reducing the time to identification of yeast from blood cultures. This study aimed to evaluate three rapid methods for the identification of yeast direct from blood cultures; Gram's stain analysis, the AdvanDX Peptide Nucleic Acid in Situ Hybridisation Yeast Traffic Light system (PNA-FISH YTL) and Bruker Sepsityper alongside matrix-assisted laser desorption ionisation time of flight mass spectrometry (MALDI-TOF MS). Fifty blood cultures spiked with a known single yeast strain were analysed by blinded operators experienced in each method. Identifications were compared with MALDI-TOF MS CHROMagar Candida culture and ITS rRNA sequence-based identifications. On first attempt, success rates of 96% (48/50) and 76% (36/50) were achieved using PNA-FISH YTL and Gram's stain respectively. MALDI-TOF MS demonstrated a success rate of 56% (28/50) when applying manufacturer's species log score thresholds and 76% (38/50) using in-house parameters, including lowering the species log score threshold to >1.5. In conclusion, PNA-FISH YTL demonstrated a high success rate successfully identifying yeast commonly encountered in fungaemia. Sepsityper(™) with MALDI-TOF MS was accurate but increased sensitivity is required. Due to the misidentification of commonly encountered yeast Gram's stain analysis demonstrated limited utility in this setting.
Grant, G A; Frison, S L; Sporns, P
Sulfamethazine (SMT) and its major metabolite, N(4)-acetylsulfamethazine (NA-SMT), were each recovered from spiked water (0.1 ppb) and 10% (w/v) aqueous suspensions of soil (1 ppb) or composted manure (1 ppb), by using a three-stage solid phase immunoextraction (SPIE) system, followed by detection with matrix-assisted laser/desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Sulfonamide recovery rates are reported for separate stages of the SPIE system and for trace-level sulfonamide SPIE extraction from the environmental samples. SPIE MALDI-TOF MS is a rapid and definitive technique with potentially better efficiency relative to other established trace-level sulfonamide analytical methods. SPIE MALDI-TOF MS required 1.5 h per batch (8-24 samples/batch) for sample enrichment, 5 min per batch for probe preparation, and 5 min per sample to acquire and process the spectrum. This is the first time MALDI-TOF MS has been reported as a potential means of detecting trace-level drug residues in complex environmental samples.
Calderaro, Adriana; Arcangeletti, Maria Cristina; Rodighiero, Isabella; Buttrini, Mirko; Montecchini, Sara; Vasile Simone, Rosita; Medici, Maria Cristina; Chezzi, Carlo; De Conto, Flora
In this study matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS), a reliable identification method for the diagnosis of bacterial and fungal infections, is presented as an innovative tool to investigate the protein profile of cell cultures infected by the most common viruses causing respiratory tract infections in humans. MALDI-TOF MS was applied to the identification of influenza A and B viruses, adenovirus C species, parainfluenza virus types 1, 2 and 3, respiratory syncytial virus, echovirus, cytomegalovirus and metapneumovirus. In this study MALDI-TOF MS was proposed as a model to be applied to the identification of cultivable respiratory viruses using cell culture as a viral proteins enrichment method to the proteome profiling of virus infected and uninfected cell cultures. The reference virus strains and 58 viruses identified from respiratory samples of subjects with respiratory diseases positive for one of the above mentioned viral agents by cell culture were used for the in vitro infection of suitable cell cultures. The isolated viral particles, concentrated by ultracentrifugation, were used for subsequent protein extraction and their spectra profiles were generated by MALDI-TOF MS analysis. The newly created library allowed us to discriminate between uninfected and respiratory virus infected cell cultures. PMID:27786297
Eibisch, Mandy; Fuchs, Beate; Schiller, Jurgen; Sub, Rosmarie; Teuber, Kristin
Matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS) is increasingly used to investigate the phospholipid (PL) compositions of tissues and body fluids, often without previous separation of the total mixture into the individual PL classes. Therefore, the questions of whether all PL classes are detectable…
Martiny, D; Cremagnani, P; Gaillard, A; Miendje Deyi, V Y; Mascart, G; Ebraert, A; Attalibi, S; Dediste, A; Vandenberg, O
The mutualisation of analytical platforms might be used to address rising healthcare costs. Our study aimed to evaluate the feasibility of networking a unique matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) system for common use in several university hospitals in Brussels, Belgium. During a one-month period, 1,055 successive bacterial isolates from the Brugmann University Hospital were identified on-site using conventional techniques; these same isolates were also identified using a MALDI-TOF MS system at the Porte de Hal Laboratory by sending target plates and identification projects via transportation and the INFECTIO_MALDI software (Infopartner, Nancy, France), respectively. The occurrence of transmission problems (<2 %) and human errors (<1 %) suggested that the system was sufficiently robust to be implemented in a network. With a median time-to-identification of 5 h and 11 min (78 min, min-max: 154-547), MALDI-TOF MS networking always provided a faster identification result than conventional techniques, except when chromogenic culture media and oxidase tests were used (p < 0.0001). However, the limited clinical benefits of the chromogenic culture media do not support their extra cost. Our financial analysis also suggested that MALDI-TOF MS networking could lead to substantial annual cost savings. MALDI-TOF MS networking presents many advantages, and few conventional techniques (optochin and oxidase tests) are required to ensure the same quality in patient care from the distant laboratory. Nevertheless, such networking should not be considered unless there is a reorganisation of workflow, efficient communication between teams, qualified technologists and a reliable IT department and helpdesk to manage potential connectivity problems.
Carrasco, Gema; de Dios Caballero, Juan; Garrido, Noelia; Valdezate, Sylvia; Cantón, Rafael; Sáez-Nieto, Juan A
Nocardia species are difficult to identify, a consequence of the ever increasing number of species known and their homogeneous genetic characteristics. 16S rRNA analysis has been the gold standard for identifying these organisms, but proteomic techniques such as matrix-assisted laser desorption ionization-time of flight (MALDI-TOF MS) and housekeeping gene analysis, have also been explored. One hundred high (n = 25), intermediate (n = 20), and low (n = 55) prevalence (for Spain) Nocardia strains belonging to 30 species were identified via 16S rRNA and MALDI-TOF MS analysis. The manufacturer-provided database MALDI Biotyper library v4.0 (5.627 entries, Bruker Daltonik) was employed. In the high prevalence group (Nocardia farcinica, N. abscessus, N. cyriacigeorgica and N. nova), the 16S rRNA and MALDI-TOF MS methods provided the same identification for 76% of the strains examined. For the intermediate prevalence group (N. brasiliensis, N. carnea, N. otitidiscaviarum and N. transvalensis complex), this figure fell to 45%. In the low-prevalence group (22 species), these two methods were concordant only in six strains at the species level. Tetra-gene multi-locus sequencing analysis (MLSA) involving the concatemer gyrB-16S rRNA-hsp65-secA1 was used to arbitrate between discrepant identifications (n = 67). Overall, the MLSA confirmed the results provided at species level by 16S rRNA analysis in 34.3% of discrepancies, and those provided by MALDI-TOF MS in 13.4%. MALDI-TOF MS could be a strong candidate for the identification of Nocardia species, but only if its reference spectrum database improves, especially with respect to unusual, recently described species and species included in the described Nocardia complexes.
Altun, Osman; Botero-Kleiven, Silvia; Carlsson, Sarah; Ullberg, Måns; Özenci, Volkan
Rapid identification of bacteria from blood cultures enables early initiation of appropriate antibiotic treatment in patients with bloodstream infections (BSI). The objective of the present study was to evaluate the use of matrix-associated laser desorption ionization-time of flight (MALDI-TOF) MS after a short incubation on solid media for rapid identification of bacteria from positive blood culture bottles. MALDI-TOF MS was performed after 2.5 and 5.5 h plate incubation of samples from positive blood cultures. Identification scores with values ≥ 1.7 were accepted as successful identification if the results were confirmed by conventional methods. Conventional methods included MALDI-TOF MS, Vitek 2, and diverse biochemical and agglutination tests after overnight culture. In total, 515 positive blood cultures with monomicrobial bacterial growth representing one blood culture per patient were included in the study. There were 229/515 (44.5%) and 286/515 (55.5%) blood culture bottles with Gram-negative bacteria (GNB) and Gram-positive bacteria (GPB), respectively. MALDI-TOF MS following short-term culture could accurately identify 300/515 (58.3%) isolates at 2.5 h, GNB being identified in greater proportion (180/229; 78.6%) than GPB (120/286; 42.0%). In an additional 124/515 bottles (24.1%), identification was successful at 5.5 h, leading to accurate identification of bacteria from 424/515 (82.3%) blood cultures after short-term culture. Interestingly, 11/24 of the isolated anaerobic bacteria could be identified after 5.5 h. The present study demonstrates, in a large number of clinical samples, that MALDI-TOF MS following short-term culture on solid medium is a reliable and rapid method for identification of bacteria from blood culture bottles with monomicrobial bacterial growth.
Kanerva, Sanna; Ketola, Raimo A.; Kitunen, Veikko; Smolander, Aino; Kotiaho, Tapio
Nitrogen (N) is usually the nutrient restricting productivity in boreal forests. Forest soils contain a great amount of nitrogen, but only a small part of it is in mineral form. Most part of soil N is bound in the structures of different organic compounds such as proteins, peptides, amino acids and more stabilized, refractory compounds. Due to the fact that soil organic N has a very important role in soil nutrient cycling and in plant nutrition, there is a need for more detailed knowledge of its chemistry in soil. Conventional methods to extract and analyze soil organic N are usually very destructive for structures of higher molecular weight organic compounds, such as proteins. The aim of this study was to characterize proteins extracted from boreal forest soil by "soft" extraction methods in order to maintain their molecular structure. The organic layer (F) from birch forest floor containing 78% of organic matter was sieved, freeze dried, pulverized, and extracted with a citrate or phosphate buffer (pH 6 or 8). Sequential extraction with the citrate or phosphate buffer and an SDS buffer (pH 6.8), slightly modified from the method of Chen et al. (2009, Proteomics 9: 4970-4973), was also done. Proteins were purified from the soil extract by extraction with buffered phenol and precipitated with methanol + 0.1M ammonium acetate at -20°C. Characterization of proteins was performed with matrix assisted laser desorption ionization - time-of-flight mass spectrometry (MALDI-TOF/MS) and the concentration of total proteins was measured using Bradford's method. Bovine serum albumin (BSA) was used as a positive control in the extractions and as a standard protein in Bradford's method. Our results showed that sequential extraction increased the amount of extracted proteins compared to the extractions without the SDS-buffer; however, it must be noted that the use of SDS-buffer very probably increased denaturization of proteins. Purification of proteins from crude soil extracts
Rodriguez, Alena M; Dutertre, Sebastien; Lewis, Richard J; Marí, Frank
The venom of cone snails is composed of highly modified peptides (conopeptides) that target a variety of ion channels and receptors. The venom of these marine gastropods represents a largely untapped resource of bioactive compounds of potential pharmaceutical value. Here, we use a combination of bioanalytical techniques to uncover the extent of venom expression variability in Conus purpurascens, a fish-hunting cone snail species. The injected venom of nine specimens of C. purpurascens was separated by reversed-phase high-performance liquid chromatography (RP-HPLC), and fractions were analyzed using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) in parallel with liquid chromatography-electrospray ionization (LC-ESI)-TripleTOF-MS to compare standard analytical protocols used in preparative bioassay-guided fractionations with a deeper peptidomic analysis. Here, we show that C. purpurascens exhibits pronounced intraspecific venom variability. RP-HPLC fractionation followed by MALDI-TOF-MS analysis of the injected venom of these nine specimens identified 463 distinct masses, with none common to all specimens. Using LC-ESI-TripleTOF-MS, the injected venom of these nine specimens yielded a total of 5517 unique masses. We also compare the injected venom of two specimens with their corresponding dissected venom. We found 2566 and 1990 unique masses for the dissected venom compared to 941 and 1959 masses in their corresponding injected venom. Of these, 742 and 1004 masses overlapped between the dissected and injected venom, respectively. The results indicate that larger conopeptide libraries can be assessed by studying multiple individuals of a given cone snail species. This expanded library of conopeptides enhances the opportunities for discovery of molecular modulators with direct relevance to human therapeutics. Graphical Abstract The venom of cone snails are extraordinarily complex mixtures of highly modified peptides. Venom
Angelini, Roberto; Lobasso, Simona; Gorgoglione, Ruggiero; Bowron, Ann; Steward, Colin G.; Corcelli, Angela
Barth syndrome (BTHS), an X-linked disease associated with cardioskeletal myopathy, neutropenia, and organic aciduria, is characterized by abnormalities of cardiolipin (CL) species in mitochondria. Diagnosis of the disease is often compromised by lack of rapid and widely available diagnostic laboratory tests. The present study describes a new method for BTHS screening based on MALDI-TOF/MS analysis of leukocyte lipids. This generates a “CL fingerprint” and allows quick and simple assay of the relative levels of CL and monolysocardiolipin species in leukocyte total lipid profiles. To validate the method, we used vector algebra to analyze the difference in lipid composition between controls (24 healthy donors) and patients (8 boys affected by BTHS) in the high-mass phospholipid range. The method of lipid analysis described represents an important additional tool for the diagnosis of BTHS and potentially enables therapeutic monitoring of drug targets, which have been shown to ameliorate abnormal CL profiles in cells. PMID:26144817
Höll, Linda; Behr, Jürgen; Vogel, Rudi F
Modified atmosphere packaging (MAP) is widely used in food industry to extend the microbiological shelf-life of meat. Typically, poultry meat has been packaged in a CO2/N2 atmosphere (with residual low O2). Recently, some producers use high O2 MAP for poultry meat to empirically reach comparable shelf lifes. In this work, we compared spoilage microbiota of skinless chicken breast in high (80% O2, 20% CO2) and low O2 MAP (65% N2 and 35% CO2). Two batches of meat were incubated in each atmosphere for 14 days at 4 °C and 10 °C. Atmospheric composition of each pack and colony forming units (25 °C, 48 h, BHI agar) of poultry samples were determined at seven timepoints. Identification of spoilage organisms was carried out by MALDI-TOF MS. Brochothrix thermosphacta, Carnobacterium sp. and Pseudomonas sp. were the main organisms found after eight days at 4 °C and 10 °C in high O2 MAP. In low O2 MAP, the main spoilage microbiota was represented by species Hafnia alvei at 10 °C, and genera Carnobacterium sp., Serratia sp., and Yersinia sp. at 4 °C. High O2 MAP is suggested as preferential gas because were less detrimental and pathogens like Yersinia were not observed.
Gonzalez-Gil, Graciela; Thomas, Ludivine; Emwas, Abdul-Hamid; Lens, Piet N L; Saikaly, Pascal E
Anaerobic granular sludge is composed of multispecies microbial aggregates embedded in a matrix of extracellular polymeric substances (EPS). Here we characterized the chemical fingerprint of the polysaccharide fraction of EPS in anaerobic granules obtained from full-scale reactors treating different types of wastewater. Nuclear magnetic resonance (NMR) signals of the polysaccharide region from the granules were very complex, likely as a result of the diverse microbial population in the granules. Using nonmetric multidimensional scaling (NMDS), the (1)H NMR signals of reference polysaccharides (gellan, xanthan, alginate) and those of the anaerobic granules revealed that there were similarities between the polysaccharides extracted from granules and the reference polysaccharide alginate. Further analysis of the exopolysaccharides from anaerobic granules, and reference polysaccharides using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) revealed that exopolysaccharides from two of the anaerobic granular sludges studied exhibited spectra similar to that of alginate. The presence of sequences related to the synthesis of alginate was confirmed in the metagenomes of the granules. Collectively these results suggest that alginate-like exopolysaccharides are constituents of the EPS matrix in anaerobic granular sludge treating different industrial wastewater. This finding expands the engineered environments where alginate has been found as EPS constituent of microbial aggregates.
Gonzalez-Gil, Graciela; Thomas, Ludivine; Emwas, Abdul-Hamid; Lens, Piet N. L.; Saikaly, Pascal E.
Anaerobic granular sludge is composed of multispecies microbial aggregates embedded in a matrix of extracellular polymeric substances (EPS). Here we characterized the chemical fingerprint of the polysaccharide fraction of EPS in anaerobic granules obtained from full-scale reactors treating different types of wastewater. Nuclear magnetic resonance (NMR) signals of the polysaccharide region from the granules were very complex, likely as a result of the diverse microbial population in the granules. Using nonmetric multidimensional scaling (NMDS), the 1H NMR signals of reference polysaccharides (gellan, xanthan, alginate) and those of the anaerobic granules revealed that there were similarities between the polysaccharides extracted from granules and the reference polysaccharide alginate. Further analysis of the exopolysaccharides from anaerobic granules, and reference polysaccharides using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) revealed that exopolysaccharides from two of the anaerobic granular sludges studied exhibited spectra similar to that of alginate. The presence of sequences related to the synthesis of alginate was confirmed in the metagenomes of the granules. Collectively these results suggest that alginate-like exopolysaccharides are constituents of the EPS matrix in anaerobic granular sludge treating different industrial wastewater. This finding expands the engineered environments where alginate has been found as EPS constituent of microbial aggregates. PMID:26391984
Wang, Bangping; Li, Zhenxing; Zheng, Lina; Liu, Yixuan; Lin, Hong
As fish is one source of the `big eight' food allergens, the prevalence of fish allergy has increased over the past few years. In order to better understand fish allergy, it is necessary to identify fish allergens. Based on the sera from fish-allergenic patients, a 28 kDa protein from local mackerel ( Scomber japonicus), which has not been reported as a fish allergen, was found to be reactive with most of the patients' sera. The 28 kDa protein was analyzed by MALDI-TOF-MS (Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry). Mascot search in NCBI database (Date: 08/07/2010) showed that the top protein matched, i.e. triosephosphate isomerase (TPI) from Xiphophorus maculatus and Poecilia reticulata, had a mowse (molecular weight search) score of 98. In addition, TPI from Epinephelus coioides also matched this mackerel protein with a mowse score of 96. Because TPI is considered as an allergen in other non-fish organisms, such as lychee, wheat, latex, archaeopotamobius ( Archaeopotamobius sibiriensis) and crangon ( Crangon crangon), we consider that it may also be an allergen in mackerel.
Šedo, Ondrej; Nemec, Alexandr; Křížová, Lenka; Kačalová, Magdaléna; Zdráhal, Zbyněk
MALDI-TOF MS is currently becoming the method of choice for rapid identification of bacterial species in routine diagnostics. Yet, this method suffers from the inability to differentiate reliably between some closely related bacterial species including those of the Acinetobacter calcoaceticus-Acinetobacter baumannii (ACB) complex, namely A. baumannii and Acinetobacter nosocomialis. In the present study, we evaluated a protocol which was different from that used in the Bruker Daltonics identification system (MALDI BioTyper) to improve species identification using a taxonomically precisely defined set of 105 strains representing the four validly named species of the ACB complex. The novel protocol is based on the change in matrix composition from alpha-cyano-4-hydroxycinnamic acid (saturated solution in water:acetonitrile:trifluoroacetic acid, 47.5:50:2.5, v/v) to ferulic acid (12.5mgml(-1) solution in water:acetonitrile:formic acid 50:33:17, v/v), while the other steps of sample processing remain unchanged. Compared to the standard protocol, the novel one extended the range of detected compounds towards higher molecular weight, produced signals with better mass resolution, and allowed the detection of species-specific signals. As a result, differentiation of A. nosocomialis and A. baumannii strains by cluster analysis was improved and 13 A. nosocomialis strains, assigned erroneously or ambiguously by using the standard protocol, were correctly identified.
Horká, Marie; Kubesová, Anna; Salplachta, Jiří; Zapletalová, Eva; Horký, Jaroslav; Slais, Karel
Microbial strains are now spreading out of their original geographical areas of incidence and previously adequate morphological identification methods often must be accompanied by a phenotypic characterization for the successful microbial identification. The fungal genus Monilinia represents a suitable example. Monilinia species represent important fruit pathogens responsible for major losses in fruit production. Four closely related spp. of Monilinia: Monilinia laxa, Monilinia fructigena, Monilinia fructicola and Monilia polystroma have been yet identified. However, the classical characterization methods are not sufficient for current requirements, especially for phytosanitary purposes. In this study, rapid and reproducible methods have been developed for the characterization of Monilinia spp. based on the utilization of five well-established analytical techniques: CZE, CIEF, gel IEF, SDS-PAGE and MALDI-TOF MS, respectively. The applicability of these techniques for the identification of unknown spores of Monilinia spp. collected from infected fruits was also evaluated. It was found that isoelectric points, migration velocities or the protein patterns can be used as the identification markers in the case of cultivated filamentous fungi. Moreover, the results obtained by capillary electromigration techniques are independent on the host origin of the spores. On the other hand, the host origin of the fungi can play an important role in the precise fungi identification by the other techniques.
Li, Yan; Yan, Bo; Deng, Chunhui; Tang, Jia; Liu, Junyan; Zhang, Xiangmin
In this study, a novel method of on-plate digestion using trypsin-immobilized magnetic nanospheres was developed followed by MALDI-TOF-MS for rapid and effective analysis and identification of proteins. We utilized a facile one-pot method for the direct preparation of amine-functionalized magnetic nanospheres with highly magnetic properties and the amino groups on the outer surface. Through the reaction of the aldehyde groups with amine groups, trypsin was simply and stably immobilized onto the magnetic nanospheres. The obtained trypsin-linked magnetic nanospheres were then applied for on-plate digestion of sample proteins (myoglobin and Cytochrome c). Moreover, after digestion, the trypsin-linked nanospheres could be easily removed from the plate due to their magnetic property, which would avoid causing contamination on the ion source chamber in MS. The effects of the temperature and incubation time on the digestion efficiency were characterized. Within only 5 min, proteins could be efficiently digested with the peptide sequence coverage higher than or equal to that of the traditional in-solution digestion for 12 h. Furthermore, RPLC fractions of rat liver extract were also successfully processed using this novel method. These results suggested that our improved on-plate digestion protocol for MALDI-MS may find further application in automated analysis of large sets of proteins.
AlMasoud, Najla; Xu, Yun; Nicolaou, Nicoletta; Goodacre, Royston
Over the past few decades there has been an increased interest in using various analytical techniques for detecting and identifying microorganisms. More recently there has been an explosion in the application of matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) for bacterial characterization, and here we optimize this approach in order to generate reproducible MS data from bacteria belonging to the genera Bacillus and Brevibacillus. Unfortunately MALDI-TOF-MS generates large amounts of data and is prone to instrumental drift. To overcome these challenges we have developed a preprocessing pipeline that includes baseline correction, peak alignment followed by peak picking that in combination significantly reduces the dimensionality of the MS spectra and corrects for instrument drift. Following this two different prediction models were used which are based on support vector machines and these generated satisfactory prediction accuracies of approximately 90%. PMID:25086893
Şamlı, Asuman; İlki, Arzu
Mycobacteria are an important cause of morbidity in humans. Rapid and accurate mycobacterial identification is important for improving patient outcomes. However, identification of Mycobacterium species is not easy, due to the slow and fastidious growth of mycobacteria. Recently, biochemical, sequencing, and probing methods have come to be used for identification. This study compared the performance of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for the identification of M.tuberculosis and non-tuberculosis Mycobacteria (NTM) to those of nucleic acid hybridization (NAH) and the MPT64 immunochromatographic test. A total of 69 isolates from Marmara University Hospital, Microbiology Laboratory obtained between 2012 and 2013 were included in our study. All strains were grown on Lowenstein-Jensen and Middlebrook 7H9 medium. Among the 69 isolates, 56 (81%) were isolated as Mycobacterium tuberculosis complex (MTC), and 13 (19%) were isolated as NTM by the MPT64 ICT. NAH was able to identify all isolates to the species level. The isolated NTM included M. intracellulare (n:5), M. lentiflavum (n:3), M. xenopi (n:2), M. malmoense (n:1), M. abscessus (n:1), and M. avium (n:1). MALDI-TOF MS identified 88% of the mycobacterial isolates. All M. tuberculosis strains were identified correctly, but the ratio was 38.5% for NTM. Mycobacterial identification using MALDI-TOF MS takes 45 minutes and costs 3 Euro/test, whereas mycobacterial identification using NAH takes 6-7 hours and costs 30 Euro/test. In conclusion, MALDI-TOF MS has the potential to identify mycobacteria in the clinical laboratory setting by reducing identification turnaround time and laboratory costs for isolate referral.
Liang, Boying; Ju, Yue; Joubert, James R.; Kaleta, Erin J.; Lopez, Rodrigo; Jones, Ian W.; Hall, Henry K.; Ratnayaka, Saliya N.; Wysocki, Vicki H.; Saavedra, S. Scott
Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) coupled with affinity capture is a well-established method to extract biological analytes from complex samples followed by label-free detection and identification. Many bioanalytes of interest bind to membrane-associated receptors, however, the matrices and high vacuum conditions inherent to MALDI-TOF MS make it largely incompatible with the use of artificial lipid membranes with incorporated receptors as platforms for detection of captured proteins and peptides. Here we show that cross-linking polymerization of a planar supported lipid bilayer (PSLB) provides the stability needed for MALDI-TOF MS analysis of proteins captured by receptors embedded in the membrane. PSLBs composed of poly(bis-SorbPC) and doped with the ganglioside receptors GM1 and GD1a were used for affinity capture of the B-subunits of cholera toxin, heat-labile enterotoxin, and pertussis toxin. The three toxins were captured simultaneously, then detected and identified by MS based on differences in their molecular weights. Poly(bis-SorbPC) PSLBs are inherently resistant to nonspecific protein adsorption, which allowed selective toxin detection to be achieved in complex matrices (bovine serum and shrimp extract). Using GM1-cholera toxin B as a model receptor-ligand pair, the minimal detectable concentration of toxin was estimated to be 4 nM. On-plate trypsin digestion of bound cholera toxin B followed by MS/MS analysis of digested peptides was performed successfully, demonstrating the feasibility of using the PSLB-based affinity capture platform for identification of unknown, membrane-associated proteins. Overall, this work demonstrates that combining a poly(lipid) affinity capture platform with MALDI-TOF MS detection is a viable approach for capture and proteomic characterization of membrane-associated proteins in a label-free manner. PMID:25694144
Background In this study mass spectrometry was used for evaluating extracted leptospiral protein samples and results were compared with molecular typing methods. For this, an extraction protocol for Leptospira spp. was independently established in two separate laboratories. Reference spectra were created with 28 leptospiral strains, including pathogenic, non-pathogenic and intermediate strains. This set of spectra was then evaluated on the basis of measurements with well-defined, cultured leptospiral strains and with 16 field isolates of veterinary or human origin. To verify discriminating peaks for the applied pathogenic strains, statistical analysis of the protein spectra was performed using the software tool ClinProTools. In addition, a dendrogram of the reference spectra was compared with phylogenetic trees of the 16S rRNA gene sequences and multi locus sequence typing (MLST) analysis. Results Defined and reproducible protein spectra using MALDI-TOF MS were obtained for all leptospiral strains. Evaluation of the newly-built reference spectra database allowed reproducible identification at the species level for the defined leptospiral strains and the field isolates. Statistical analysis of three pathogenic genomospecies revealed peak differences at the species level and for certain serovars analyzed in this study. Specific peak patterns were reproducibly detected for the serovars Tarassovi, Saxkoebing, Pomona, Copenhageni, Australis, Icterohaemorrhagiae and Grippotyphosa. Analysis of the dendrograms of the MLST data, the 16S rRNA sequencing, and the MALDI-TOF MS reference spectra showed comparable clustering. Conclusions MALDI-TOF MS analysis is a fast and reliable method for species identification, although Leptospira organisms need to be produced in a time-consuming culture process. All leptospiral strains were identified, at least at the species level, using our described extraction protocol. Statistical analysis of the three genomospecies L. borgpetersenii
Pérez-Sancho, Marta; Vela, Ana I.; García-Seco, Teresa; González, Sergio; Domínguez, Lucas; Fernández-Garayzábal, Jose Francisco
The application of MALDI-TOF MS for identifying streptococcal isolates recovered from clinical specimens of diseased pigs was evaluated. For this proposal, the MALDI BDAL Database (Bruker Daltoniks, Germany) was supplemented with the main spectrum profiles (MSP) of the reference strains of S. porci, S. porcorum and S. plurextorum associated with pneumonia and septicemia. Although these three species showed similar MALDI profiles, several peaks were recognized that can be useful for their differentiation: S. porci (4113, 6133, 7975 and 8228 m/z Da), S. plurextorum (3979, 4078, 4665, 6164, 6491, 6812, 7959 and 9330 m/z Da) and S. porcorum (3385, 3954, 4190, 6772, 7908, and 8381 m/z Da). After adding these MSPs, an evaluation was conducted to determine the accuracy of MALDI-TOF MS for the identification of streptococci from diseased pigs using 74 field isolates. Isolates were identified as S. suis, S. porcinus, S. dysgalactiae, S. hyovaginalis, S. porcorum, S. alactolyticus, S. hyointestinalis and S. orisratti. This is the first time that the latter three species have been reported from clinical specimens of pigs. Overall, there was good concordance (95.9%) between the results obtained from MALDI-TOF MS identification (best hint) and those from genotyping. Our results demonstrate the good performance of MALDI-TOF MS (100% sensitivity and specificity) for identifying most of the species of streptococci that can frequently be isolated from diseased pigs. However, conflicting results were observed in the correct identification of some isolates of S. dysgalactiae and S. alactolyticus. PMID:28125697
Danesi, Patrizia; Drigo, Ilenia; Iatta, Roberta; Firacative, Carolina; Capelli, Gioia; Cafarchia, Claudia; Meyer, Wieland
Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) offers an effective alternative to phenotypic and molecular methods for the rapid identification of microorganisms. Our aim in this study was to create an in-house library for a set of strains of nine uncommonly reported human and animal cryptococcal species, including Cryptococcus adeliensis, C. albidosimilis, C. albidus, C. aureus, C. carnescens, C. laurentii, C. magnus, C. victoriae and C. uniguttulatus, and to use this library to make timely and correct identifications using MALDI-TOF MS for use in routine laboratory diagnostics. Protein extracts obtained via the formic acid extraction method of 62 veterinary non-C. neoformans-C. gattii cryptococcal isolates were studied. The obtained mass spectra correctly grouped all 62 studied isolates according to species identification previously obtained by internal transcribe spacer sequence analysis. The in-house database was than exported and successfully uploaded to the Microflex LT (Maldi Biotyper; Bruker Daltonics) instrument at a different diagnostic laboratory in Italy. Scores >2.7 obtained from isolates reanalyzed in the latter laboratory supported the high reproducibility of the method. The possibility of creating and transferring an in-house library adds to the usefulness MALDI-TOF MS an important tool for the rapid and inexpensive identification of pathogenic and saprophytic fungi as required for differential diagnosis of human and animal mycoses.
Background The poor reproducibility of matrix-assisted desorption/ionization time-of-flight (MALDI-TOF) spectra limits the effectiveness of the MALDI-TOF MS-based identification of filamentous fungi with highly heterogeneous phenotypes in routine clinical laboratories. This study aimed to enhance the MALDI-TOF MS-based identification of filamentous fungi by assessing several architectures of reference spectrum libraries. Results We established reference spectrum libraries that included 30 filamentous fungus species with various architectures characterized by distinct combinations of the following: i) technical replicates, i.e., the number of analyzed deposits for each culture used to build a reference meta-spectrum (RMS); ii) biological replicates, i.e., the number of RMS derived from the distinct subculture of each strain; and iii) the number of distinct strains of a given species. We then compared the effectiveness of each library in the identification of 200 prospectively collected clinical isolates, including 38 species in 28 genera. Identification effectiveness was improved by increasing the number of both RMS per strain (p<10-4) and strains for a given species (p<10-4) in a multivariate analysis. Conclusion Addressing the heterogeneity of MALDI-TOF spectra derived from filamentous fungi by increasing the number of RMS obtained from distinct subcultures of strains included in the reference spectra library markedly improved the effectiveness of the MALDI-TOF MS-based identification of clinical filamentous fungi. PMID:23565856
Curtoni, Antonio; Cipriani, Raffaella; Marra, Elisa Simona; Barbui, Anna Maria; Cavallo, Rossana; Costa, Cristina
Matrix-assisted laser-desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) is a useful tool for rapid identification of microorganisms. Unfortunately, its direct application to positive blood culture is still lacking standardized procedures. In this study, we evaluated an easy- and rapid-to-perform protocol for MALDI-TOF MS direct identification of microorganisms from positive blood culture after a short-term incubation on solid medium. This protocol was used to evaluate direct identification of microorganisms from 162 positive monomicrobial blood cultures; at different incubation times (3, 5, 24 h), MALDI-TOF MS assay was performed from the growing microorganism patina. Overall, MALDI-TOF MS concordance with conventional methods at species level was 60.5, 80.2, and 93.8% at 3, 5, and 24 h, respectively. Considering only bacteria, the identification performances at species level were 64.1, 85.0, and 94.1% at 3, 5, and 24 h, respectively. This protocol applied to a commercially available MS typing system may represent, a fast and powerful diagnostic tool for pathogen direct identification and for a promptly and pathogen-driven antimicrobial therapy in selected cases.
Dolatabadi, Somayeh; Kolecka, Anna; Versteeg, Matthijs; de Hoog, Sybren G; Boekhout, Teun
This study addresses the usefulness of matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) MS for reliable identification of the two most frequently occurring clinical species of Rhizopus, namely Rhizopus arrhizus with its two varieties, arrhizus and delemar, and Rhizopus microsporus. The test-set comprised 38 isolates of clinical and environmental origin previously identified by internal transcribed spacer (ITS) sequencing of rDNA. Multi-locus sequence data targeting three gene markers (ITS, ACT, TEF ) showed two monophylic clades for Rhizopus arrhizus and Rhizopus microsporus (bootstrap values of 99 %). Cluster analysis confirmed the presence of two distinct clades within Rhizopus arrhizus representing its varieties arrhizus and delemar. The MALDI Biotyper 3.0 Microflex LT platform (Bruker Daltonics) was used to confirm the distinction between Rhizopus arrhizus and Rhizopus microsporus and the presence of two varieties within the species Rhizopus arrhizus. An in-house database of 30 reference main spectra (MSPs) was initially tested for correctness using commercially available databases of Bruker Daltonics. By challenging the database with the same strains of which an in-house database was created, automatic identification runs confirmed that MALDI-TOF MS is able to recognize the strains at the variety level. Based on principal component analysis, two MSP dendrograms were created and showed concordance with the multi-locus tree; thus, MALDI-TOF MS is a useful tool for diagnostics of mucoralean species.
Müller, Roland; Marchetti-Deschmann, Martina; Elgass, Helmut; Breiteneder, Heimo; Kratzmeier, Martin; Allmaier, Günter
The molecular weights (MW) of seven (glyco)proteins, of which five were plasma-derived, with MWs higher than 200 kDa were determined with three techniques: CGE-on-a-chip, SDS-PAGE and MALDI-TOF-MS. While the analysis of medium to high MW proteins with SDS-PAGE was an already well-established technique, the usefulness of MALDI-TOF-MS for the exact MW determination of high mass proteins was only partly described in literature so far. CGE-on-a-chip is the newest of all three applied techniques and was so far not applicable. Therefore, it was not evaluated for high MW (glyco)proteins. All proteins were analyzed under nonreducing as well as reducing conditions. In this work, it was demonstrated that all three described techniques were capable of determining the MW of all high molecular weight (glyco)proteins. The noncommercial CGE-on-a-chip assay allowed for the first time the electrophoretic separation of proteins in the MW range from 14 to 1000 kDa. MW assignment was limited to 500 kDa in the case of SDS-PAGE and 660 kDa in the case of the high MW CGE-on-a-chip assay. With the proper matrix and sample preparation, analysis with a standard MALDI-TOF-MS provided accurate MWs for all high MW proteins up to 1 MDa.
Mizukado, Junji; Sato, Hiroaki; Chen, Liang; Suzuki, Yasumasa; Yamane, Shogo; Aoyama, Yoshinori; Suda, Hiroyuki
High-resolution matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry (TOF MS) was used for the analysis of the low-molecular-weight products from the photo-oxidation of poly(3-hexylthiophene) (P3HT) in solution and thin film. Eight new peak series were observed in the low-mass range of the mass spectra of the products degraded in solution, and the formulas of the eight components were determined from the accurate mass. From SEC/MALDI-TOF MS, two components were identified as the degraded products, and the other six components were derived from the fragmentation of the degraded products during the MALDI process. A mechanism for the formation of these components was proposed on the basis of the results of MALDI-TOF MS. For the thin film degradation, a part of products in the solution degradation were observed, which supports that the oxidation of P3HT in solution and thin film proceeded in the same mechanism. This study shows that high-resolution MALDI-TOF MS is effective for the analysis of the low-molecular-weight products from P3HT photo-oxidation and expected to be feasible for the degradation analyses of other polymers. Copyright © 2015 John Wiley & Sons, Ltd.
Barbano, Duane; Diaz, Regina; Zhang, Lin; Sandrin, Todd; Gerken, Henri; Dempster, Thomas
Current molecular methods to characterize microalgae are time-intensive and expensive. Matrix Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) may represent a rapid and economical alternative approach. The objectives of this study were to determine whether MALDI-TOF MS can be used to: 1) differentiate microalgae at the species and strain levels and 2) characterize simple microalgal mixtures. A common protein extraction sample preparation method was used to facilitate rapid mass spectrometry-based analysis of 31 microalgae. Each yielded spectra containing between 6 and 56 peaks in the m/z 2,000 to 20,000 range. The taxonomic resolution of this approach appeared higher than that of 18S rDNA sequence analysis. For example, two strains of Scenedesmus acutus differed only by two 18S rDNA nucleotides, but yielded distinct MALDI-TOF mass spectra. Mixtures of two and three microalgae yielded relatively complex spectra that contained peaks associated with members of each mixture. Interestingly, though, mixture-specific peaks were observed at m/z 11,048 and 11,230. Our results suggest that MALDI-TOF MS affords rapid characterization of individual microalgae and simple microalgal mixtures. PMID:26271045
Barbano, Duane; Diaz, Regina; Zhang, Lin; Sandrin, Todd; Gerken, Henri; Dempster, Thomas
Current molecular methods to characterize microalgae are time-intensive and expensive. Matrix Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) may represent a rapid and economical alternative approach. The objectives of this study were to determine whether MALDI-TOF MS can be used to: 1) differentiate microalgae at the species and strain levels and 2) characterize simple microalgal mixtures. A common protein extraction sample preparation method was used to facilitate rapid mass spectrometry-based analysis of 31 microalgae. Each yielded spectra containing between 6 and 56 peaks in the m/z 2,000 to 20,000 range. The taxonomic resolution of this approach appeared higher than that of 18S rDNA sequence analysis. For example, two strains of Scenedesmus acutus differed only by two 18S rDNA nucleotides, but yielded distinct MALDI-TOF mass spectra. Mixtures of two and three microalgae yielded relatively complex spectra that contained peaks associated with members of each mixture. Interestingly, though, mixture-specific peaks were observed at m/z 11,048 and 11,230. Our results suggest that MALDI-TOF MS affords rapid characterization of individual microalgae and simple microalgal mixtures.
Tseng, Mei-Chun; Obena, Rofeamor; Lu, Ying-Wei; Lin, Po-Chiao; Lin, Ping-Yu; Yen, Yung-Sheng; Lin, Jiann-Tsuen; Huang, Li-De; Lu, Kuang-Lieh; Lai, Long-Li; Lin, Chun-Cheng; Chen, Yu-Ju
Efficient structural characterization is important for quality control when developing novel materials. In this study, we demonstrated the soft ionization capability of the hybrid of immobilized silica and 2,5-dihydrobenzoic acid (DHB) on iron oxide magnetic nanoparticles in MALDI-TOF MS with a clean background. The ratio between SiO(2) and DHB was examined and was found to affect the surface immobilization of DHB on the nanoparticle, critically controlling the ionization efficiency and interference background. Compared with commercial DHB, the functionalized nanoparticle-assisted MALDI-TOF MS provided superior soft ionization with production of strong molecular ions within 5 ppm mass accuracy on a variety of new types of synthetic materials used for solar cells, light emitting devices, dendrimers, and glycolipids, including analytes with either thermally labile structures or poor protonation tendencies. In addition, the enhancements of the molecular ion signal also provided high-quality product-ion spectra allowing structural characterization and unambiguous small molecule identification. Using this technique, the structural differences among the isomers were distinguished through their characteristic fragment ions and comprehensive fragmentation patterns. With the advantages of long-term stability and simple sample preparation by deposition on a regular sample plate, the use of DHB-functionalized nanoparticles combined with high-resolution MALDI-TOF MS provides a generic platform for rapid and unambiguous structure determination of small molecules.
Yamamoto, Naomi; Takahashi, Hajime; Tamura, Hiroto
The genetic lineages of Listeria monocytogenes and other species of the genus Listeria are correlated with pathogenesis in humans. Although matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has become a prevailing tool for rapid and reliable microbial identification, the precise discrimination of Listeria species and lineages remains a crucial issue in clinical settings and for food safety. In this study, we constructed an accurate and reliable MS database to discriminate the lineages of L. monocytogenes and the species of Listeria (L. monocytogenes, L. innocua, L. welshimeri, L. seeligeri, L. ivanovii, L. grayi, and L. rocourtiae) based on the S10-spc-alpha operon gene encoded ribosomal protein mass spectrum (S10-GERMS) proteotyping method, which relies on both genetic information (genomics) and observed MS peaks in MALDI-TOF MS (proteomics). The specific set of eight biomarkers (ribosomal proteins L24, L6, L18, L15, S11, S9, L31 type B, and S16) yielded characteristic MS patterns for the lineages of L. monocytogenes and the different species of Listeria, and led to the construction of a MS database that was successful in discriminating between these organisms in MALDI-TOF MS fingerprinting analysis followed by advanced proteotyping software Strain Solution analysis. We also confirmed the constructed database on the proteotyping software Strain Solution by using 23 Listeria strains collected from natural sources. PMID:27442502
Su, Kang-Yi; Yan, Bo-Shiun; Chiu, Hao-Chieh; Yu, Chong-Jen; Chang, So-Yi; Jou, Ruwen; Liu, Jia-Long; Hsueh, Po-Ren; Yu, Sung-Liang
The increasing incidence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) Mycobacterium tuberculosis (MTB) adds further urgency for rapid and multiplex molecular testing to identify the MTB complex and drug susceptibility directly from sputum for disease control. A nucleotide matrix-assisted-laser-desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS)-based assay was developed to identify MTB (MTBID panel) and 45 chromosomal mutations for resistance to eight antibiotics (MTBDR panel). We conducted a 300 case trial from outpatients to evaluate this platform. An MTBID panel specifically identified MTB with as few as 10 chromosome DNA copies. The panel was 100% consistent with an acid-fast stain and culture for MTB, nontuberculous mycobacteria, and non-mycobacteria bacteria. The MTBDR panel was validated using 20 known MDR-MTB isolates. In a 64-case double-blind clinical isolates test, the sensitivity and specificity were 83% and 100%, respectively. In a 300-case raw sputum trial, the MTB identification sensitivity in smear-negative cases using MALDI-TOF MS was better than the COBAS assay (61.9% vs. 46.6%). Importantly, the failure rate of MALDI-TOF MS was better than COBAS (11.3% vs. 26.3%). To the best of our knowledge, the test described herein is the only multiplex test that predicts resistance for up to eight antibiotics with both sensitivity and flexibility. PMID:28134321
Kondori, Nahid; Erhard, Marcel; Welinder-Olsson, Christina; Groenewald, Marizeth; Verkley, Gerard; Moore, Edward R B
Conventional mycological identifications based on the recognition of morphological characteristics can be problematic. A relatively new methodology applicable for the identification of microorganisms is based on the exploitation of taxon- specific mass patterns recorded from abundant cell proteins directly from whole-cell preparations, using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). This study reports the application of MALDI-TOF MS for the differentiation and identifications of black yeasts, isolated from the respiratory tracts of patients with cystic fibrosis (CF). Initial phenotypic and DNA sequence-based analyses identified these isolates to be Exophiala dermatitidis. The type strains of E. dermatitidis (CBS 207.35(T)) and other species of Exophiala were included in the MALDI-TOF MS analyses to establish the references for comparing the mass spectra of the clinical isolates of Exophiala. MALDI-TOF MS analyses exhibited extremely close relationships among the clinical isolates and with the spectra generated from the type strain of E. dermatitidis. The relationships observed between the E. dermatitidis strains from the MALDI-TOF MS profiling analyses were supported by DNA sequence-based analyses of the rRNA ITS1 and ITS2 regions. These data demonstrated the applicability of MALDI-TOF MS as a reliable, rapid and cost-effective method for the identification of isolates of E. dermatitidis and other clinically relevant fungi and yeasts that typically are difficult to identify by conventional methods.
Berrazeg, Meryem; Diene, Seydina M.; Drissi, Mourad; Kempf, Marie; Richet, Hervé; Landraud, Luce; Rolain, Jean-Marc
Background Klebsiella pneumoniae is one of the most important pathogens responsible for nosocomial outbreaks worldwide. Epidemiological analyses are useful in determining the extent of an outbreak and in elucidating the sources and the spread of infections. The aim of this study was to investigate the epidemiological spread of K. pneumoniae strains using a MALDI-TOF MS approach. Methods Five hundred and thirty-five strains of K. pneumoniae were collected between January 2008 and March 2011 from hospitals in France and Algeria and were identified using MALDI-TOF. Antibiotic resistance patterns were investigated. Clinical and epidemiological data were recorded in an Excel file, including clustering obtained from the MSP dendrogram, and were analyzed using PASW Statistics software. Results Antibiotic susceptibility and phenotypic tests of the 535 isolates showed the presence of six resistance profiles distributed unequally between the two countries. The MSP dendrogram revealed five distinct clusters according to an arbitrary cut-off at the distance level of 500. Data mining analysis of the five clusters showed that K. pneumoniae strains isolated in Algerian hospitals were significantly associated with respiratory infections and the ESBL phenotype, whereas those from French hospitals were significantly associated with urinary tract infections and the wild-type phenotype. Conclusions MALDI-TOF was found to be a promising tool to identify and differentiate between K. pneumoniae strains according to their phenotypic properties and their epidemiological distribution. This is the first time that MALDI-TOF has been used as a rapid tool for typing K. pneumoniae clinical isolates. PMID:23620754
Wattal, C; Oberoi, J K
The study addresses the utility of Matrix Assisted Laser Desorption/Ionisation Time-Of-Flight mass spectrometry (MALDI-TOF MS) using VITEK MS and the VITEK 2 antimicrobial susceptibility testing (AST) system for direct identification (ID) and timely AST from positive blood culture bottles using a lysis-filtration method (LFM). Between July and December 2014, a total of 140 non-duplicate mono-microbial blood cultures were processed. An aliquot of positive blood culture broth was incubated with lysis buffer before the bacteria were filtered and washed. Micro-organisms recovered from the filter were first identified using VITEK MS and its suspension was used for direct AST by VITEK 2 once the ID was known. Direct ID and AST results were compared with classical methods using solid growth. Out of the 140 bottles tested, VITEK MS resulted in 70.7 % correct identification to the genus and/ or species level. For the 103 bottles where identification was possible, there was agreement in 97 samples (94.17 %) with classical culture. Compared to the routine method, the direct AST resulted in category agreement in 860 (96.5 %) of 891 bacteria-antimicrobial agent combinations tested. The results of direct ID and AST were available 16.1 hours before those of the standard approach on average. The combined use of VITEK MS and VITEK 2 directly on samples from positive blood culture bottles using a LFM technique can result in rapid and reliable ID and AST results in blood stream infections to result in early institution of targeted treatment. The combination of LFM and AST using VITEK 2 was found to expedite AST more reliably.
Abbady, A Q; Al-Daoude, A; Al-Mariri, A; Zarkawi, M; Muyldermans, S
The deployment of today's antibodies that are able to distinguish Brucella from the closely similar pathogens, such as Yersinia, is still considered a great challenge since both pathogens share identical LPS (lipopolysaccharide) O-ring epitopes. In addition, because of the great impact of Brucella on health and economy in many countries including Syria, much effort is going to the development of next generation vaccines, mainly on the identification of new immunogenic proteins of this pathogen. In this context, Brucella-specific nanobodies (Nbs), camel genetic engineered heavy-chain antibody fragments, could be of great value. Previously, a large Nb library was constructed from a camel immunized with heat-killed Brucella. Phage display panning of this 'immune' library with Brucella total lysate resulted in a remarkable fast enrichment for a Nb referred to as NbBruc02. In the present work, we investigated the main characteristics of this Nb that can efficiently distinguish under well-defined conditions the Brucella from other bacteria including Yersinia. NbBruc02 showed a strong and specific interaction with its antigen within the crude lysate as tested by a surface plasmon resonance (SPR) biosensor and it was also able to pull down its cognate antigen from such lysate by immuno-capturing. Using matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS), NbBruc02 specific antigen was identified as chaperonin GroEL, also known as heat shock protein of 60 kDa (HSP-60), which represents a Brucella immunodominant antigen responsible of maintaining proteins folding during stress conditions. Interestingly, the antigen recognition by NbBruc02 was found to be affected by the state of GroEL folding. Thus, the Nb technology applied in the field of infectious diseases, e.g. brucellosis, yields two outcomes: (1) it generates specific binders that can be used for diagnosis, and perhaps treatment, and (2) it identifies the immunogenic candidate
Pence, M A; McElvania TeKippe, E; Wallace, M A; Burnham, C-A D
The rapid identification of yeast is essential for the optimization of antifungal therapy. The objective of our study was to evaluate two matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) platforms, the bioMérieux VITEK MS (IVD Knowledgebase v.2.0) and Bruker Biotyper (software version 3.1), for the rapid identification of medically relevant yeast. One hundred and seventeen isolates, representing six genera and 18 species, were analyzed using multiple direct smear methods to optimize identification. Sequence analysis was the gold standard for comparison. Isolates were analyzed with VITEK MS using the direct smear method +/- a 25 % formic acid on-plate extraction. For Biotyper, isolates were analyzed using direct smear without formic acid, and with 25 % and 100 % formic acid on-plate extractions. When all methods were included, VITEK MS correctly identified 113 (96.6 %) isolates after 24 h with one misidentification, and Biotyper correctly identified 77 (65.8 %) isolates using a threshold of ≥2.0 with no misidentifications. Using a revised threshold of ≥1.7, Biotyper correctly identified 103 (88.0 %) isolates, with 3 (2.6 %) misidentifications. For both platforms, the number of identifications was significantly increased using a formic acid overlay (VITEK MS, p < 0.01; Biotyper, p < 0.001), and reducing the Biotyper threshold from ≥2.0 to ≥1.7 significantly increased the rate of identification (p < 0.001). The data in this study demonstrate that the direct smear method with on-plate formic acid extraction can be used for yeast identification on both MS platforms, and more isolates are identified using the VITEK MS system (p < 0.01).
Tsai, Chia-Ju; Lin, Ying-Chi; Chen, Yen-Ling; Feng, Chia-Hsien
Lipoic acid (LA) is an essential cofactor in mitochondrial enzymes and an ideal antioxidant in prokaryotic and eukaryotic cells. Capillary liquid chromatography coupled with ultraviolet detection (CapLC-UV) and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) are two environmentally friendly methods for determining LA. In this study, a pre-column microwave-assisted derivatization with 4-bromomethyl-6,7-dimethoxycoumarin enhanced the UV absorbance of LA and was monitored at 345 nm by CapLC-UV. Gradient separation was performed using a reversed-phase C18 column with a mobile phase consisting of acetonitrile-0.1% formic acid solution. The ionization of LA was increased, and the LA derivative was detected by MALDI-TOF MS at m/z 683 with an α-cyano-4-hydroxycinnamic acid matrix. The linear response ranged from 0.1 to 40 μM with a correlation coefficient of 0.999. The CapLC-UV and MALDI-TOF MS had detection limits of 5 and 4 fmol, respectively. These methods effectively detected LA in dietary supplements and cosmetics. Cellular proteomes of a human keratinocyte cell line (HaCaT) irradiated with UV radiation were also compared with and without LA treatment. The cellular proteomes were identified by nanoultra performance LC with LTQ Orbitrap system after trypsin digestion. Protein identification was performed by simultaneous peptide sequencing and MASCOT search. The analysis revealed changes in several proteins, including CDC42, TPI1, HNRPA2B1, PRDX1, PTGES3 and MYL6.
Liang, Boying; Ju, Yue; Joubert, James R; Kaleta, Erin J; Lopez, Rodrigo; Jones, Ian W; Hall, Henry K; Ratnayaka, Saliya N; Wysocki, Vicki H; Saavedra, S Scott
Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) coupled with affinity capture is a well-established method to extract biological analytes from complex samples followed by label-free detection and identification. Many bioanalytes of interest bind to membrane-associated receptors; however, the matrices and high-vacuum conditions inherent to MALDI-TOF MS make it largely incompatible with the use of artificial lipid membranes with incorporated receptors as platforms for detection of captured proteins and peptides. Here we show that cross-linking polymerization of a planar supported lipid bilayer (PSLB) provides the stability needed for MALDI-TOF MS analysis of proteins captured by receptors embedded in the membrane. PSLBs composed of poly(bis-sorbylphosphatidylcholine) (poly(bis-SorbPC)) and doped with the ganglioside receptors GM1 and GD1a were used for affinity capture of the B subunits of cholera toxin, heat-labile enterotoxin, and pertussis toxin. The three toxins were captured simultaneously, then detected and identified by MS on the basis of differences in their molecular weights. Poly(bis-SorbPC) PSLBs are inherently resistant to nonspecific protein adsorption, which allowed selective toxin detection to be achieved in complex matrices (bovine serum and shrimp extract). Using GM1-cholera toxin subunit B as a model receptor-ligand pair, we estimated the minimal detectable concentration of toxin to be 4 nM. On-plate tryptic digestion of bound cholera toxin subunit B followed by MS/MS analysis of digested peptides was performed successfully, demonstrating the feasibility of using the PSLB-based affinity capture platform for identification of unknown, membrane-associated proteins. Overall, this work demonstrates that combining a poly(lipid) affinity capture platform with MALDI-TOF MS detection is a viable approach for capture and proteomic characterization of membrane-associated proteins in a label-free manner.
Heras Cañas, V.; Pérez Ramirez, M.D.; Bermudez Jiménez, F.; Rojo Martin, M.D.; Miranda Casas, C.; Marin Arriaza, M.; Navarro Marí, J.M.
Lactococcus garvieae is a Gram-positive, catalase negative coccus arranged in pairs or short chains, well-known as a fish pathogen. We report a case of Infective Endocarditis (IE) by L. garvieae in a native valve from a 68-year-old male with unknown history of contact with raw fish and an extensive history of heart disease. This case highlights the reliability of MALDI-TOF MS compared to conventional methods in the identification of rare microorganisms like this. PMID:25949815
Sugawara, Ryota; Yamada, Sayumi; Tu, Zhihao; Sugawara, Akiko; Suzuki, Kousuke; Hoshiba, Toshihiro; Eisaka, Sadao; Yamaguchi, Akihiro
Mushrooms are a favourite natural food in many countries. However, some wild species cause food poisoning, sometimes lethal, due to misidentification caused by confusing fruiting bodies similar to those of edible species. The morphological inspection of mycelia, spores and fruiting bodies have been traditionally used for the identification of mushrooms. More recently, DNA sequencing analysis has been successfully applied to mushrooms and to many other species. This study focuses on a simpler and more rapid methodology for the identification of wild mushrooms via protein profiling based on matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF MS). A preliminary study using 6 commercially available cultivated mushrooms suggested that a more reproducible spectrum was obtained from a portion of the cap than from the stem of a fruiting body by the extraction of proteins with a formic acid-acetonitrile mixture (1 + 1). We used 157 wild mushroom-fruiting bodies collected in the centre of Hokkaido from June to November 2014. Sequencing analysis of a portion of the ribosomal RNA gene provided 134 identifications of mushrooms by genus or species, however 23 samples containing 10 unknown species that had lower concordance rate of the nucleotide sequences in a BLAST search (less than 97%) and 13 samples that had unidentifiable poor or mixed sequencing signals remained unknown. MALDI-TOF MS analysis yielded a reproducible spectrum (frequency of matching score ≥ 2.0 was ≥6 spectra from 12 spectra measurements) for 114 of 157 samples. Profiling scores that matched each other within the database gave correct species identification (with scores of ≥2.0) for 110 samples (96%). An in-house prepared database was constructed from 106 independent species, except for overlapping identifications. We used 48 wild mushrooms that were collected in autumn 2015 to validate the in-house database. As a result, 21 mushrooms were identified at the species level with
Background Cholera is an acute diarrheal disease caused by Vibrio cholerae. Outbreaks are caused by a genetically homogenous group of strains from serogroup O1 or O139 that are able to produce the cholera toxin. Rapid detection and identification of these epidemic strains is essential for an effective response to cholera outbreaks. Results The use of ferulic acid as a matrix in a new MALDI-TOF MS assay increased the measurable mass range of existing MALDI-TOF MS protocols for bacterial identification. The assay enabled rapid discrimination between epidemic V. cholerae O1/O139 strains and other less pathogenic V. cholerae strains. OmpU, an outer membrane protein whose amino acid sequence is highly conserved among epidemic strains of V. cholerae, appeared as a discriminatory marker in the novel MALDI-TOF MS assay. Conclusions The extended mass range of MALDI-TOF MS measurements obtained by using ferulic acid improved the screening for biomarkers in complex protein mixtures. Differences in the mass of abundant homologous proteins due to variation in amino acid sequences can rapidly be examined in multiple samples. Here, a rapid MALDI-TOF MS assay was developed that could discriminate between epidemic O1/O139 strains and other less pathogenic V. cholerae strains based on differences in mass of the OmpU protein. It appeared that the amino acid sequence of OmpU from epidemic V. cholerae O1/O139 strains is unique and highly conserved. PMID:24943244
Suzuki, Takashi; Maeda, Tomoo; Grant, Suzanne; Grant, Gordon; Sporns, Peter
Accumulation of Fructans was confirmed in asparagus tissues that had been cultured for 2 days on media supplemented with glucose. It is very common that Fructans are biosynthesized from sucrose. We hypothesized however that Fructans could also be biosynthesized from glucose. Stem tissues of in vitro-cultured asparagus were subcultured for 72 h on a medium containing 0.5M of [1-(13)C]glucose. A medium containing 0.5M of normal ((12)C) glucose was used as control. Carbohydrates were extracted from the tissues and analyzed using HPLC, MALDI-TOF MS and ESI-MS. HPLC results indicated that the accumulation of short-chain Fructans was similar in both (13)C-labelled and control samples. Short-chain Fructans of DP=3-7 were detected using MALDI-TOF MS. The molecular mass of each oligomer in the (13)C-labelled sample was higher than the mass of the natural sample by 1 m/z unit per sugar moiety. The results of ESI-MS on the HPLC fractions of neokestose and 1-kestose showed that these oligomers (DP=3) were biosynthesized from exogenous glucose added to the medium. We conclude that not only exogenous sucrose but glucose can induce Fructan biosynthesis; fructans of both inulin type and inulin neoseries are also biosynthesized from glucose accumulated in asparagus tissues; the glucose molecules (or its metabolic products) were incorporated into Fructans as structural monomers.
Erler, René; Wichels, Antje; Heinemeyer, Ernst-August; Hauk, Gerhard; Hippelein, Martin; Reyes, Nadja Torres; Gerdts, Gunnar
Mesophilic marine bacteria of the family Vibrionaceae, specifically V. cholerae, V. parahaemolyticus and V. vulnificus, are considered to cause severe illness in humans. Due to climate-change-driven temperature increases, higher Vibrio abundances and infections are predicted for Northern Europe, which in turn necessitates environmental surveillance programs to evaluate this risk. We propose that whole-cell matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) profiling is a promising tool for the fast and reliable species classification of environmental isolates. Because the reference database does not contain sufficient Vibrio spectra we generated the VibrioBase database in this study. Mass spectrometric data were generated from 997 largely environmental strains and filed in this new database. MALDI-TOF MS clusters were assigned based on the species classification obtained by analysis of partial rpoB (RNA polymerase beta-subunit) sequences. The affiliation of strains to species-specific clusters was consistent in 97% of all cases using both approaches, and the extended VibrioBase generated more specific species identifications with higher matching scores compared to the commercially available database. Therefore, we have made the VibrioBase database freely accessible, which paves the way for detailed risk assessment studies of potentially pathogenic Vibrio spp. from marine environments.
Zhang, Lin; Smart, Sonja; Sandrin, Todd R
MALDI-TOF MS profiling has been shown to be a rapid and reliable method to characterize pure cultures of bacteria. Currently, there is keen interest in using this technique to identify bacteria in mixtures. Promising results have been reported with two- or three-isolate model systems using biomarker-based approaches. In this work, we applied MALDI-TOF MS-based methods to a more complex model mixture containing six bacteria. We employed: 1) a biomarker-based approach that has previously been shown to be useful in identification of individual bacteria in pure cultures and simple mixtures and 2) a similarity coefficient-based approach that is routinely and nearly exclusively applied to identification of individual bacteria in pure cultures. Both strategies were developed and evaluated using blind-coded mixtures. With regard to the biomarker-based approach, results showed that most peaks in mixture spectra could be assigned to those found in spectra of each component bacterium; however, peaks shared by two isolates as well as peaks that could not be assigned to any individual component isolate were observed. For two-isolate blind-coded samples, bacteria were correctly identified using both similarity coefficient- and biomarker-based strategies, while for blind-coded samples containing more than two isolates, bacteria were more effectively identified using a biomarker-based strategy. PMID:26537565
Horká, Marie; Salplachta, Jiří; Karásek, Pavel; Kubesová, Anna; Horký, Jaroslav; Matoušková, Hana; Slais, Karel; Roth, Michal
This study was undertaken to investigate feasibility of a combination of capillary isoelectric focusing (CIEF) in a tapered fused silica (FS) capillary with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for a rapid and reliable identification of bacteria taken from plant-tissue-containing samples. Eight strains representing different species of the genus Dickeya were selected on the basis of close proximity of their isoelectric points: D. chrysanthemi, D. chrysanthemi bv. parthenii, D. chrysanthemi bv. chrysanthemi, D. dadantii, D. paradisiaca, D. solani, D. diffenbachiae, and D. dianthicola. Because the Dickeya species (spp.) cannot be easily discriminated from each other when CIEF is performed in a cylindrical FS capillary (commonly used in CIEF) even if a narrow pH gradient is used, a tapered FS capillary was employed instead, which enabled satisfactory discrimination of the examined bacteria due to enhanced separation efficiency of CIEF in the tapered FS capillary. CIEF in the tapered FS capillary was also successfully used for the detection and characterization of Dickeya spp. in a plant-tissue-containing sample. Then an off-line combination of CIEF with MALDI-TOF MS was employed for rapid and reliable identification of Dickeya spp. in the plant-tissue-containing sample. It was found that the presence of plant tissue did not affect the results, making the proposed procedure very promising with respect to the fast and reliable detection and identification of bacteria in plant-tissue-containing samples.
Rivas, Daniel; Ginebreda, Antoni; Pérez, Sandra; Quero, Carmen; Barceló, Damià
Degradation of solid polymers in the aquatic environment encompasses a variety of biotic and abiotic processes giving rise to heterogeneous patterns across the surface of the material, which cannot be investigated using conventional Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) that only renders an "average" picture of the sample. In that context, MALDI-TOF MS Imaging (MALDI MSI) provides a rapid and efficient tool to study 2D spatial changes occurred in the chemical composition of the polymer surface. Commercial polycaprolactone diol (average molecular weight of 1250Da) was selected as test material because it had been previously known to be amenable to biological degradation. The test oligomer probe was incubated under aerobic and denitrifying conditions using synthetic water and denitrifying mixed liquor obtained from a wastewater treatment plant respectively. After ca. seven days of exposure the mass spectra obtained by MALDI MSI showed the occurrence of chemical modifications in the sample surface. Observed heterogeneity across the probe's surface indicated significant degradation and suggested the contribution of biotic processes. The results were investigated using different image processing tools. Major changes on the oligomer surface were observed when exposed to denitrifying conditions.
Gholipour, Yousef; Nonami, Hiroshi; Erra-Balsells, Rosa
Single-cell cytoplasm sap (1-10 pL) was extracted by using a pressure probe glass microcapillary tip from tulip leaf and bulb and analyzed by UV-MALDI-TOF MS for free underivatized carbohydrate content. Three matrices including 2,5-dihydroxybenzoic acid (DHB), 2,4,6-trihydroxyacetophenone (THAP), and carbon nanotubes (CNTs) in positive ion mode were selected for analysis because of acceptable carbohydrate-related signal reproducibility. Disaccharide and oligosaccharide (up to 15 Hex when THAP was used, 11 Hex with DHB, and 7 Hex with CNTs) were detected in tulip bulb cell cytoplasm sample. When DHB was used as matrix, neutral carbohydrates were more abundantly detected as sodiated cations; the sugar-related signals, however, appeared as dominant potassiated cations when THAP and CNTs were used. Small amount of monosaccharide was also detected in bulb cell cytoplasm with CNTs as matrix. UV-MALDI-TOF MS of leaf cell extract resulted in high-resolution detection of hexose and disaccharide with DHB, THAP, and CNTs.
Development of a Rapid and Accurate Identification Method for Citrobacter Species Isolated from Pork Products Using a Matrix-Assisted Laser-Desorption Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS).
Kwak, Hye-Lim; Han, Sun-Kyung; Park, Sunghoon; Park, Si Hong; Shim, Jae-Yong; Oh, Mihwa; Ricke, Steven C; Kim, Hae-Yeong
Previous detection methods for Citrobacter are considered time consuming and laborious. In this study, we have developed a rapid and accurate detection method for Citrobacter species in pork products, using matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS). A total of 35 Citrobacter strains were isolated from 30 pork products and identified by both MALDI-TOF MS and 16S rRNA gene sequencing approaches. All isolates were identified to the species level by the MALDI-TOF MS, while 16S rRNA gene sequencing results could not discriminate them clearly. These results confirmed that MALDI-TOF MS is a more accurate and rapid detection method for the identification of Citrobacter species.
Penny, Christian; Grothendick, Beau; Zhang, Lin; Borror, Connie M.; Barbano, Duane; Cornelius, Angela J.; Gilpin, Brent J.; Fagerquist, Clifton K.; Zaragoza, William J.; Jay-Russell, Michele T.; Lastovica, Albert J.; Ragimbeau, Catherine; Cauchie, Henry-Michel; Sandrin, Todd R.
MALDI-TOF MS has been utilized as a reliable and rapid tool for microbial fingerprinting at the genus and species levels. Recently, there has been keen interest in using MALDI-TOF MS beyond the genus and species levels to rapidly identify antibiotic resistant strains of bacteria. The purpose of this study was to enhance strain level resolution for Campylobacter jejuni through the optimization of spectrum processing parameters using a series of designed experiments. A collection of 172 strains of C. jejuni were collected from Luxembourg, New Zealand, North America, and South Africa, consisting of four groups of antibiotic resistant isolates. The groups included: (1) 65 strains resistant to cefoperazone (2) 26 resistant to cefoperazone and beta-lactams (3) 5 strains resistant to cefoperazone, beta-lactams, and tetracycline, and (4) 76 strains resistant to cefoperazone, teicoplanin, amphotericin, B and cephalothin. Initially, a model set of 16 strains (three biological replicates and three technical replicates per isolate, yielding a total of 144 spectra) of C. jejuni was subjected to each designed experiment to enhance detection of antibiotic resistance. The most optimal parameters were applied to the larger collection of 172 isolates (two biological replicates and three technical replicates per isolate, yielding a total of 1,031 spectra). We observed an increase in antibiotic resistance detection whenever either a curve based similarity coefficient (Pearson or ranked Pearson) was applied rather than a peak based (Dice) and/or the optimized preprocessing parameters were applied. Increases in antimicrobial resistance detection were scored using the jackknife maximum similarity technique following cluster analysis. From the first four groups of antibiotic resistant isolates, the optimized preprocessing parameters increased detection respective to the aforementioned groups by: (1) 5% (2) 9% (3) 10%, and (4) 2%. An additional second categorization was created from the
Kumar, Ramadhar; Sripriya, R.; Balaji, S.; Senthil Kumar, M.; Sehgal, P. K.
In this study, we report on physical and in vitro biological characterization of succinylated collagen (SC). SC was prepared by succinylation of type I bovine tendon collagen. SC swells and dissolves in physiological pH buffers (pH 7.4) Biocompatibility of SC to collagen for fibroblasts was comparable but L6 myoblasts showed pronounced proliferation and differentiation with SC. Using the MALDI-TOF/MS technique, SC was found with increased molecular mass by 16,359 Da per molecule which corresponds to about 54 succinyl groups covalently linked to the collagen strand. Raman spectroscopy revealed the retention of triple helical structure conformation in the presence of linked succinyl groups. New peaks near 1737, 1675 and 1420 cm -1 and decreased intensities near 2440 and 488 cm -1 provides the most convenient marker bands for succinylation of collagen. The intense band regions near 2856-2934, 2724, and 1445 cm -1 also confirms the existence of succinyl groups.
Enjalbal, Christine; Ribière, Patrice; Lamaty, Frédéric; Yadav-Bhatnagar, Neerja; Martinez, Jean; Aubagnac, Jean-Louis
Macromolecules of tunable solubility, used to mimic inert insoluble materials while maintaining solution conditions, allowed the performance of efficient supported organic chemistry and facilitated in situ reaction monitoring. To satisfy the high throughput requirements of automated synthetic processes, organic syntheses carried out on bifunctional polyethylene glycol polymers (PEG(3400)-OH) were monitored step-by-step by matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). A protocol was designed to control the ionization mechanism of such polymers exhibiting high affinity for alkali metal cations. Automated, rapid, and reliable data interpretation was performed by an in-house developed visual basic application relying on the sodiated ion accurate monoisotopic mass measurement. The methodology was illustrated through the monitoring of a six-step synthetic scheme.
Wei, Shu-Dong; Zhou, Hai-Chao; Lin, Yi-Ming; Liao, Meng-Meng; Chai, Wei-Ming
The structures of the condensed tannins from leaf, stem bark and root bark of Acacia confusa were characterized by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis, and their antioxidant activities were measured using 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging and ferric reducing/antioxidant power (FRAP) assays. The results showed that the condensed tannins from stem bark and root bark include propelargonidin and procyanidin, and the leaf condensed tannins include propelargonidin, procyanidin and prodelphinidin, all with the procyanidin dominating. The condensed tannins had different polymer chain lengths, varying from trimers to undecamers for leaf and root bark and to dodecamers for stem bark. The condensed tannins extracted from the leaf, stem bark and root bark all showed a very good DPPH radical scavenging activity and ferric reducing power.
Firacative, Carolina; Trilles, Luciana; Meyer, Wieland
Background The Cryptococcus neoformans/C. gattii species complex comprises two sibling species that are divided into eight major molecular types, C. neoformans VNI to VNIV and C. gattii VGI to VGIV. These genotypes differ in host range, epidemiology, virulence, antifungal susceptibility and geographic distribution. The currently used phenotypic and molecular identification methods for the species/molecular types are time consuming and expensive. As Matrix-Assisted Laser Desorption Ionization-Time-of-Flight Mass Spectrometry (MALDI-TOF MS) offers an effective alternative for the rapid identification of microorganisms, the objective of this study was to examine its potential for the identification of C. neoformans and C. gattii strains at the intra- and inter-species level. Methodology Protein extracts obtained via the formic acid extraction method of 164 C. neoformans/C. gattii isolates, including four inter-species hybrids, were studied. Results The obtained mass spectra correctly identified 100% of all studied isolates, grouped each isolate according to the currently recognized species, C. neoformans and C. gattii, and detected potential hybrids. In addition, all isolates were clearly separated according to their major molecular type, generating greater spectral differences among the C. neoformans molecular types than the C. gattii molecular types, most likely reflecting a closer phylogenetic relationship between the latter. The number of colonies used and the incubation length did not affect the results. No spectra were obtained from intact yeast cells. An extended validated spectral library containing spectra of all eight major molecular types was established. Conclusions MALDI-TOF MS is a rapid identification tool for the correct recognition of the two currently recognized human pathogenic Cryptococcus species and offers a simple method for the separation of the eight major molecular types and the detection of hybrid strains within this species complex in the
Kim, Eiseul; Cho, Youngjae; Lee, Yoonju; Han, Sun-Kyung; Kim, Chang-Gyeom; Choo, Dong-Won; Kim, Young-Rok; Kim, Hae-Yeong
Weissella are obligate heterofermentative lactic acid bacteria belonging to the Leuconostocaceae family. Some Weissella can be found in salted and fermented foods, such as kimchi and jeotgal, and plays an important role in the fermentation process. In the present study, for the first time, a rapid and accurate identification method for Weissella species from kimchi and jeotgal was developed based on MALDI-TOF MS, supplemented with an in-house database. Of the 135 Weissella spectra aligned with the MALDI bioTyper database, 56 isolates (41.5%) yielded no reliable identification results with low log scores (<1.7). After registering the spectra of six Weissella reference strains, all of the isolates were correctly identified, of which 113 (83.7%) and 22 (16.3%) were identified at the species and genus level, respectively. Moreover, a dendrogram generated by protein profiles of the different Weissella species clearly presented distinctive clusters, and PCA analysis separated the spectra of Weissella species into four clusters. In comparing food origins, different Weissella species were identified from two fermented foods. W. soli and W. cibaria were isolated from kimchi, while W. thailandensis and W. halotolerans were isolated from jeotgal. The results of our proteomic approach confirm that the MALDI bioTyper database, with our in-house Weissella database, is sufficient for Weissella identification. The MALDI-TOF MS method provides fast and reliable discrimination between different species in the genus Weissella and, therefore, will be useful for safety control in fish farms or in the production of fermented foods. This method can also be applied to the control of opportunistic pathogenic Weissella in human clinical infections.
Dybwad, Marius; van der Laaken, Anton L; Blatny, Janet Martha; Paauw, Armand
Rapid and reliable identification of Bacillus anthracis spores in suspicious powders is important to mitigate the safety risks and economic burdens associated with such incidents. The aim of this study was to develop and validate a rapid and reliable laboratory-based matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis method for identifying B. anthracis spores in suspicious powder samples. A reference library containing 22 different Bacillus sp. strains or hoax materials was constructed and coupled with a novel classification algorithm and standardized processing protocol for various powder samples. The method's limit of B. anthracis detection was determined to be 2.5 × 10(6) spores, equivalent to a 55-μg sample size of the crudest B. anthracis-containing powder discovered during the 2001 Amerithrax incidents. The end-to-end analysis method was able to successfully discriminate among samples containing B. anthracis spores, closely related Bacillus sp. spores, and commonly encountered hoax materials. No false-positive or -negative classifications of B. anthracis spores were observed, even when the analysis method was challenged with a wide range of other bacterial agents. The robustness of the method was demonstrated by analyzing samples (i) at an external facility using a different MALDI-TOF MS instrument, (ii) using an untrained operator, and (iii) using mixtures of Bacillus sp. spores and hoax materials. Taken together, the observed performance of the analysis method developed demonstrates its potential applicability as a rapid, specific, sensitive, robust, and cost-effective laboratory-based analysis tool for resolving incidents involving suspicious powders in less than 30 min.
Ratcliffe, Paul; Fang, Hong; Thidholm, Ellinor; Boräng, Stina; Westling, Katarina; Özenci, Volkan
Granulicatella and Abiotrophia spp. were known as nutritionally variant streptococci (NVS). Such strains have caused major diagnostic difficulties due to fastidious culturing and unspecific colony morphology. The present study is aimed at comparing the performance of laboratory available diagnostic methods for NVS isolates and determining the antimicrobial susceptibility of these isolates. Fourteen clinical invasive isolates, consisting of 10 Granulicatella adiacens, 1 Granulicatella elegans, and 3 Abiotrophia defectiva were in parallel analyzed by 2 matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) systems, i.e., Bruker MS and Vitek MS, as well as Vitek 2 for the species determination. 16S rRNA gene sequencing was applied as a reference method. The Vitek MS gave correct identification for all 14 isolates. The Bruker MS could correctly identify 8/10 G. adiacens, 0/1 G. elegans, and 3/3 A. defectiva isolates at the first analysis occasion, and all 14 isolates became identifiable after repeated tests. The Vitek 2 system could identify 6/10 G. adiacens, 1/1 G. elegans, and 2/3 A. defectiva isolates at the species level. Antimicrobial susceptibilities of 11 antibiotics were determined by Etest. Resistance against ciprofloxacin, ceftriaxone, rifampicin, and tetracycline were observed in 4, 10, 4, and 1 isolates, respectively. In conclusion, MALDI-TOF MS is a useful tool for the rapid diagnosis of NVS. Phenotypic testing by Vitek 2 is only partially effective for the accurate identification of such strains. The emergence of resistant NVS isolates indicates the necessity of monitoring antimicrobial susceptibilities of such uncommon pathogens.
Huang, Chien-Hsun; Huang, Lina; Chang, Mu-Tzu; Chen, Kuo-Lung
Members of the Bacillus subtilis group (BSG) possess industrial applicability; unfortunately, B. subtilis and its phylogenetically closest species are indistinguishable from one another using 16S rDNA sequencing, physiological and biochemical tests. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is a relatively novel technique for the fast and reliable identification of microorganisms. The aim of this study was to construct a unique analytical in-house database (IHDB) for BSG discrimination based on whole-cell protein fingerprinting using MALDI-TOF MS, as well as to discover biomarkers from the MS peaks to generate a classification model for further differentiation using the ClinProTools software. Type strains of 12 species (included five subspecies) of the BSG were used to build a main spectrum profile (MSP) to create an IHDB under the optimized parameters. The BSG isolates obtained from partial recA gene sequencing were used for IHDB validation. A total of 84 (100%) isolates were correctly identified to the species level and had high score values (mean score: 2.52). However, the IHDB had ambiguous identification at the subspecies level of Bacillus amyloliquefaciens. After implementation of the classification models, the strains could be clearly differentiated. We have successfully developed a rapid, accurate and cost-effective platform for the species- and subspecies-level discrimination of BSG based on the implementation of the IHDB and coupled with ClinProTools, which can be employed as an alternative technology to DNA sequencing and applied for efficient quality control of the microbial agent.
Chao, Qiao-Ting; Lee, Tai-Fen; Teng, Shih-Hua; Peng, Li-Yun; Chen, Ping-Hung; Teng, Lee-Jene; Hsueh, Po-Ren
We assessed the accuracy of species-level identification of two commercially available matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) systems (Bruker Biotyper and Vitek MS) and two conventional phenotypic methods (Phoenix 100 YBC and Vitek 2 Yeast ID) with that of rDNA gene sequencing analysis among 200 clinical isolates of commonly encountered yeasts. The correct identification rates of the 200 yeast isolates to species or complex (Candida parapsilosis complex, C. guilliermondii complex and C. rugosa complex) levels by the Bruker Biotyper, Vitek MS (using in vitro devices [IVD] database), Phoenix 100 YBC and Vitek 2 Yeast ID (Sabouraud's dextrose agar) systems were 92.5%, 79.5%, 89%, and 74%, respectively. An additional 72 isolates of C. parapsilosis complex and 18 from the above 200 isolates (30 in each of C. parapsilosis, C. metapsilosis, and C. orthopsilosis) were also evaluated separately. Bruker Biotyper system could accurately identify all C. parapsilosis complex to species level. Using Vitek 2 MS (IVD) system, all C. parapsilosis but none of C. metapsilosis, or C. orthopsilosis could be accurately identified. Among the 89 yeasts misidentified by the Vitek 2 MS (IVD) system, 39 (43.8%), including 27 C. orthopsilosis isolates, could be correctly identified Using the Vitek MS Plus SARAMIS database for research use only. This resulted in an increase in the rate of correct identification of all yeast isolates (87.5%) by Vitek 2 MS. The two species in C. guilliermondii complex (C. guilliermondii and C. fermentati) isolates were correctly identified by cluster analysis of spectra generated by the Bruker Biotyper system. Based on the results obtained in the current study, MALDI-TOF MS systems present a promising alternative for the routine identification of yeast species, including clinically commonly and rarely encountered yeast species and several species belonging to C. parapsilosis complex, C. guilliermondii complex
Veloo, A C M; Elgersma, P E; Friedrich, A W; Nagy, E; van Winkelhoff, A J
With matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), bacteria can be identified quickly and reliably. This accounts especially for anaerobic bacteria. Because growth rate and oxygen sensitivity differ among anaerobic bacteria, we aimed to study the influence of incubation time, exposure to oxygen and sample preparation on the quality of the spectrum using the Bruker system. Also, reproducibility and inter-examiner variability were determined. Twenty-six anaerobic species, representing 17 genera, were selected based on gram-stain characteristics, growth rate and colony morphology. Inter-examiner variation showed that experience in the preparation of the targets can be a significant variable. The influence of incubation time was determined between 24 and 96 h of incubation. Reliable species identification was obtained after 48 h of incubation for gram-negative anaerobes and after 72 h for gram-positive anaerobes. Exposure of the cultures to oxygen did not influence the results of the MALDI-TOF MS identifications of all tested gram-positive species. Fusobacterium necrophorum and Prevotella intermedia could not be identified after >24 h and 48 h of exposure to oxygen, respectively. Other tested gram-negative bacteria could be identified after 48 h of exposure to oxygen. Most of the tested species could be identified using the direct spotting method. Bifidobacterium longum and Finegoldia magna needed on-target extraction with 70% formic acid in order to obtain reliable species identification and Peptoniphilus ivorii a full extraction. Spectrum quality was influenced by the amount of bacteria spotted on the target, the homogeneity of the smear and the experience of the examiner.
Fernández, Javier; Rodríguez-Lucas, Carlos; Fernández-Suárez, Jonathan; Vazquez, Fernando; Rodicio, M Rosario
A new protocol for Enterobacteriaceae identification and detection of carbapenemase-producing isolates from blood cultures by combining MALDI-TOF MS and the Carba NP test has been evaluated. Bacterial identification was correct in 129 of the 130 isolates tested while the Carba NP detected 28 out of the 29 carbapenemase producers.
Background Low-molecular-weight glutenin subunits (LMW-GS) play a crucial role in determining end-use quality of common wheat by influencing the viscoelastic properties of dough. Four different methods - sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), two-dimensional gel electrophoresis (2-DE, IEF × SDS-PAGE), matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and polymerase chain reaction (PCR), were used to characterize the LMW-GS composition in 103 cultivars from 12 countries. Results At the Glu-A3 locus, all seven alleles could be reliably identified by 2-DE and PCR. However, the alleles Glu-A3e and Glu-A3d could not be routinely distinguished from Glu-A3f and Glu-A3g, respectively, based on SDS-PAGE, and the allele Glu-A3a could not be differentiated from Glu-A3c by MALDI-TOF-MS. At the Glu-B3 locus, alleles Glu-B3a, Glu-B3b, Glu-B3c, Glu-B3g, Glu-B3h and Glu-B3j could be clearly identified by all four methods, whereas Glu-B3ab, Glu-B3ac, Glu-B3ad could only be identified by the 2-DE method. At the Glu-D3 locus, allelic identification was problematic for the electrophoresis based methods and PCR. MALDI-TOF-MS has the potential to reliably identify the Glu-D3 alleles. Conclusions PCR is the simplest, most accurate, lowest cost, and therefore recommended method for identification of Glu-A3 and Glu-B3 alleles in breeding programs. A combination of methods was required to identify certain alleles, and would be especially useful when characterizing new alleles. A standard set of 30 cultivars for use in future studies was chosen to represent all LMW-GS allelic variants in the collection. Among them, Chinese Spring, Opata 85, Seri 82 and Pavon 76 were recommended as a core set for use in SDS-PAGE gels. Glu-D3c and Glu-D3e are the same allele. Two new alleles, namely, Glu-D3m in cultivar Darius, and Glu-D3n in Fengmai 27, were identified by 2-DE. Utilization of the suggested standard cultivar set, seed of
Grosse-Herrenthey, Anke; Maier, Thomas; Gessler, Frank; Schaumann, Reiner; Böhnel, Helge; Kostrzewa, Markus; Krüger, Monika
Diverse techniques were applied to effect the identification and classification of isolated clostridial strains. Nevertheless, the correct identification of clostridial strains remains a laborious, time-consuming task which entails a not inconsiderable degree of expertise. In addition to this, traditional methods based on the metabolic properties of the bacteria require rigorously standardized media and growth conditions to assure the attainment of reproducible results. Although DNA-based methods, like the PCR of a species specific gene, are known to yield precise and reproducible results, their degree of effectivity is circumscribed by the fact that even the incidence of a toxin encoding gene is not necessarily linked to nor consequently indicative of the presence of an infectious disease. Moreover, most of these methods postulate an initial assumption concerning the expected bacterial species involved before the choice of PCR primer for use can be made. Consequently, the scope of these methods is restricted to that of targeted analyses. The 16S rDNA sequencing which is assumed to be the gold standard for bacterial classification having the unequivocal advantage of being capable of determining even uncultivable bacteria is nonetheless a time-consuming and costly technique. In the present study we describe the utilization of matrix-assisted laser desorption and ionization-time-of-flight mass spectrometry (MALDI-TOF MS) for whole cell fingerprinting in combination with a dedicated bioinformatic software tool to distinguish between various clostridial species. Total 64 clostridial strains of 31 different species each displayed a mass spectrum unique to the strain involved, to the effect that it was also possible not only to differentiate between the strains examined, but also to establish to which species the individual strains belonged to. Starting with a single colony it was possible to correctly identify a Clostridium species within minutes. It was even possible
Lévesque, Simon; Dufresne, Philippe J; Soualhine, Hafid; Domingo, Marc-Christian; Bekal, Sadjia; Lefebvre, Brigitte; Tremblay, Cécile
Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has emerged as a rapid, highly accurate, and cost-effective method for routine identification of a wide range of microorganisms. We carried out a side by side comparative evaluation of the performance of Bruker Biotyper versus VITEK MS for identification of a large and diverse collection of microorganisms. Most difficult and/or unusual microorganisms, as well as commonly encountered microorganisms were selected, including Gram-positive and negative bacteria, mycobacteria, actinomycetes, yeasts and filamentous fungi. Six hundred forty two strains representing 159 genera and 441 species from clinical specimens previously identified at the Laboratoire de santé publique du Québec (LSPQ) by reference methods were retrospectively chosen for the study. They included 254 Gram-positive bacteria, 167 Gram-negative bacteria, 109 mycobacteria and aerobic actinomycetes and 112 yeasts and moulds. MALDI-TOF MS analyses were performed on both systems according to the manufacturer's instructions. Of the 642 strains tested, the name of the genus and / or species of 572 strains were referenced in the Bruker database while 406 were present in the VITEK MS IVD database. The Biotyper correctly identified 494 (86.4%) of the strains, while the VITEK MS correctly identified 362 (92.3%) of the strains (excluding 14 mycobacteria that were not tested). Of the 70 strains not present in the Bruker database at the species level, the Biotyper correctly identified 10 (14.3%) to the genus level and 2 (2.9%) to the complex/group level. For 52 (74.2%) strains, we obtained no identification, and an incorrect identification was given for 6 (8.6%) strains. Of the 178 strains not present in the VITEK MS IVD database at the species level (excluding 71 untested mycobacteria and actinomycetes), the VITEK MS correctly identified 12 (6.8%) of the strains each to the genus and to the complex/group level. For 97 (54
Peng, Yanchun; Yu, Zitong; Islam, Shahidul; Zhang, Yujuan; Wang, Xiaolong; Lei, Zhensheng; Yu, Kan; Sun, Dongfa; Ma, Wujun
Low molecular weight glutenin subunits are important components of wheat storage proteins, which play an important role in determining end-use quality of common wheat. A newly established matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) procedure was used to analyze 478 landraces of bread wheat collected from the Yangtze-River region in China. Results indicated that 17 alleles at three loci: Glu-A3, Glu-B3 and Glu-D3 were identified, resulting in 87 different allele combinations. Of the 17 alleles detected at all the Glu-3 loci, five belonged to Glu-A3, seven to Glu-B3 and five to Glu-D3 locus. MALDI-TOF-MS indicated Glu-A3a/c was present in 72.8%, Glu-A3b in 8.4%, Glu-A3d in 8.4%, Glu-A3f in 5.2% and Glu-A3e in 3.6% lines. Seven types of alleles were identified at the Glu-B3 locus: Glu-B3d/i (25.5%), Glu-B3b (21.3%), Glu-B3c (16.9%), Glu-B3h (13.8%), Glu-B3f (8.4%), Glu-B3a (8.2%), and Glu-B3g (5.2%). Five types of Glu-D3 alleles were detected: Glu-D3a (58.4%), Glu-D3c (22.6%), Glu-D3d (15.5%), Glu-D3b (3.3%) and Glu-D3f (0.2%). Four new alleles that showed abnormal MALDI-TOF spectrum patterns were identified at the Glu-A3 and Glu-B3 loci. A detailed study is needed to further characterize these alleles and their potential usage for wheat improvement. PMID:27795690
Lévesque, Simon; Dufresne, Philippe J.; Soualhine, Hafid; Domingo, Marc-Christian; Bekal, Sadjia; Lefebvre, Brigitte; Tremblay, Cécile
Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) has emerged as a rapid, highly accurate, and cost-effective method for routine identification of a wide range of microorganisms. We carried out a side by side comparative evaluation of the performance of Bruker Biotyper versus VITEK MS for identification of a large and diverse collection of microorganisms. Most difficult and/or unusual microorganisms, as well as commonly encountered microorganisms were selected, including Gram-positive and negative bacteria, mycobacteria, actinomycetes, yeasts and filamentous fungi. Six hundred forty two strains representing 159 genera and 441 species from clinical specimens previously identified at the Laboratoire de santé publique du Québec (LSPQ) by reference methods were retrospectively chosen for the study. They included 254 Gram-positive bacteria, 167 Gram-negative bacteria, 109 mycobacteria and aerobic actinomycetes and 112 yeasts and moulds. MALDI-TOF MS analyses were performed on both systems according to the manufacturer’s instructions. Of the 642 strains tested, the name of the genus and / or species of 572 strains were referenced in the Bruker database while 406 were present in the VITEK MS IVD database. The Biotyper correctly identified 494 (86.4%) of the strains, while the VITEK MS correctly identified 362 (92.3%) of the strains (excluding 14 mycobacteria that were not tested). Of the 70 strains not present in the Bruker database at the species level, the Biotyper correctly identified 10 (14.3%) to the genus level and 2 (2.9%) to the complex/group level. For 52 (74.2%) strains, we obtained no identification, and an incorrect identification was given for 6 (8.6%) strains. Of the 178 strains not present in the VITEK MS IVD database at the species level (excluding 71 untested mycobacteria and actinomycetes), the VITEK MS correctly identified 12 (6.8%) of the strains each to the genus and to the complex/group level. For 97
Verroken, Alexia; Defourny, Lydwine; le Polain de Waroux, Olivier; Belkhir, Leïla; Laterre, Pierre-François; Delmée, Michel; Glupczynski, Youri
Shortening the turn-around time (TAT) of positive blood culture (BC) identification (ID) and susceptibility results is essential to optimize antimicrobial treatment in patients with sepsis. We aimed to evaluate the impact on antimicrobial prescription of a modified workflow of positive BCs providing ID and partial susceptibility results for Enterobacteriaceae (EB), Pseudomonas aeruginosa and Staphylococcus aureus on the day of BC positivity detection. This study was divided into a pre-intervention period (P0) with a standard BC workflow followed by 2 intervention periods (P1, P2) with an identical modified workflow. ID was performed with MALDI-TOF MS from blood, on early or on overnight subcultures. According to ID results, rapid phenotypic assays were realized to detect third generation cephalosporin resistant EB/P. aeruginosa or methicillin resistant S. aureus. Results were transmitted to the antimicrobial stewardship team for patient’s treatment revision. Times to ID, to susceptibility results and to optimal antimicrobial treatment (OAT) were compared across the three study periods. Overall, 134, 112 and 154 positive BC episodes in P0, P1 and P2 respectively were included in the analysis. Mean time to ID (28.3 hours in P0) was reduced by 65.3% in P1 (10.2 hours) and 61.8% in P2 (10.8 hours). Mean time to complete susceptibility results was reduced by 27.5% in P1 and 27% in P2, with results obtained after 32.4 and 32.6 hours compared to 44.7 hours in P0. Rapid tests allowed partial susceptibility results to be obtained after a mean time of 11.8 hours in P1 and 11.7 hours in P2. Mean time to OAT was decreased to 21.6 hours in P1 and to 17.9 hours in P2 compared to 36.1 hours in P0. Reducing TAT of positive BC with MALDI-TOF MS ID and rapid susceptibility testing accelerated prescription of targeted antimicrobial treatment thereby potentially improving the patients’ clinical outcome. PMID:27228001
Verroken, Alexia; Defourny, Lydwine; le Polain de Waroux, Olivier; Belkhir, Leïla; Laterre, Pierre-François; Delmée, Michel; Glupczynski, Youri
Shortening the turn-around time (TAT) of positive blood culture (BC) identification (ID) and susceptibility results is essential to optimize antimicrobial treatment in patients with sepsis. We aimed to evaluate the impact on antimicrobial prescription of a modified workflow of positive BCs providing ID and partial susceptibility results for Enterobacteriaceae (EB), Pseudomonas aeruginosa and Staphylococcus aureus on the day of BC positivity detection. This study was divided into a pre-intervention period (P0) with a standard BC workflow followed by 2 intervention periods (P1, P2) with an identical modified workflow. ID was performed with MALDI-TOF MS from blood, on early or on overnight subcultures. According to ID results, rapid phenotypic assays were realized to detect third generation cephalosporin resistant EB/P. aeruginosa or methicillin resistant S. aureus. Results were transmitted to the antimicrobial stewardship team for patient's treatment revision. Times to ID, to susceptibility results and to optimal antimicrobial treatment (OAT) were compared across the three study periods. Overall, 134, 112 and 154 positive BC episodes in P0, P1 and P2 respectively were included in the analysis. Mean time to ID (28.3 hours in P0) was reduced by 65.3% in P1 (10.2 hours) and 61.8% in P2 (10.8 hours). Mean time to complete susceptibility results was reduced by 27.5% in P1 and 27% in P2, with results obtained after 32.4 and 32.6 hours compared to 44.7 hours in P0. Rapid tests allowed partial susceptibility results to be obtained after a mean time of 11.8 hours in P1 and 11.7 hours in P2. Mean time to OAT was decreased to 21.6 hours in P1 and to 17.9 hours in P2 compared to 36.1 hours in P0. Reducing TAT of positive BC with MALDI-TOF MS ID and rapid susceptibility testing accelerated prescription of targeted antimicrobial treatment thereby potentially improving the patients' clinical outcome.
Matrix-assisted Laser Desorption Ionization-Time-of-Flight Mass Spectrometry (MALDI-TOF MS) as a Reliable Tool to Identify Species of Catalase-negative Gram-positive Cocci not Belonging to the Streptococcus Genus
Almuzara, Marisa; Barberis, Claudia; Velázquez, Viviana Rojas; Ramirez, Maria Soledad; Famiglietti, Angela; Vay, Carlos
Objective: To evaluate the performance of matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) by using 190 Catalase-negative Gram-Positive Cocci (GPC) clinical isolates. Methods: All isolates were identified by conventional phenotypic tests following the proposed scheme by Ruoff and Christensen and MALDI-TOF MS (Bruker Daltonics, BD, Bremen, Germany). Two different extraction methods (direct transfer formic acid method on spot and ethanol formic acid extraction method) and different cut-offs for genus/specie level identification were used. The score cut-offs recommended by the manufacturer (≥ 2.000 for species-level, 1.700 to 1.999 for genus level and <1.700 no reliable identification) and lower cut-off scores (≥1.500 for genus level, ≥ 1.700 for species-level and score <1.500 no reliable identification) were considered for identification. A minimum difference of 10% between the top and next closest score was required for a different genus or species. MALDI-TOF MS identification was considered correct when the result obtained from MS database agreed with the phenotypic identification result. When both methods gave discordant results, the 16S rDNA or sodA genes sequencing was considered as the gold standard identification method. The results obtained by MS concordant with genes sequencing, although discordant with conventional phenotyping, were considered correct. MS results discordant with 16S or sodA identification were considered incorrect. Results: Using the score cut-offs recommended by the manufacturer, 97.37% and 81.05% were correctly identified to genus and species level, respectively. On the other hand, using lower cut-off scores for identification, 97.89% and 94.21% isolates were correctly identified to genus and species level respectively by MALDI-TOF MS and no significant differences between the results obtained with two extraction methods were obtained. Conclusion: The results obtained suggest that MALDI-TOF
The ability to predict protein function from structure is becoming increasingly important; hence, elucidation and determination of protein structure become the major steps in proteomics. The present study was undertaken for identification of metalloprotease produced by Bacillus cereus B80 and recognition of characteristics that can be industrially exploited. The enzyme was purified in three steps combining precipitation and chromatographic methods resulting in 33.5% recovery with 13.1-fold purification of enzyme which was detected as a single band with a molecular mass of 26 kDa approximately in SDS-PAGE and zymogram. The MALDI-TOF MS showed that the enzyme exhibited 70–93% similarity with zinc metalloproteases from various strains Bacillus sp. specifically from Bacillus cereus group. The sequence alignment revealed the presence of zinc-binding region VVVHEMCHMV in the most conserved C terminus region. Secondary structure of the enzyme was obtained by CD spectra and I-TASSER. The enzyme kinetics revealed a Michaelis constant (Km) of 0.140 μmol/ml and Vmax of 2.11 μmol/min. The application studies showed that the enzyme was able to hydrolyze various proteins with highest affinity towards casein followed by BSA and gelatin. The enzyme exhibited strong fibrinolytic, collagenolytic, and gelatinolytic properties and stability in various organic solvents. PMID:25802851
Fuchs, B; Bondzio, A; Wagner, U; Schiller, J
Alterations of the phospholipid (PL) compositions of body fluids are assumed to be indicative of inflammatory diseases, e.g. rheumatoid arthritis (RA). Recently, we have shown that particularly the phosphatidylcholine/lysophosphatidylcholine (PC/LPC) ratio determined in human synovial fluids (SF) and sera represents a reliable measure of the inflammatory state in RA patients. However, it is not yet clear to what extent the PC/LPC ratio is also affected by nutrition habits. In the present study, the PL and the corresponding acyl chain compositions of human body fluids (SF and serum of RA patients as well as serum from healthy volunteers) are compared with those of two other mammalian species (horses and dogs suffering from degenerative joint diseases as well as healthy controls) by high-resolution 31P-nuclear magnetic resonance (NMR) spectroscopy and matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS). The most important result of this study is that the PL compositions of SF and serum of horse and dog are comparable with those of human body fluids. Compared with humans, however, the horse body fluid contains less PCs with highly unsaturated arachidonoyl residues, while that of dogs possesses the highest content of arachidonoyl-containing PC. These species-related differences stem primarily from different nutrition habits (meat vs. plants).
Angeletti, Silvia; Lo Presti, Alessandra; Cella, Eleonora; Dicuonzo, Giordano; Crea, Francesca; Palazzotti, Bernardetta; Dedej, Etleva; Ciccozzi, Massimo; De Florio, Lucia
Clinical Candida isolates from two different hospitals in Rome were identified and clustered by MALDI-TOF MS system and their origin and evolution estimated by Bayesian phylogenetic analysis. The different species of Candida were correctly identified and clustered separately, confirming the ability of these techniques to discriminate between different Candida species. Focusing MALDI-TOF analysis on a single Candida species, Candida albicans and Candida parapsilosis strains clustered differently for hospital setting as well as for period of isolation than Candida glabrata and Candida tropicalis isolates. The evolutionary rates of C. albicans and C. parapsilosis (1.93×10(-2) and 1.17×10(-2)substitutions/site/year, respectively) were in agreement with a higher rate of mutation of these species, even in a narrow period, than what was observed in C. glabrata and C. tropicalis strains (6.99×10(-4) and 7.52×10(-3)substitutions/site/year, respectively). C. albicans resulted as the species with the highest between and within clades genetic distance values in agreement with the temporal-related clustering found by MALDI-TOF and the high evolutionary rate 1.93×10(-2)substitutions/site/year.
Kumar, Jitendra; Sharma, Vijay K.; Singh, Dheeraj K.; Mishra, Ashish; Gond, Surendra K.; Verma, Satish K.; Kumar, Anuj; Kharwar, Ravindra Nath
The endophytic Streptomyces coelicolor strain AZRA 37 was isolated from the surface sterilized root of Azadirachta indica A. Juss., commonly known as neem plant in India. Since only a few reports are available regarding epigenetic modulations of microbial entities, S. coelicolor was treated with different concentrations of 5-azacytidine for this purpose and evaluated for its antibacterial potential against five human pathogenic bacteria (Aeromonas hydrophila IMS/GN11, Enterococcus faecalis IMS/GN7, Salmonella typhi MTCC 3216, Shigella flexneri ATCC 12022 and Staphylococcus aureus ATCC 25923). The crude extract obtained from cultures treated with 25 μM concentration of 5-azacytidine, was found effective against all five pathogenic bacteria tested while the untreated control was only active against 3 pathogenic bacteria. HPLC analysis of crude compounds from treated cultures showed a greater number of compounds than that of the control. Extraction of whole cell protein and its SDS PAGE analysis showed an additional major protein band in 25 μM 5-azacytidine treated culture and MALDI TOF MS/MS analysis revealed that this protein belongs to the porin family. PMID:26844762
Vallejo, Juan Andrés; Miranda, Patricia; Flores-Félix, José David; Sánchez-Juanes, Fernando; Ageitos, José M; González-Buitrago, José Manuel; Velázquez, Encarna; Villa, Tomás G
Chicha is a drink prepared in several Andean countries from Inca's times by maize fermentation. Currently this fermentation is carried out in familiar artesanal "chicherías" that make one of the most known types of chicha, the "chicha de jora". In this study we isolate and identify the yeasts mainly responsible of the fermentation process in this type of chicha in 10 traditional "chicherías" in Cusco region in Peru. We applied by first time MALDI-TOF MS analysis for the identification of yeast of non-clinic origin and the results showed that all of yeast strains isolated belong to the species Saccharomyces cerevisiae. These results agree with those obtained after the analysis of the D1/D2 and 5.8S-ITS regions. However the chicha strains have a phenotypic profile that differed in more than 40% as compared to that of current S. cerevisiae strains. To the best of our knowledge this is the first report concerning the yeasts involved in chicha fermentation.
Zhang, Jing; Zheng, Xiaoling; Ni, Yanli
In this study, a 4-vinylphenylboronic acid-functionalized graphene oxide (GO) material was prepared via atom-transfer radical polymerization (ATRP) method and applied for the first time as a novel matrix for the selective enrichment and analysis of small-molecule compounds with vicinal diols, which have been the focus of intense research in the field of life science, by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) in positive-ion mode. There are two main factors playing a decisive role in assisting laser D/I process comparing to some traditional matrices: (1) GO provides π-conjugated system by itself for laser absorption and energy transfer; (2) the modified 4-vinylphenylboronic acid can selectively capture small-molecule compounds with vicinal diols. The results demonstrate that the novel material has distinct advantages over previously reported matrices in enriching and assisting the highly efficient ionization of target molecules for mass spectrometry analysis. This work indicates a new application branch for graphene-based matrices and provides an alternative solution for small-molecules analysis.
Zhang, Jing; Zheng, Xiaoling; Ni, Yanli
In this study, a 4-vinylphenylboronic acid-functionalized graphene oxide (GO) material was prepared via atom-transfer radical polymerization (ATRP) method and applied for the first time as a novel matrix for the selective enrichment and analysis of small-molecule compounds with vicinal diols, which have been the focus of intense research in the field of life science, by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) in positive-ion mode. There are two main factors playing a decisive role in assisting laser D/I process comparing to some traditional matrices: (1) GO provides π-conjugated system by itself for laser absorption and energy transfer; (2) the modified 4-vinylphenylboronic acid can selectively capture small-molecule compounds with vicinal diols. The results demonstrate that the novel material has distinct advantages over previously reported matrices in enriching and assisting the highly efficient ionization of target molecules for mass spectrometry analysis. This work indicates a new application branch for graphene-based matrices and provides an alternative solution for small-molecules analysis.
Zeng, Xin-Hua; Huang, He-Qing; Chen, Dong-Shi; Jin, Hong-Wei; Huang, Hui-Ying
Human serum has been widely employed clinically for diagnosing various fatal diseases. However, the concentration of most proteins in human serum is too low to be directly measured using normal analytical methods. In order to obtain reliable analytical results from proteomic analysis of human serum, appropriate sample preparation is essential. A combined off-line analytical technique of gel chromatography and matrix-assisted laser desorption ionization/time of flight mass spectrometry (MALDI-TOF MS) has been successfully established to separate proteins for MS analysis. Using these combined techniques, 176 mass signal peaks of proteins/peptides were found in 6 of 18 fractions from normal male serum (NMS) in the presence of buffer consisting of NH4HCO3 and acetonitrile. A simple gel chromatography column packed with Sephadex G-50 removed most signal-suppressing compounds such as salts and high abundance proteins (HAP). The molecular mass to charge (m/z) ratios of differential peptides revealed in serum of male patient with liver-cancer (LCMPS) compared to NMS were 5365, 5644 and 6462, and these peptides can be used as biomarkers to clinically diagnose liver-cancer. The simple and convenient chromatographic method described here is not only superior to recently described HPLC separation for MS analysis, but also reveals many novel and significant serum biomarkers for the clinical diagnosis of various diseases.
Weller, Simon A; Stokes, Margaret G M; Lukaszewski, Roman A
A chemical (ethanol; formic acid; acetonitrile) protein extraction method for the preparation of bacterial samples for matrix assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF MS) identification was evaluated for its ability to inactivate bacterial species. Initial viability tests (with and without double filtration of the extract through 0.2 μM filters), indicated that the method could inactivate Escherichia coli MRE 162 and Klebsiella pneumoniae ATCC 35657, with or without filtration, but that filtration was required to exclude viable, avirulent, Bacillus anthracis UM23CL2 from extracts. Multiple, high stringency, viability experiments were then carried out on entire filtered extracts prepared from virulent B. anthracis Vollum vegetative cells and spores ranging in concentration from 10(6)-10(8) cfu per extract. B. anthracis was recovered in 3/18 vegetative cell extracts and 10/18 spore extracts. From vegetative cell extracts B. anthracis was only recovered from extracts that had undergone prolonged Luria (L)-broth (7 day) and L-agar plate (a further 7 days) incubations. We hypothesise that the recovery of B. anthracis in vegetative cell extracts is due to the escape of individual sub-lethally injured cells. We discuss our results in view of working practises in clinical laboratories and in the context of recent inadvertent releases of viable B. anthracis.
Maniwa, Jiro; Izumi, Shunsuke; Isobe, Naoki; Terada, Takato
Background The objective of this study was to identify substantially increased proteins in bovine cystic follicular fluid (FF) in order to clarify the pathology and etiology of bovine ovarian follicular cysts (BOFC). Methods Proteins in normal and cystic FF samples were subjected to two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and were compared using silver stained gel images with PDQuest image analysis software. Peptides from these increased spots were analyzed by matrix assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS), and were identified based on the NCBI database by a peptide mass fingerprinting method. Results Comparative proteomic analysis showed 8 increased protein spots present in cystic FF. MS analysis and database searching revealed that the increased proteins in cystic FF were bovine mitochondrial f1-atpase (BMFA), erythroid associated factor (EAF), methionine synthase (MeS), VEGF-receptor, glyceraldehydes 3-phosphate dehydrogenase (GAPDH), heat shock protein 70 (HSP70), β-lactoglobulin (BLG) and succinate dehydrogenase Ip subunit (SD). Conclusion Our results suggest that these proteins are overexpressed in BOFC, and that they may play important roles in the pathogenesis of BOFC. Furthermore, these proteins in the FF could be useful biomarkers for BOFC. PMID:15941490
Rapid and accurate identification of microorganisms, a prerequisite for appropriate patient care and infection control, is a critical function of any clinical microbiology laboratory. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is a quick and reliable method for identification of microorganisms, including bacteria, yeast, molds, and mycobacteria. Indeed, there has been a revolutionary shift in clinical diagnostic microbiology. In the present review, the state of the art and advantages of MALDI-TOF MS-based bacterial identification are described. The potential of this innovative technology for use in strain typing and detection of antibiotic resistance is also discussed. This article is part of a Special Issue entitled: Medical Proteomics.
Almuhayawi, Mohammed; Altun, Osman; Abdulmajeed, Adam Dilshad; Ullberg, Måns; Özenci, Volkan
Detection and identification of anaerobic bacteria in blood cultures (BC) is a well-recognized challenge in clinical microbiology. We studied 100 clinical anaerobic BC isolates to evaluate the performance of BacT/ALERT-FN, -FN Plus (BioMérieux), BACTEC-Plus and -Lytic (Becton Dickinson BioSciences) BC bottles in detection and time to detection (TTD) of anaerobic bacteria. BACTEC Lytic had higher detection rate (94/100, 94%) than BacT/ALERT FN Plus (80/100, 80%) (p<0.01) in the studied material. There was no significant difference in detection of anaerobic bacteria among the remaining bottle types. The 67 anaerobic bacteria that signalled positive in all four bottle types were analyzed to compare the time to detection (TTD) and isolates were directly identified by MALDI-TOF MS. There was a significant difference in TTD among the four bottle types (p<0.0001). The shortest median TTD was 18 h in BACTEC Lytic followed by BacT/ALERT FN (23.5 h), BACTEC Plus (27 h) and finally BacT/ALERT FN Plus (38 h) bottles. In contrast, MALDI-TOF MS performed similarly in all bottle types with accurate identification in 51/67 (76%) BacT/ALERT FN, 51/67 (76%) BacT/ALERT FN Plus, 53/67 (79%) BACTEC Plus and 50/67 (75%) BACTEC Lytic bottles. In conclusion, BACTEC Lytic bottles have significantly better detection rates and shorter TTD compared to the three other bottle types. The anaerobic BC bottles are equally suitable for direct MALDI-TOF MS for rapid and reliable identification of common anaerobic bacteria. Further clinical studies are warranted to investigate the performance of anaerobic BC bottles in detection of anaerobic bacteria and identification by direct MALDI-TOF MS.
Su, Huai-Hsin; Chuang, Lea-Yea; Tseng, Wei-Lung; Lu, Chi-Yu
Spermine and spermidine are multiple-nitrogen compounds found in many foods. Both compounds are essential for cell growth and human health. This study established a simple and fast method of detecting spermine and spermidine in food samples by matrix-assisted laser desorption/ionization combined with time-of-flight mass spectrometry (MALDI-TOF MS). After a simple sample preparation procedure, spermine and spermidine were directly detected by MALDI-TOF MS with no additional purification procedure. The calibration curves for spermine and spermidine ranged from 0.1 to 10 μg/mL. In intra- and inter-batch assays of three different concentrations of spermine and spermidine, all relative standard deviations and relative errors were below 18.9%. These experimental results confirmed the practicability and effectiveness of the proposed MALDI-TOF MS method for fast determination of spermine and spermidine in food samples. Furthermore, since spermine and spermidine have important roles in apoptosis, up-regulation and down-regulation of spermine and spermidine during apoptosis were analyzed. After treating NRK-52E cells with spermine and spermidine, the cells were lysed, and cell proteins were collected, and digested. Apoptosis-related proteins were then identified by tandem MS.
Direct identification of microorganisms from positive blood cultures using the lysis-filtration technique and matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS): a multicentre study.
Farina, Claudio; Arena, Fabio; Casprini, Patrizia; Cichero, Paola; Clementi, Massimo; Cosentino, Marina; Degl'Innocenti, Roberto; Giani, Tommaso; Luzzaro, Francesco; Mattei, Romano; Mauri, Carola; Nardone, Maria; Rossolini, Gian Maria; Serna Ortega, Paula Andrea; Vailati, Francesca
Microbial identification from blood cultures is essential to institute optimal antibiotic therapy and improve survival possibilities. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has been successfully applied to identify bacteria and yeasts from positive blood cultures broths. The aim of this multicentre study was to evaluate the reliability of the lysis-filtration technique associated with MALDI-TOF MS to directly identify microorganisms from 765 positive blood cultures collected in six Italian hospitals. Overall, 675/765 (78.1%) blood isolates were correctly identified at the species level, with significant differences between Gram-negative and Gram-positive bacteria (92.6%, and 69.8%, respectively). Some difficulties arise in identifying Streptococcus pneumoniae, Staphylococcus aureus, yeasts and anaerobes. The lysis-filtration protocol is a suitable procedure in terms of performance in identifying microorganisms, but it is quite expensive and technically time-consuming since the time of filtration is not regular for all the samples. The application of the MALDI-TOF MS technique to the direct microbial identification from positive blood cultures is a very promising approach, even if more experience must be gained to minimize errors and costs.
Background Diabetes like many diseases and biological processes is not mono-causal. On the one hand multi-factorial studies with complex experimental design are required for its comprehensive analysis. On the other hand, the data from these studies often include a substantial amount of redundancy such as proteins that are typically represented by a multitude of peptides. Coping simultaneously with both complexities (experimental and technological) makes data analysis a challenge for Bioinformatics. Results We present a comprehensive work-flow tailored for analyzing complex data including data from multi-factorial studies. The developed approach aims at revealing effects caused by a distinct combination of experimental factors, in our case genotype and diet. Applying the developed work-flow to the analysis of an established polygenic mouse model for diet-induced type 2 diabetes, we found peptides with significant fold changes exclusively for the combination of a particular strain and diet. Exploitation of redundancy enables the visualization of peptide correlation and provides a natural way of feature selection for classification and prediction. Classification based on the features selected using our approach performs similar to classifications based on more complex feature selection methods. Conclusions The combination of ANOVA and redundancy exploitation allows for identification of biomarker candidates in multi-dimensional MALDI-TOF MS profiling studies with complex experimental design. With respect to feature selection our method provides a fast and intuitive alternative to global optimization strategies with comparable performance. The method is implemented in R and the scripts are available by contacting the corresponding author. PMID:21554713
Wang, Weiping; Xi, Haiyan; Huang, Mei; Wang, Jie; Fan, Ming; Chen, Yong; Shao, Haifeng
Background Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is an emerging technology newly applied to identifying bacterial and yeast strains. The aim of this study was to evaluate the clinical performance of the VITEK® MS system in the identification of bacteria and yeast strains routinely isolated from clinical samples. Methods We prospectively analyzed routine MALDI-TOF mass spectrometry identification in parallel with conventional phenotypic identification of bacteria and yeasts regardless of phylum or source of isolation. Discordant results were resolved with 16S rDNA or internal transcribed spacer (ITS) gene sequencing. Colonies (a single deposit on a MALDI disposable target without any prior extraction step) were analyzed using the VITEK® MS system. Peptide spectra acquired by the system were compared with the VITEK® MS IVD database Version 2.0, and the identification scores were recorded. Results Of the 1,181 isolates (1,061 bacterial isolates and 120 yeast isolates) analyzed, 99.5% were correctly identified by MALDI-TOF mass spectrometry; 95.7% identified to the species level, 3.6% identified to the genus level, and 0.3% identified within a range of species belonging to different genera. Conversely, 0.1% of isolates were misidentified and 0.4% were unidentified, partly because the species were not included in the database. Re-testing using a second deposit provided a successful identification for 0.5% of isolates unidentified with the first deposit. Our results show that the VITEK® MS system has exceptional performance in identifying bacteria and yeast by comparing acquired peptide spectra to those contained in its database. Conclusions MALDI-TOF mass spectrometry is a rapid, accurate, and relatively inexpensive method for bacterial and yeast identification. Our results demonstrate that the VITEK® MS system is a fast and reliable technique, and has the potential to replace conventional phenotypic
Characterization of Multidrug Resistant E. faecalis Strains from Pigs of Local Origin by ADSRRS-Fingerprinting and MALDI -TOF MS; Evaluation of the Compatibility of Methods Employed for Multidrug Resistance Analysis.
Nowakiewicz, Aneta; Ziółkowska, Grażyna; Zięba, Przemysław; Gnat, Sebastian; Trościańczyk, Aleksandra; Adaszek, Łukasz
The aim of this study was to characterize multidrug resistant E. faecalis strains from pigs of local origin and to analyse the relationship between resistance and genotypic and proteomic profiles by amplification of DNA fragments surrounding rare restriction sites (ADSRRS-fingerprinting) and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI -TOF MS). From the total pool of Enterococcus spp. isolated from 90 pigs, we selected 36 multidrug resistant E. faecalis strains, which represented three different phenotypic resistance profiles. Phenotypic resistance to tetracycline, macrolides, phenicols, and lincomycin and high-level resistance to aminoglycosides were confirmed by the occurrence of at least one corresponding resistance gene in each strain. Based on the analysis of the genotypic and phenotypic resistance of the strains tested, five distinct resistance profiles were generated. As a complement of this analysis, profiles of virulence genes were determined and these profiles corresponded to the phenotypic resistance profiles. The demonstration of resistance to a wide panel of antimicrobials by the strains tested in this study indicates the need of typing to determine the spread of resistance also at the local level. It seems that in the case of E. faecalis, type and scope of resistance strongly determines the genotypic pattern obtained with the ADSRRS-fingerprinting method. The ADSRRS-fingerprinting analysis showed consistency of the genetic profiles with the resistance profiles, while analysis of data with the use of the MALDI- TOF MS method did not demonstrate direct reproduction of the clustering pattern obtained with this method. Our observations were confirmed by statistical analysis (Simpson's index of diversity, Rand and Wallace coefficients). Even though the MALDI -TOF MS method showed slightly higher discrimination power than ADSRRS-fingerprinting, only the latter method allowed reproduction of the clustering pattern of
Khennouchi, Nour Chems el Houda; Loucif, Lotfi; Boutefnouchet, Nafissa; Allag, Hamoudi
Enterobacter cloacae is among the most important pathogens responsible for nosocomial infections and outbreaks. In this study, 77 Enterobacter isolates were collected: 27 isolates from Algerian hospitals (in Constantine, Annaba, and Skikda) and 50 isolates from Marseille, France. All strains were identified by matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS). Antibiotic susceptibility testing was performed by the disk diffusion method. PCR was used to detect extended-spectrum-beta-lactamase (ESBL)-encoding, fluoroquinolone resistance-encoding, and aminoglycoside-modifying enzyme (AME) genes. Epidemiological typing was performed using MALDI-TOF MS with data mining approaches, along with multilocus sequence typing (MLST). Sixty-eight isolates (27 from Algeria, 41 from Marseille) were identified by MALDI-TOF MS as E. cloacae. Resistance to antibiotics in the Algerian isolates was significantly higher than that in the strains from Marseille, especially for beta-lactams and aminoglycosides. Eighteen of the 27 Algerian isolates and 11 of the 41 Marseille isolates possessed at least one ESBL-encoding gene: blaCTX-M and/or blaTEM. AME genes were detected in 20 of the 27 Algerian isolates and 8 of the 41 Marseille isolates [ant(2″)-Ia, aac(6′)-Ib-cr, aadA1, aadA2, and armA]. Conjugation experiments showed that armA was carried on a transferable plasmid. MALDI-TOF typing showed three separate clusters according to the geographical distribution and species level. An MLST-based phylogenetic tree showed a clade of 14 E. cloacae isolates from a urology unit clustering together in the MALDI-TOF dendrogram, suggesting the occurrence of an outbreak in this unit. In conclusion, the ability of MALDI-TOF to biotype strains was confirmed, and surveillance measures should be implemented, especially for Algerian patients hospitalized in France. PMID:26239991
Khennouchi, Nour Chems el Houda; Loucif, Lotfi; Boutefnouchet, Nafissa; Allag, Hamoudi; Rolain, Jean-Marc
Enterobacter cloacae is among the most important pathogens responsible for nosocomial infections and outbreaks. In this study, 77 Enterobacter isolates were collected: 27 isolates from Algerian hospitals (in Constantine, Annaba, and Skikda) and 50 isolates from Marseille, France. All strains were identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Antibiotic susceptibility testing was performed by the disk diffusion method. PCR was used to detect extended-spectrum-beta-lactamase (ESBL)-encoding, fluoroquinolone resistance-encoding, and aminoglycoside-modifying enzyme (AME) genes. Epidemiological typing was performed using MALDI-TOF MS with data mining approaches, along with multilocus sequence typing (MLST). Sixty-eight isolates (27 from Algeria, 41 from Marseille) were identified by MALDI-TOF MS as E. cloacae. Resistance to antibiotics in the Algerian isolates was significantly higher than that in the strains from Marseille, especially for beta-lactams and aminoglycosides. Eighteen of the 27 Algerian isolates and 11 of the 41 Marseille isolates possessed at least one ESBL-encoding gene: blaCTX-M and/or blaTEM. AME genes were detected in 20 of the 27 Algerian isolates and 8 of the 41 Marseille isolates [ant(2″)-Ia, aac(6')-Ib-cr, aadA1, aadA2, and armA]. Conjugation experiments showed that armA was carried on a transferable plasmid. MALDI-TOF typing showed three separate clusters according to the geographical distribution and species level. An MLST-based phylogenetic tree showed a clade of 14 E. cloacae isolates from a urology unit clustering together in the MALDI-TOF dendrogram, suggesting the occurrence of an outbreak in this unit. In conclusion, the ability of MALDI-TOF to biotype strains was confirmed, and surveillance measures should be implemented, especially for Algerian patients hospitalized in France.
Characterization of Multidrug Resistant E. faecalis Strains from Pigs of Local Origin by ADSRRS-Fingerprinting and MALDI -TOF MS; Evaluation of the Compatibility of Methods Employed for Multidrug Resistance Analysis
Nowakiewicz, Aneta; Ziółkowska, Grażyna; Zięba, Przemysław; Gnat, Sebastian; Trościańczyk, Aleksandra; Adaszek, Łukasz
The aim of this study was to characterize multidrug resistant E. faecalis strains from pigs of local origin and to analyse the relationship between resistance and genotypic and proteomic profiles by amplification of DNA fragments surrounding rare restriction sites (ADSRRS-fingerprinting) and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI -TOF MS). From the total pool of Enterococcus spp. isolated from 90 pigs, we selected 36 multidrug resistant E. faecalis strains, which represented three different phenotypic resistance profiles. Phenotypic resistance to tetracycline, macrolides, phenicols, and lincomycin and high-level resistance to aminoglycosides were confirmed by the occurrence of at least one corresponding resistance gene in each strain. Based on the analysis of the genotypic and phenotypic resistance of the strains tested, five distinct resistance profiles were generated. As a complement of this analysis, profiles of virulence genes were determined and these profiles corresponded to the phenotypic resistance profiles. The demonstration of resistance to a wide panel of antimicrobials by the strains tested in this study indicates the need of typing to determine the spread of resistance also at the local level. It seems that in the case of E. faecalis, type and scope of resistance strongly determines the genotypic pattern obtained with the ADSRRS-fingerprinting method. The ADSRRS-fingerprinting analysis showed consistency of the genetic profiles with the resistance profiles, while analysis of data with the use of the MALDI- TOF MS method did not demonstrate direct reproduction of the clustering pattern obtained with this method. Our observations were confirmed by statistical analysis (Simpson’s index of diversity, Rand and Wallace coefficients). Even though the MALDI -TOF MS method showed slightly higher discrimination power than ADSRRS-fingerprinting, only the latter method allowed reproduction of the clustering pattern of
Rodrigues, Naiara Miranda Bento; Bronzato, Greiciane França; Santiago, Gabrielli Stefaninni; Botelho, Larissa Alvarenga Batista; Moreira, Beatriz Meurer; Coelho, Irene da Silva; Souza, Miliane Moreira Soares de; Coelho, Shana de Mattos de Oliveira
Mastitis adversely affects milk production and in general cows do not regain their full production levels post recovery, leading to considerable economic losses. Moreover the percentage decrease in milk production depends on the specific pathogen that caused the infection and enterobacteria are responsible for this greater reduction. Phenotypic tests are among the currently available methods used worldwide to identify enterobacteria; however they tend to misdiagnose the species despite the multiple tests carried out. On the other hand The Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) technique has been attracting attention for its precise identification of several microorganisms at species level. In the current study, 183 enterobacteria were detected in milk (n=47) and fecal samples (n=94) from cows, and samples from water (n=23) and milk lines (n=19). All these samples were collected from a farm in Rio de Janeiro with the specific purpose of presenting the MALDI-TOF MS technique as an efficient methodology to identify Enterobacteriaceae from bovine environments. The MALDI-TOF MS technique results matched the biochemical test results in 92.9% (170/183) of the enterobacteria species and the gyrB sequencing confirmed 100% of the proteomic technique results. The amino acid decarboxylation test made the most misidentifications and Enterobacter spp. was the most misidentified genus (76.9%, 10/13). These results aim to clarify the current biochemical errors in enterobacteria identification, considering isolates from a bovine environment, and show the importance for more careful readings of phenotypic tests which are often used in veterinary microbiology laboratories.
McElvania TeKippe, E; Burnham, C-A D
The purpose of this investigation was to evaluate the analytical performance characteristics of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for the identification of unusual organisms. We evaluated the accuracy of two MALDI-TOF MS systems, bioMérieux VITEK MS (database v2.0) and Bruker Biotyper (software version 3.0), for the identification of the most difficult and/or unusual microorganisms isolated from clinical specimens. Our study included 174 bacterial isolates recovered from clinical cultures at Barnes-Jewish Hospital, St. Louis, MO, from 2009 to 2013, representing 50 genera and 52 species. MS identifications were compared to the identification reported by the reference laboratory. Discrepancies were resolved using molecular methods, including 16S rRNA gene sequencing and additional molecular methods. When performed, molecular methods were considered the gold standard. Of the 168 isolates resolved to the genus level, VITEK MS identified 145 (86.3 %), and of the 114 isolates resolved to the species level, 97 (85.1 %) were correctly identified. Bruker Biotyper identified 155 (92.3 %) of 168 isolates to the genus level and 97 (85.1 %) of 114 isolates to the species level. VITEK MS and Bruker Biotyper provided no identification for 17 (10.1 %) and 12 (7.1 %) organisms, respectively, and misidentified six (3.6 %) and one (0.6 %) isolate, respectively. Six isolates (3.6 %) were not resolvable to the genus level and were excluded from data analysis due to the lack of a gold standard for comparison. There was no significant difference in the number of organisms identified to the genus level, species level, unidentified, or misidentified by the two MALDI-TOF MS systems (p = 0.11, 1.0, 0.44, and 0.12, respectively).
Girard, Victoria; Mailler, Sandrine; Welker, Martin; Arsac, Maud; Cellière, Béatrice; Cotte-Pattat, Pierre-Jean; Chatellier, Sonia; Durand, Géraldine; Béni, Anne-Marie; Schrenzel, Jacques; Miller, Elizabeth; Dussoulier, Rahima; Dunne, W Michael; Butler-Wu, Susan; Saubolle, Michael A; Sussland, Den; Bell, Melissa; van Belkum, Alex; Deol, Parampal
Identification of microorganisms by MALDI-TOF MS has been widely accepted in clinical microbiology. However, for Mycobacterium spp. and Nocardia spp. such identification has not yet reached the optimal level of routine testing. Here we describe the development of an identification tool for 49 and 15 species of Mycobacterium spp. and Nocardia spp., respectively. During database construction, a number of ambiguous reference identifications were revealed and corrected via molecular analyses. Eventually, more than 2000 individual mass spectra acquired from 494 strains were included in a reference database and subjected to bio-statistical analyses. This led to correct species identification and correct combination of species into several complexes or groups, such as the Mycobacterium tuberculosis complex. With the Advanced Spectrum Classifier algorithm, class-specific bin weights were determined and tested by cross-validation experiments with good results. When challenged with independent isolates, overall identification performance was 90% for identification of Mycobacterium spp. and 88% for Nocardia spp. However, for a number of Mycobacterium sp. isolates, no identification could be achieved and in most cases, this could be attributed to the production of polymers that masked the species-specific protein peak patterns. For the species where >20 isolates were tested, correct identification reached 95% or higher. With the current spectral database, the identification of Mycobacterium spp. and Nocardia spp. by MALDI-TOF MS can be performed in routine clinical diagnostics although in some complicated cases verification by sequencing remains mandatory.
A statistical design of experiments for optimizing the MALDI-TOF-MS sample preparation of polymers. An application in the assessment of the thermo-mechanical degradation mechanisms of poly (ethylene terephthalate).
Badía, J D; Strömberg, E; Ribes-Greus, A; Karlsson, S
The sample preparation procedure for MALDI-TOF MS of polymers is addressed in this study by the application of a statistical Design of Experiments (DoE). Industrial poly (ethylene terephthalate) (PET) was chosen as model polymer. Different experimental settings (levels) for matrixes, analyte/matrix proportions and concentrations of cationization agent were considered. The quality parameters used for the analysis were signal-to-noise ratio and resolution. A closer inspection of the statistical results provided the study not only with the best combination of factors for the MALDI sample preparation, but also with a better understanding of the influence of the different factors, individually or in combination, to the signal. The application of DoE for the improvement of the MALDI measure of PET stated that the best combination of factors and levels was the following: matrix (dithranol), proportion analyte/matrix/cationization agent (1/15/1, V/V/V), and concentration of cationization agent (2 g L(-1)). In a second part, multiple processing by means of successive injection cycles was used to simulate the thermo-mechanical degradation effects on the oligomeric distribution of PET under mechanical recycling. The application of MALDI-TOF-MS showed that thermo-mechanical degradation primarily affected initially predominant cyclic species. Several degradation mechanisms were proposed, remarking intramolecular transesterification and hydrolysis. The ether links of the glycol unit in PET were shown to act as potential reaction sites, driving the main reactions of degradation.
The Technical and Biological Reproducibility of Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) Based Typing: Employment of Bioinformatics in a Multicenter Study
Oberle, Michael; Wohlwend, Nadia; Jonas, Daniel; Maurer, Florian P.; Jost, Geraldine; Tschudin-Sutter, Sarah; Vranckx, Katleen; Egli, Adrian
Background The technical, biological, and inter-center reproducibility of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI TOF MS) typing data has not yet been explored. The aim of this study is to compare typing data from multiple centers employing bioinformatics using bacterial strains from two past outbreaks and non-related strains. Material/Methods Participants received twelve extended spectrum betalactamase-producing E. coli isolates and followed the same standard operating procedure (SOP) including a full-protein extraction protocol. All laboratories provided visually read spectra via flexAnalysis (Bruker, Germany). Raw data from each laboratory allowed calculating the technical and biological reproducibility between centers using BioNumerics (Applied Maths NV, Belgium). Results Technical and biological reproducibility ranged between 96.8–99.4% and 47.6–94.4%, respectively. The inter-center reproducibility showed a comparable clustering among identical isolates. Principal component analysis indicated a higher tendency to cluster within the same center. Therefore, we used a discriminant analysis, which completely separated the clusters. Next, we defined a reference center and performed a statistical analysis to identify specific peaks to identify the outbreak clusters. Finally, we used a classifier algorithm and a linear support vector machine on the determined peaks as classifier. A validation showed that within the set of the reference center, the identification of the cluster was 100% correct with a large contrast between the score with the correct cluster and the next best scoring cluster. Conclusions Based on the sufficient technical and biological reproducibility of MALDI-TOF MS based spectra, detection of specific clusters is possible from spectra obtained from different centers. However, we believe that a shared SOP and a bioinformatics approach are required to make the analysis robust and reliable. PMID:27798637
Proton sponge-functionalized silica as high performance adsorbents for solid-phase extraction of trace perfluoroalkyl sulfonates in the environmental water samples and their direct analysis by MALDI-TOF-MS.
Cao, Dong; Hu, Ming; Han, Chunguang; Yu, Jiyao; Cui, Lin; Liu, Yongxue; Wang, Hailin; Cai, Yaqi; Kang, Yuehui; Zhou, Yiqi
1,8-Bis(dimethylamino)naphthalene (DMAN), a classical 'proton sponge', was functionalized on silica particles as a novel solid-phase extraction (SPE) adsorbent (DMAN@silica) for extracting perfluoroalkyl sulfonates (PFSs). High reproducibility and excellent extraction capability for PFSs were obtained in a wide pH range (3.0~8.5). The adsorbed PFSs on DMAN@silica sorbents could be efficiently eluted by 1,8-bis(tetramethylguanidino)naphthalene (TMGN) solution which is a proton sponge with higher proton affinity than DMAN. The elution could be directly analyzed by MALDI-TOF-MS using TMGN as matrix. Clear mass spectra for the PFSs were obtained due to no matrix ions interference observed. Furthermore, a novel strategy based on the DMAN@silica-SPE enrichment, followed by MALDI-TOF-MS analysis, was proposed and applied for PFSs quantification in environmental water samples. The calibration curves of each of the target analytes showed a wide linear dynamic range of response (0.1-10 ng L(-1) for perfluorooctane sulfonate (PFOS), perfluorohexyl sulfonate (PFHxS) and perfluorobutylsulfonate (PFBS)), which were over 2 orders of magnitude. The detection limits for PFOS, PFHxS, and PFBS were 0.021, 0.016, and 0.013 ng L(-1), respectively (S/N = 3). Recoveries of PFOS, PFHxS, and PFBS are in the ranges of 92-104%, 95-102%, and 98-109% for spiked river water samples. These results indicated that the prepared DMAN@silica adsorbents could efficiently enrich PFSs and that the proposed method is reliable.
Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for detection and identification of albumin phosphylation by organophosphorus pesticides and G- and V-type nerve agents.
John, Harald; Breyer, Felicitas; Thumfart, Jörg Oliver; Höchstetter, Hans; Thiermann, Horst
Toxic organophosphorus compounds (OPC), e.g., pesticides and nerve agents (NA), are known to phosphylate distinct endogenous proteins in vivo and in vitro. OPC adducts of butyrylcholinesterase and albumin are considered to be valuable biomarkers for retrospective verification of OPC exposure. Therefore, we have detected and identified novel adducts of human serum albumin (HSA) by means of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Pure albumin and plasma were incubated with numerous pesticides and NA of the V- and G-type in different molar ratios. Samples were prepared either by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by in-gel enzymatic cleavage using endoproteinase Glu-C (Glu-C) or by combining highly albumin-selective affinity extraction with ultrafiltration followed by reduction, carbamidomethylation, and enzymatic cleavage (Glu-C) prior to MALDI-TOF MS analysis. Characteristic mass shifts for phosphylation revealed tyrosine adducts at Y(411) (Y(401)KFQNALLVRY(411)TKKVPQVSTPTLVE(425)), Y(148) and Y(150) (I(142)ARRHPY(148)FY(150)APE(153), single and double labeled), and Y(161) (L(154)LFFAKRY(161)KAAFTE(167)) produced by original NA (tabun, sarin, soman, cyclosarin, VX, Chinese VX, and Russian VX) as well as by chlorpyrifos-oxon, diisopropyl fluorophosphate (DFP), paraoxon-ethyl (POE), and profenofos. MALDI-MS/MS of the single-labeled I(142)-E(153) peptide demonstrated that Y(150) was phosphylated with preference to Y(148). Aged albumin adducts were not detected. The procedure described was reproducible and feasible for detection of adducts at the most reactive Y(411)-residue (S/N ≥ 3) when at least 1% of total albumin was labeled. This was achieved by incubating plasma with molar HSA/OPC ratios ranging from approximately 1:0.03 (all G-type NA, DFP, and POE) to 1:3 (V-type NA, profenofos). Relative signal intensity of the Y(411) adduct correlated well with the spotted relative
Application of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) in preparation of chitosan oligosaccharides (COS) with degree of polymerization (DP) 5-12 containing well-distributed acetyl groups
Chen, Mian; Zhu, Xiqiang; Li, Zhiming; Guo, Xueping; Ling, Peixue
COS have many biological activities, and have been widely used as a health food. Molecular size is considered as a key parameter for COS' activities. However, many criteria are used practically, and true qualities of COS from different producers may not be always comparable. This can partly explain the disagreement in COS' functional researches, as resulting in COS, even with astonish effects, have not been further developed as a drug for tumor patients. As anti-tumor activities have been studied based on DP in pharmacological researches, we employed MALDI-TOF-MS to monitor fine structure, including DP, in COS' preparation and comparison. Then one of the COS products was analyzed with the composition of DP 5-12, mainly 7-10. Moreover, that COS' product contains well-distributed acetyl groups, while typical Commercial COS sample nearly contains no acetyl groups. As fresh precise parameters, the DP and the number of acetyl groups matching with special DP can be introduced in COS' further study on structure-activity relationships (SARs) as a new drug.
Shen, Qing; Dong, Wei; Yang, Mei; Li, Linqiu; Cheung, Hon-Yeung; Zhang, Zhifeng
A matrix solid-phase dispersion (MSPD) procedure with titanium dioxide (TiO2) nanoparticles (NP) as sorbent was developed for the selective extraction of phospholipids from almond samples, and matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF/MS) was employed for analysis. A remarkable increase in the signals of phospholipid accompanied by a decrease in those of triacylglycerols and diacylglycerols was observed in the relevant mass spectra. The proposed method was applied to five batches of almonds originating from four geographical areas, whereas principal component analysis (PCA) was utilized to normalize the relative amounts of the identified phospholipid species. The results indicated that the lipidomic fingerprint of almonds was successfully established by the negative ion mode spectrum, and the ratio of m/z 833.6 to 835.6 as well as m/z 821.6 could be introduced as potential markers for the differentiation of the tested almonds with different geographical origins. The whole method is of great promise for selective separation of phospholipids from nonphospholipids, especially the glycerides, and superior in fast screening and characterization of phospholipids in almond samples.
Núñez, Cristina; Bastida, Rufina; Macías, Alejandro; Valencia, Laura; Neuman, Nicolás I; Rizzi, Alberto C; Brondino, Carlos D; González, Pablo J; Capelo, José Luis; Lodeiro, Carlos
The Py(2)N(4)S(2) octadentate coordinating ligand afforded dinuclear cobalt, copper and zinc complexes and the corresponding mixed metal compounds. The overall geometry and bonding modes have been deduced on the basis of elemental analysis data, MALDI-TOF-MS, IR, UV-vis and EPR spectroscopies, single-crystal X-Ray diffraction, conductivity and magnetic susceptibility measurements. In the copper and zinc complexes, a μ-hydroxo bridge links the two metal ions. In both cases, the coordination geometry is distorted octahedral. Magnetic and EPR data reveal weakly antiferromagnetic high spin Co(II) ions, compatible with a dinuclear structure. The magnetic characterization of the dinuclear Cu(II) compound indicates a ferromagnetically coupled dimer with weak antiferromagnetic intermolecular interactions. The intra-dimer ferromagnetic behaviour was unexpected for a Cu(II) dimer with such μ-hydroxo bridging topology. We discuss the influence on the magnetic properties of non-covalent interactions between the bridging moiety and the lattice free water molecules.
Analysis of Wheat Prolamins, the Causative Agents of Celiac Sprue, Using Reversed Phase High Performance Liquid Chromatography (RP-HPLC) and Matrix-Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (MALDI-TOF-MS)
Mejías, Jaime H.; Lu, Xiaoqiao; Osorio, Claudia; Ullman, Jeffrey L.; von Wettstein, Diter; Rustgi, Sachin
Wheat prolamins, commonly known as “gluten”, are a complex mixture of 71–78 proteins, which constitute ~80% of the proteins in the wheat grains and supply 50% of the global dietary protein demand. Prolamins are also responsible for numerous gluten-induced disorders and determine the unique visco-elastic properties of the wheat dough. These properties necessitate the reliable determination of the prolamin composition in wheat grains and their derived products. Therefore, this study examined the impact of HPLC conditions, including column type, column temperature, flow rate, and the gradient of polar and non-polar solvents in the mobile phase, to improve the analytical resolution of prolamins. The following conditions were found optimal for analyses: column temperature 60 °C, flow rate 1.0 mL/min and an elution gradient of 20%–60% of 0.1% trifluoroacetic acid + acetonitrile in 60 min. For further improvement of resolution, gliadin and glutenin extracts were analyzed using MALDI-TOF-MS in combination with HPLC fractionation. Two semi-quantitative methods, densitometry of stained polyacrylamide gels and HPLC, were used to determine relative prolamin quantities and the correspondence between the methods was established. The combinatorial gluten analyses approach developed during the present study was used to analyze prolamin profiles of wheat transformants expressing DEMETER silencing artificial microRNA, and the results are discussed. PMID:24739977
van der Werf, Inez D; Calvano, Cosima D; Palmisano, Francesco; Sabbatini, Luigia
A simple protocol, based on Bligh-Dyer (BD) extraction followed by MALDI-TOF-MS analysis, for fast identification of paint binders in single microsamples is proposed. For the first time it is demonstrated that the BD method is effective for the simultaneous extraction of lipids and proteins from complex, and atypical matrices, such as pigmented paint layers. The protocol makes use of an alternative denaturing anionic detergent (RapiGest™) in order to improve efficiency of protein digestion and purification step. Detection of various lipid classes, such as triacylglycerols (TAGs) and phospholipids (PLs), and their oxidation by-products was accomplished, whereas proteins could be identified by peptide mass fingerprinting. The effect of pigments on ageing of lipids and proteins was also investigated. Finally, the proposed protocol was successfully applied to the study of a late-15th century Italian panel painting allowing the identification of various proteinaceous and lipid sections in organic binders, such as egg yolk, egg white, animal glue, casein, and drying oil.
Parolin, Carola; Giordani, Barbara; Compri, Monica; Cevenini, Roberto; Vitali, Beatrice
Lactobacilli represent a wide range of bacterial species with several implications for the human host. They play a crucial role in maintaining the ecological equilibrium of different biological niches and are essential for fermented food production and probiotic formulation. Despite the consensus about the ‘health-promoting’ significance of Lactobacillus genus, its genotypic and phenotypic characterization still poses several difficulties. The aim of this study was to assess the integration of different approaches, genotypic (16S rRNA gene sequencing), proteomic (MALDI-TOF MS) and metabolomic (1H-NMR), for the taxonomic and metabolic characterization of Lactobacillus species. For this purpose we analyzed 40 strains of various origin (intestinal, vaginal, food, probiotics), belonging to different species. The high discriminatory power of MALDI-TOF for species identification was underlined by the excellent agreement with the genotypic analysis. Indeed, MALDI-TOF allowed to correctly identify 39 out of 40 Lactobacillus strains at the species level, with an overall concordance of 97.5%. In the perspective to simplify the MALDI TOF sample preparation, especially for routine practice, we demonstrated the perfect agreement of the colony-picking from agar plates with the protein extraction protocol. 1H-NMR analysis, applied to both culture supernatants and bacterial lysates, identified a panel of metabolites whose variations in concentration were associated with the taxonomy, but also revealed a high intra-species variability that did not allow a species-level identification. Therefore, despite not suitable for mere taxonomic purposes, metabolomics can be useful to correlate particular biological activities with taxonomy and to understand the mechanisms related to the antimicrobial effect shown by some Lactobacillus species. PMID:28207855
Pashkova, Anna; Chen, Hsuan-Shen; Rejtar, Tomas; Zang, Xin; Giese, Roger; Andreev, Victor; Moskovets, Eugene; Karger, Barry L
The goal of this study was the development of N-terminal tags to improve peptide identification using high-throughput MALDI-TOF/TOF MS. Part 1 of the study was focused on the influence of derivatization on the intensities of MALDI-TOF MS signals of peptides. In part 2, various derivatization approaches for the improvement of peptide fragmentation efficiency in MALDI-TOF/TOF MS are explored. We demonstrate that permanent cation tags, while significantly improving signal intensity in the MS mode, lead to severe suppression of MS/MS fragmentation, making these tags unsuitable for high-throughput MALDI-TOF/TOF MS analysis. In the present work, it was found that labeling with Alexa Fluor 350, a coumarin tag containing a sulfo group, along with guanidation of epsilon-amino groups of Lys, could enhance unimolecular fragmentation of peptides with the formation of a high-intensity y-ion series, while the peptide intensities in the MS mode were not severely affected. LC-MALDI-TOF/TOF MS analysis of tryptic peptides from the SCX fractions of an E. coli lysate revealed improved peptide scores, a doubling of the total number of peptides, and a 30% increase in the number of proteins identified, as a result of labeling. Furthermore, by combining the data from native and labeled samples, confidence in correct identification was increased, as many proteins were identified by different peptides in the native and labeled data sets. Additionally, derivatization was found not to impair chromatographic behavior of peptides. All these factors suggest that labeling with Alexa Fluor 350 is a promising approach to the high-throughput LC-MALDI-TOF/TOF MS analysis of proteomic samples.
Núñez, Cristina; Bastida, Rufina; Macías, Alejandro; Valencia, Laura; Ribas, Joan; Capelo, José Luis; Lodeiro, Carlos
A series of dinuclear Ni(II) and Fe(II) complexes with a Py(2)N(4)S(2) coordinating octadentate macrocyclic ligand L prepared by direct reactions have been studied. The overall geometry and bonding mode have been deduced on the basis of elemental analysis data, infrared, MALDI-TOF-MS, UV-vis spectroscopy, X-ray diffraction, conductivity and magnetic susceptibility measurements. In general both M(2+) centres are sited into the macrocyclic cavity coordinated to a pyridinic nitrogen atom, one sulfur atom, two secondary amine groups from the macrocyclic backbone and completing the coordination spheres with two solvent or anionic molecules in a distorted octahedral geometry, except in the case of [Ni(2)L(mu-Cl)(H(2)O)(2)](BF(4))(3).2H(2)O, where the metal ions are sited in the macrocyclic cavity coordinated to a pyridinic nitrogen atom, one sulfur atom, two secondary amine groups from the macrocyclic backbone, one water molecule and one chloride ion acting as a bridge between the two centres in a distorted octahedral geometry. The magnetic properties of the nickel(II) complexes, [Ni(2)L(CH(3)CN)(4)](BF(4))(4).3.5CH(3)CN (11) and [Ni(2)L(mu-Cl)(H(2)O)(2)](BF(4))(3).2H(2)O (12) has been recorded in the solid state and indicates an unexpected ferromagnetic exchange in both cases, especially in compound 11 because no similar systems are previously reported in the literature presenting this magnetic behaviour. Further complexes with similar ligands are in progress to corroborate this unexpected ferromagnetic behaviour.
Crecelius, Anna; Clench, Malcolm R; Richards, Don S; Parr, Vic
A quantitative thin-layer chromatography (TLC)-matrix-assisted laser desorption (MALDI) TOF mass spectrometry (MS) method for the determination of Piroxicam has been developed. Following preliminary experiments three different approaches to the incorporation of the internal standard (Tenoxicam) into the TLC plates were investigated. These were: (a) adding the internal standard to the mobile phase and pre-developing the plate, (b) coating the plate with internal standard by electrospraying prior to matrix application and finally, (c) mixing the internal standard into the matrix solution and electrospraying both. The most successful method was that where the internal standard was pre-developed over the plate. For this method linearity was observed over the range between 400 and 800ng of Piroxicam. The precision was found to be in the range of 1-9% R.S.D. from the average detected value (n = 5), dependent on the amount of analyte on the TLC plate. The proposed method was accurate with +/-2% deviation from the known amount of Piroxicam in the sample spot.
Najam-Ul-Haq, M; Rainer, M; Heigl, N; Szabo, Z; Vallant, R; Huck, C W; Engelhardt, H; Bischoff, K-D; Bonn, G K
In the bioanalytical era, novel nano-materials for the selective extraction, pre-concentration and purification of biomolecules prior to analysis are vital. Their application as affinity binding in this regard is needed to be authentic. We report here the comparative application of derivatised materials and surfaces on the basis of nano-crystalline diamond, carbon nanotubes and fullerenes for the analysis of marker peptides and proteins by material enhanced laser desorption ionisation mass spectrometry MELDI-MS. In this particular work, the emphasis is placed on the derivatization, termed as immobilised metal affinity chromatography (IMAC), with three different support materials, to show the effectiveness of MELDI technique. For the physicochemical characterisation of the phases, near infrared reflectance spectroscopy (NIRS) is used, which is a well-established method within the analytical chemistry, covering a wide range of applications. NIRS enables differentiation between silica materials and different fullerenes derivatives, in a 3-dimensional factor-plot, depending on their derivatizations and physical characteristics. The method offers a physicochemical quantitative description in the nano-scale level of particle size, specific surface area, pore diameter, pore porosity, pore volume and total porosity with high linearity and improved precision. The measurement takes only a few seconds while high sample throughput is guaranteed.
Pardo, C-A; Tan, R N; Hennequin, C; Beyrouthy, R; Bonnet, R; Robin, F
Plasmid-mediated quinolone resistance mechanisms have become increasingly prevalent among Enterobacteriaceae strains since the 1990s. Among these mechanisms, AAC(6')-Ib-cr is the most difficult to detect. Different detection methods have been developed, but they require expensive procedures such as Sanger sequencing, pyrosequencing, polymerase chain reaction (PCR) restriction, or the time-consuming phenotypic method of Wachino. In this study, we describe a simple matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) method which can be easily implemented in clinical laboratories that use the MALDI-TOF technique for bacterial identification. We tested 113 strains of Enterobacteriaceae, of which 64 harbored the aac(6')-Ib-cr gene. We compared two MALDI-TOF strategies, which differed by their norfloxacin concentration (0.03 vs. 0.5 g/L), and the method of Wachino with the PCR and sequencing strategy used as the reference. The MALDI-TOF strategy, performed with 0.03 g/L norfloxacin, and the method of Wachino yielded the same high performances (Se = 98 %, Sp = 100 %), but the turnaround time of the MALDI-TOF strategy was faster (<5 h), simpler, and inexpensive (<1 Euro). Our study shows that the MALDI-TOF strategy has the potential to become a major method for the detection of many different enzymatic resistance mechanisms.
Starostin, Konstantin V.; Demidov, Evgeny A.; Bryanskaya, Alla V.; Efimov, Vadim M.; Rozanov, Alexey S.; Peltek, Sergey E.
Microorganism identification by MALDI TOF mass-spectrometry is based on the comparison of the mass spectrum of the studied organism with those of reference strains. It is a rapid and reliable method. However, commercial databases and programs are mostly designed for identification of clinically important strains and can be used only for particular mass spectrometer models. The need for open platforms and reference databases is obvious. In this study we describe a geometric approach for microorganism identification by mass spectra and demonstrate its capabilities by analyzing 24 strains belonging to the Bacillus pumilus group. This method is based on representing mass spectra as points on a multidimensional space, which allows us to use geometric distances to compare the spectra. Delimitation of microorganisms performed by geometric approach correlates well with the results of molecular phylogenetic analysis and clustering using Biotyper 3.1. All three methods used allowed us to reliably divide the strains into two groups corresponding to closely related species, Bacillus pumilus and Bacillus altitudinis. The method developed by us will be implemented in a Web interface designed for using open reference databases for microorganism identification. The data is available at http://www.bionet.nsc.ru/mbl/database/database.html. PMID:26592761
De Ceglie, Cristina; Calvano, Cosima D; Zambonin, Carlo G
High protein content sport nutritional supplements are found as powder products containing, as ingredients, amino acids and proteins with important nutritional values as milk, soy and egg proteins. An EU Food Supplements Directive (2002) requires that supplements should be safe, both in dosages and in purity. It is important, then, to develop rapid and sensitive methods to be employed for the quality control of these substances. In this work, we apply, for the first time, matrix-assisted laser desorption ionization-mass spectrometry as a fast, reproducible and sensitive method for the quality control of sport nutritional supplements based on proteins. To this aim, several commercial egg- and/or milk-based powder products have been processed by in gel or in solution digestion and analyzed in comparison to pure standard products. This strategy allowed to assess the reliability of the indications on proteins (as caseins, whey proteins and ovalbumin) declared in the label of several sport nutritional supplements.
Mediavilla-Gradolph, María Concepción; De Toro-Peinado, Inmaculada; Bermúdez-Ruiz, María Pilar; García-Martínez, María de los Ángeles; Ortega-Torres, María; Montiel Quezel-Guerraz, Natalia; Palop-Borrás, Begoña
The aim of this study was to compare the results obtained for identification by MALDI-TOF of nontuberculous mycobacteria (NTM) isolated in clinical samples with those obtained by GenoType Mycobacterium CM/AS (common mycobacteria/additional species). A total of 66 Mycobacterium isolates from various clinical specimens (mainly respiratory) were tested in this study. They were identified using MALDI-TOF Bruker from strains isolated in Lowenstein, following the recommended protocol of heat inactivation and extraction, and were simultaneously analyzed through hybridization by GenoType Mycobacterium from liquid culture MGIT. Our results showed that identification by MALDI-TOF was correct in 98.4% (65/66) of NTM isolated in our clinical practice (M. avium, M. intracellulare, M. abscessus, M. chelonae, M. fortuitum, M. mucogenicum, M. kansasii, and M. scrofulaceum). MALDI-TOF was found to be an accurate, rapid, and cost-effective system for identification of mycobacteria species. PMID:26106617
Sharma, Divakar; Lata, Manju; Singh, Rananjay; Deo, Nirmala; Venkatesan, Krishnamurthy; Bisht, Deepa
Emergence of extensively drug resistant tuberculosis (XDR-TB) is the consequence of the failure of second line TB treatment. Aminoglycosides are the important second line anti-TB drugs used to treat the multi drug resistant tuberculosis (MDR-TB). Main known mechanism of action of aminoglycosides is to inhibit the protein synthesis by inhibiting the normal functioning of ribosome. Primary target of aminoglycosides are the ribosomal RNA and its associated proteins. Various mechanisms have been proposed for aminoglycosides resistance but still some are unsolved. As proteins are involved in most of the biological processes, these act as a potential diagnostic markers and drug targets. In the present study we analyzed the purely cytosolic proteome of amikacin (AK) and kanamycin (KM) resistant Mycobacterium tuberculosis isolates by proteomic and bioinformatic approaches. Twenty protein spots were found to have over expressed in resistant isolates and were identified. Among these Rv3208A, Rv2623, Rv1360, Rv2140c, Rv1636, and Rv2185c are six proteins with unknown functions or undefined role. Docking results showed that AK and KM binds to the conserved domain (DUF, USP-A, Luciferase, PEBP and Polyketidecyclase/dehydrase domain) of these hypothetical proteins and over expression of these proteins might neutralize/modulate the effect of drug molecules. TBPred and GPS-PUP predicted cytoplasmic nature and potential pupylation sites within these identified proteins, respectively. String analysis also suggested that over expressed proteins along with their interactive partners might be involved in aminoglycosides resistance. Cumulative effect of these over expressed proteins could be involved in AK and KM resistance by mitigating the toxicity, repression of drug target and neutralizing affect. These findings need further exploitation for the expansion of newer therapeutics or diagnostic markers against AK and KM resistance so that an extreme condition like XDR-TB can be prevented. PMID:27895634
Reynolds, J G; Shields, S J; Roos, J W
Combustion chamber deposits (CCDs) in internal combustion engines have been studied by various techniques to understand the relationship of performance degradation with deposit quantity and structure. XPS, XAS, NMR, and elemental analysis have offered insight into the bulk structure of C, H, N, O and metal components . MS has offered some information about compound structure, but results are limited due to the insolubility and complexity of the materials. Recent advances in MS have opened new possibilities for analysis of CCDs. Here we report initial findings on the carbon structure of these deposits determined by ESI-TOF-MS and MADLI-TOF-MS.
An, Y; Goldman, R
The dynamic range of complex biological samples represents a challenge for mass spectrometric characterization. Removal of high abundant proteins is a prerequisite for a successful mass spectrometric analysis of low abundant analytes. In particular, plasma and serum proteome span at least ten orders of magnitude and represent a major challenge for biomarker discovery. Immunoaffinity depletion is the most common methods of removal of high abundant proteins. Here we describe coupling of denaturing ultrafiltration, an alternative depletion strategy, with reverse phase fractionation and mass spectrometry for characterization of low molecular weight proteins and peptides. PMID:23765617
Tsarbopoulos, A.; Bahr, U.; Pramanik, B. N.; Karas, M.
Matrix-assisted laser desorption-ionization (MALDI)-MS analysis of glycoprotein samples is usually affected by metastable fragmentation, particularly in reflectron instruments. Use of delayed extraction (DE) is shown to minimize the observed metastable fragmentation. In the case of the CHO IL-4, which contains complex-type N-linked glycans, the disialylated component gives the most abundant MALDI signal with a dramatically improved resolution and mass accuracy over the non-DE linear TOF analysis. This increased resolution is advantageous for the 30 kDa Sf9-derived IL-4 receptor, where it is possible to differentiate the individual glycans, but not for higher mass and more heterogeneous glycoproteins. It is also shown that MALDI analysis of proteins with labile functional groups is more superior and reliable with linear DE-TOF systems rather than reflectron TOF analyzers. Further structural information on the sequence and disulfide mapping, as well as glycopeptide identification can be obtained by post-source decay (PSD) analysis of peptide and glycopeptide isolated fractions. The PSD formation of a characteristic triplet of ions separated by 33 Da in mass can be used to identify disulfide-paired peptides, even from complex digest mixtures of proteins.
Cryptosporidium parvum is a protozoan parasite, and it causes a potentially fatal gastrointestinal illness. This water borne pathogen has been the subject of several high profile disease outbreaks in the US and abroad. C. parvum presents challenges for both compliance monitorin...
Cryptosporidium parvum is a protozoan parasite, and it causes a potentially fatal gastrointestinal illness. This water borne pathogen has been the subject of several high profile disease outbreaks in the US and abroad. C. parvum presents challenges for both compliance monitoring ...
Binita, Kumari; Kumar, Sanjay; Sharma, Vinay Kumar; Sharma, Veena; Yadav, Savita
Syzygium cumini is traditionally used medicinal plant. The different part of the plant such as bark, leaves, seed and fruits are widely used as an alternative medicine in various diseases. Although the scientific community has a strong interest on S. cumini seed biochemistry focusing on metabolite composition, proteins have not yet been investigated. In the present study, we have applied a proteomic approach to study the proteome of the S. cumini seed using phenol extraction method for protein isolation, which were never analysed before. Fifteen brightly silver stained protein spots were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry after resolving on two-dimensional gel electrophoresis. These proteins have been found to involve in various functions such as antifungal, sulphur metabolism, carbohydrate metabolism, fruit ripening and softening, dormancy breaking and seed germination, hormone signalling, secondary metabolite transport, defence and stress response, nitrogen metabolism, synthesis and stabilization. Amongst the identified protein, lactoferrin was a mammalian origin protein with high nutritious and pharmaceutical value, which was purified by different types of chromatographic techniques and confirmed by western blotting. The antibacterial activity of lactoferrin was assessed by disc diffusion assay. We suggest that the protein constituents of S. cumini may have role in various functions required for plant physiology and its dietary values.
Sehgal, P; Rao, M K; Kumar, O; Vijayaraghavan, R
Ricin is a toxic protein present in the seeds of castor bean plant. It can be inactivated by heat; therefore characterization of denatured ricin is essential to differentiate it from native ricin and to avoid any ambiguity in its identification. In this study, potential of mass spectrometry using MALDI—TOF/MS has been exploited to investigate the effects of heat treatment on ricin and spiked food matrices. The molecular weights of ricin, ricin A (A1 and A2) and B chain were found to be 62.8 kDa, 31.2 kDa, 32.5 kDa and 32 kDa respectively. The mass spectrum revealed a polypeptide chain of 11.1 kDa for denatured ricin. The peptide mass fingerprinting showed 24 peptides, six were common both in native and denatured ricin. The differentiating peptide at position 294—318 (m/z 934.533) was observed only in denatured ricin. The three selected marker peptides m/z 1013.6, 1310.7, 1728.9 are chosen for identification of ricin inactivated by heat in spiked apple juice and milk samples by immunocapture analysis. There is always a probability of denatured non— toxic ricin being confused with native (toxic) ricin to create unnecessary panic. Keeping this probability in mind, our study will be of immense value in minimising such risk.
Starostin, Konstantin V.; Demidov, Evgeny A.; Bryanskaya, Alla V.; Efimov, Vadim M.; Rozanov, Alexey S.; Peltek, Sergey E.
Microorganism identification by MALDI TOF mass-spectrometry is based on the comparison of the mass spectrum of the studied organism with those of reference strains. It is a rapid and reliable method. However, commercial databases and programs are mostly designed for identification of clinically important strains and can be used only for particular mass spectrometer models. The need for open platforms and reference databases is obvious. In this study we describe a geometric approach for microorganism identification by mass spectra and demonstrate its capabilities by analyzing 24 strains belonging to the Bacillus pumilus group. This method is based on representing mass spectra as points on a multidimensional space, which allows us to use geometric distances to compare the spectra. Delimitation of microorganisms performed by geometric approach correlates well with the results of molecular phylogenetic analysis and clustering using Biotyper 3.1. All three methods used allowed us to reliably divide the strains into two groups corresponding to closely related species, Bacillus pumilus and Bacillus altitudinis. The method developed by us will be implemented in a Web interface designed for using open reference databases for microorganism identification. The data is available at http://www.bionet.nsc.ru/mbl/database/database.html.
Qiao, Liang; Roussel, Christophe; Wan, Jingjing; Yang, Pengyuan; Girault, Hubert H; Liu, Baohong
An on-plate specific enrichment method is presented for the direct analysis of peptides phosphorylation. An array of sintered TiO 2 nanoparticle spots was prepared on a stainless steel plate to provide porous substrate with a very large specific surface and durable functions. These spots were used to selectively capture phosphorylated peptides from peptide mixtures, and the immobilized phosphopeptides could then be analyzed directly by MALDI MS after washing away the nonphosphorylated peptides. beta-Casein and protein mixtures were employed as model samples to investigate the selection efficiency. In this strategy, the steps of phosphopeptide capture, purification, and subsequent mass spectrometry analysis are all successfully accomplished on a single target plate, which greatly reduces sample loss and simplifies analytical procedures. The low detection limit, small sample size, and rapid selective entrapment show that this on-plate strategy is promising for online enrichment of phosphopeptides, which is essential for the analysis of minute amount of samples in high-throughput proteome research.
Gerakines, Perry A.; Wdowiak, Thomas J.
If Europa is to be of primary exobiological interest, namely as a habitat for extant life, it is obvious that: (i) a hydrosphere must prevail beneath the cryosphere for a long time, (ii) internal energy sources must be present in a sufficient state of activity, and (iii) a reasonable technical means must be available for assessing if indeed life does exist in the hypothesized hydrosphere. This discussion focuses on technological issues, because the compounding evidence about Europa indicates that the first two are highly likely to be true. We present a consideration of time-of-flight mass spectroscopy (TOF MS) conducted in-situ on the cryosphere surface of Europa during a landed robotic mission. We assert that this is a reasonable technical means not only for exploring the composition of the cryosphere itself, but also for locating any biomolecular indicators of extant life brought to the surface through cryosphere activity. We also describe a MALDI (MAtrix Laser Desorption and Ionization) TOF MS system that we are constructing as a proof-of-concept prototype for conducting TOF MS measurements on Europa.
Wang, Sheng; Bao, Huimin; Zhang, Luyan; Yang, Pengyuan; Chen, Gang
In this report, infrared (IR)-assisted on-plate proteolysis has been developed for rapid peptide mapping. Protein solutions containing trypsin were allowed to digest directly on the spots of matrix-assisted laser desorption/ionization (MALDI) plates under IR radiation. The feasibility and performance of the novel proteolysis approach were investigated by the digestion of bovine serum albumin (BSA) and cytochrome c (Cyt-c). It was demonstrated that IR radiation substantially enhanced the efficiency of proteolysis and the digestion time was significantly reduced to 5 min. The digests were identified by MALDI time-of-flight mass spectrometry with sequence coverages of 55 (BSA) and 75% (Cyt-c) that were comparable to those obtained by using conventional in-solution tryptic digestion. The suitability of IR-assisted on-plate proteolysis to complex proteins was demonstrated by digesting human serum and casein extracted from commercially available milk sample. The present proteolysis strategy is simple and efficient, offering great promise for high-throughput protein identification.
Zhang, Yan; Shuang, Shaomin; Dong, Chuan; Lo, Chung Keung; Paau, Man Chin; Choi, Martin M F
Samples of polydisperse gold nanoclusters (AuNCs) protected with monolayers of N-acetyl-L-cysteine (NAC) have been chromatographically separated by a C18 column (4.6 mm x 250 mm) using a gradient elution program with a mobile phase of methanol (MeOH)/water containing tetrabutylammonium fluoride (Bu(4)N(+)F(-)) and sodium chloride. The effects of Bu(4)N(+)F(-) and MeOH on the separation have been investigated in detail. In conjunction with absorbance-based, fluorescence, and electrochemical detectors, the elution order of these water-soluble AuNCs is confirmed according to their core size in an ascending order. The onset oxidation potential closely follows the core size of AuNC. The separated fractions from high-performance liquid chromatography (HPLC) were collected and analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry to determine the number of Au atoms in the fractions. The sizes of AuNCs in some selected HPLC fractions were also assessed by transmission electron microscopy and high-resolution transmission electron microscopy. Photoluminescence spectra of the fractions show that the luminescent shift in the visible/near-infrared region does not follow with the core size of AuNC. More importantly, the proposed HPLC methodology has been successfully applied to analyze various polydisperse AuNC products synthesized from different gold-to-ligand mole ratios (Au/NAC) and reaction temperatures. The results confirm that larger Au/NAC and higher temperature will produce larger core size AuNCs products with narrower dispersity. In addition, AuNC samples obtained from various synthesis reaction times were analyzed by our HPLC methodology, demonstrating that the reaction's behavior follows the nucleation-growth-disintegration process.
Introduction: Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS)is increasingly utilized as a rapid technique to identify microorganisms including pathogenic bacteria. However, little attention has been paid to the significant proteomic information encoded in ...
Boyer, Anne E; Gallegos-Candela, Maribel; Quinn, Conrad P; Woolfitt, Adrian R; Brumlow, Judith O; Isbell, Katherine; Hoffmaster, Alex R; Lins, Renato C; Barr, John R
Inhalation anthrax has a rapid progression and high fatality rate. Pathology and death from inhalation of Bacillus anthracis spores are attributed to the actions of secreted protein toxins. Protective antigen (PA) binds and imports the catalytic component lethal factor (LF), a zinc endoprotease, and edema factor (EF), an adenylyl cyclase, into susceptible cells. PA-LF is termed lethal toxin (LTx) and PA-EF, edema toxin. As the universal transporter for both toxins, PA is an important target for vaccination and immunotherapeutic intervention. However, its quantification has been limited to methods of relatively low analytic sensitivity. Quantification of LTx may be more clinically relevant than LF or PA alone because LTx is the toxic form that acts on cells. A method was developed for LTx-specific quantification in plasma using anti-PA IgG magnetic immunoprecipitation of PA and quantification of LF activity that co-purified with PA. The method was fast (<4 h total time to detection), sensitive at 0.033 ng/mL LTx in plasma for the fast analysis (0.0075 ng/mL LTx in plasma for an 18 h reaction), precise (6.3-9.9% coefficient of variation), and accurate (0.1-12.7%error; n ≥ 25). Diagnostic sensitivity was 100% (n = 27 animal/clinical cases). Diagnostic specificity was 100% (n = 141). LTx was detected post-antibiotic treatment in 6/6 treated rhesus macaques and 3/3 clinical cases of inhalation anthrax and as long as 8 days post-treatment. Over the course of infection in two rhesus macaques, LTx was first detected at 0.101 and 0.237 ng/mL at 36 h post-exposure and increased to 1147 and 12,107 ng/mL in late-stage anthrax. This demonstrated the importance of LTx as a diagnostic and therapeutic target. This method provides a sensitive, accurate tool for anthrax toxin detection and evaluation of PA-directed therapeutics.
Ekström, Simon; Wallman, Lars; Helldin, Göran; Nilsson, Johan; Marko-Varga, György; Laurell, Thomas
A polymer microfabricated proteomic sample preparation and MALDI MS sample presentation device, the integrated selective enrichment target (ISET), comprising an array of perforated nanovials is reported. Each perforated nanovial can be filled with selective extraction media (microbeads) for purification and concentration of protein/peptides prior to matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS). The main areas covered are the influence of the molding-process-induced surface roughness and how to address the lack of inherent conductivity in the polyetheretherketone (PEEK) material for optimal MALDI MS readout. Application of the disposable polymeric ISET devices for solid-phase extraction and phosphopeptide capture is also demonstrated.
Liu, Wei; Hong, Wen-Xu; Zhang, Yanfang; Huang, Peiwu; Yang, Xifei; Ren, Xiaohu; Huang, Haiyan; Liu, Jianjun
Trichloroethylene (TCE) has long been well known as a major pollutant that affects both occupational and general environments. Occupational medicamentosa-like dermatitis induced by TCE (OMLDT) is an autoimmune disease, which has become one of the critical occupational health issues in China. In this study, we analyzed 18 OMLDT patients and 29 professional TCE contact people on serum proteomic analysis by matrix-assisted laser desorption ionization time-of-flight mass spectrometry and ClinProTools bioinformatics software. The intensities of 35 protein/peptide peaks were significantly different between TCE contact controls and OMLDT patients. A pattern of six peaks (m/z 1,450.33, 1,866.16, 3,262.39, 4,109.55, 5,064.85 and 5,956.57) were selected to construct a diagnostic model to discriminate the OMLDT patients from controls with sensitivity and specificity of both 93.8 %. Our findings provide an alternative proteomic approach to differentiate the OMLDT patients from TCE contact workers with high sensitivity and high specificity, which will be of potential value in clinical diagnosis for occupational disease.
Rizzardi, Kristina; Åkerlund, Thomas
Clostridium difficile strains were typed by a newly developed MALDI-TOF method, high molecular weight typing, and compared to PCR ribotyping. Among 500 isolates representing 59 PCR ribotypes a total of 35 high molecular weight types could be resolved. Although less discriminatory than PCR ribotyping, the method is extremely fast and simple, and supports for cost-effective screening of isolates during outbreak situations.
Zhang, Xueyang; Zhu, Shaochun; Deng, Chunhui; Zhang, Xiangmin
An aptamer microarray was directly fabricated on a MALDI target plate for high-throughput insulin detection. High sensitivities were observed both in standard solutions (5 ng mL(-1), 0.86 nM) and serum sample (20 ng mL(-1), 3.4 nM). This method shows great promise in the field of biomarker detection.
Książczyk, Marta; Kuczkowski, Maciej; Dudek, Bartłomiej; Korzekwa, Kamila; Tobiasz, Anna; Korzeniowska-Kowal, Agnieszka; Paluch, Emil; Wieliczko, Alina; Bugla-Płoskońska, Gabriela
In diagnostic microbiology as well as in microbiological research, the identification of a microorganism is a crucial and decisive stage. A broad choice of methods is available, based on both phenotypic and molecular properties of microbes. The aim of this study was to compare the application of phenotypic and molecular tools in bacterial identification on the example of Gram-negative intestine rod with an ambiguous phenotype. Different methods of identification procedure, which based on various properties of bacteria, were applied, e.g., microscopic observation of single-bacterial cells, macroscopic observation of bacterial colonies morphology, the automated system of microorganism identification (biochemical tests), the mass spectrometry method (analysis of bacterial proteome), and genetic analysis with PCR reactions. The obtained results revealed discrepancies in the identification of the tested bacterial strain with an atypical phenotype: mucous morphology of colonies, not characteristic for either E. coli and Citrobacter spp., mass spectrometry analysis of proteome initially assigned the tested strain to Citrobacter genus (C. freundii) and biochemical profiles pointed to Escherichia coli. A decisive method in the current study was genetic analysis with PCR reactions which identified conserved genetic sequences highly specific to E. coli species in the genome of the tested strain.
Takeuchi, H; Yasuda, A; Yasuda-Kamatani, Y; Kubo, T; Nakajima, T
Using a combination of MALDI-TOF and on-line capillary HPLC/Q-Tof mass spectroscopy, we identified and determined the amino acid sequence of a novel neuropeptide in the brain of the honeybee Apis mellifera L., termed AmTRP peptide (Apis mellifera tachykinin-related peptide), related to insect tachykinin. A cDNA for a prepro-protein (prepro-AmTRP) of AmTRP was isolated and determined to encode seven AmTRPs 1-7. Northern blot analysis indicated that the prepro-AmTRP gene is expressed differentially in the nurse bee, forager, queen and drone heads. Strong expression was detected in the queen and forager heads, while weak and almost no significant expression was detected in the nurse and drone heads, respectively. These results suggest that AmTRP peptide functions as a neuromodulator and/or hormone, associated with sex-specific or age/division of labour-selective behaviour and/or physiology of the honeybees.
Gopal, Judy; Wu, Hui-Fen; Lee, Chia-Hsun; Manikandan, Muthu
Ants and humans coexist closely and for the most part happily. We consider ants to be harmless, small beings--we have no problem picking them out of our tea cups or sugar jars, throwing them away and continuing to consume the food. This paper is an eye-opener that these ants are not as harmless as they may seem. In particular, our relationship with those present in bacteria-rich environments (e.g. a microbiological lab) need to be reconsidered. From an analytical point of view we have applied the physical preconcentration coupled ZnO NPs assisted MALDI-MS (PP-MALDI-MS) as a novel and sensitive technique for detecting bacteria on the surface of a species of ant present in our laboratory. The preconcentration methods consist of simple techniques comprising of vortex combined with centrifugation or ultrasonication resulting in increasing sample concentration up to the MALDI-MS detection limit. ZnO NPs were used to further enhance the bacterial signals for culture free rapid analysis using MALDI-MS. The importance of a vortex-combined centrifugation approach, using a large number of samples (large number of ants) and decreasing the suspension volume and addition of sample to ZnO NPs (3.5g L(-1)) were found to be crucial prerequisites for increasing MALDI-MS detection of bacteria on ants. We were able to identify the pathogenic clinically important Staphylococcus aureus on the surface of the ants. The bacterial identification was validated using ClinPro 2.1.
Urbanova, Iva; Svec, Frantisek
Superhydrophobic monolithic porous polymer layers supported onto glass plates with a gradient of hydrophobicity have been prepared and used for 2-D thin layer chromatography of peptides. The 50 μm-thin poly(glycidyl methacrylate-co-ethylene dimethacrylate) layers prepared using UV-initiated polymerization in a simple mold were first hydrolyzed using dilute sulfuric acid and then hydrophilized via two-step grafting of poly(ethylene glycol) methacrylate to obtain superhydrophilic plates. The hydrophobicity was then formed by photografting of lauryl methacrylate. The exposure to UV light that initiates photografting was spatially controlled using moving shutter that enabled forming of the diagonal gradient of hydrophobicity. This new concept enables the solutes to encounter the gradient for each of the two sequential developments. Practical application of our novel plates was demonstrated with a rapid 2-D separation of a mixture of model peptides gly-tyr, val-tyr-val, leucine enkephalin, and oxytocin in dual reversed-phase mode using different mobile phases in each direction. Detection of fluorescent-labeled peptides was achieved through UV light visualization while separation of native leucine enkephalin and oxytocin was monitored directly using MALDI mass spectrometry.
Nicolau, Rudy; Leloup, Maud; Lachassagne, Delphine; Pinault, Emilie; Feuillade-Cathalifaud, Geneviève
This work is focused on the development of an analytical procedure for the improvement of the Organic Matter structure characterization, particularly the algal matter. Two fractions of algal organic matter from laboratory cultures of algae (Euglena gracilis) and cyanobacteria (Microcystis aeruginosa) were extracted with XAD resins. The fractions were studied using laser desorption ionization (LDI) and Matrix-Assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF). A comparison with the natural organic matter characteristics from commercial humic acids and fulvic acids extracted from Suwannee River was performed. Results show that algal and natural organic matters have unique quasi-polymeric structures. Significant repeating patterns were identified. Different fractions extracted from organic matter with common origin had common structures. Thus, 44, 114 and 169Da peaks separation for fractions from E. gracilis organic matter and 28, 58 and 100Da for M. aeruginosa ones were clearly observed. Using the developed protocol, a structural scheme and organic matter composition were obtained. The range 600-2000Da contained more architectural composition differences than the range 100-600Da, suggesting that organic matter is composed of an assembly of common small molecules. Associated to specific monomers, particular patterns were common to all samples but assembly and resulting structure were unique for each organic matter. Thus, XAD fractionation coupled to mass spectroscopy allowed determining a specific fingerprint for each organic matter.
Peter, Jochen F.; Otto, Angela M.; Wolf, Bernhard
Tumor cells change their genetic expression pattern as they progress to states of increasing malignancy. Investigations at the DNA and RNA level alone cannot provide all the information resulting after the translation and processing of the corresponding proteins, which is one reason for a poor correlation between mRNA and the respective protein abundance. In diagnostics, differentially expressed peptides or proteins are important markers for the early detection of cancer. Unfortunately, tumor cells secrete peptides and proteins in only very low amounts, making mass spectrometric determination very difficult. In this publication, methods have been developed for the effective enrichment and cleanup of substances secreted by cultivated cancer cells. To obviate peptides from fetal calf serum used in cell culture, a serum surrogate was developed, which maintained growth of the cancer cells. After the binding of substances from cell-culture supernatants to custom-made magnetic reversed-phase particles, the substances were eluted and separated by capillary high-performance liquid chromatography. Fractions were spotted directly on a MALDI target, and MALDI-TOF mass spectrometric data acquisition was performed in automatic mode. This technology was used to detect substances secreted by two mammary carcinoma cell lines differing in their malignancy (MCF-7, MDA-MB231). Unequivocal differences in the peptide secretion patterns were observed. In conclusion, this system allows the sensitive investigation of peptides secreted by cancer cells in culture and provides a valuable tool for the investigation of cancer cells in different states of malignancy. PMID:18166672
Teng, Jade L L; Huang, Yi; Tse, Herman; Chen, Jonathan H K; Tang, Ying; Lau, Susanna K P; Woo, Patrick C Y
Streptococcus sinensis is a recently discovered human pathogen isolated from blood cultures of patients with infective endocarditis. Its phylogenetic position, as well as those of its closely related species, remains inconclusive when single genes were used for phylogenetic analysis. For example, S. sinensis branched out from members of the anginosus, mitis, and sanguinis groups in the 16S ribosomal RNA gene phylogenetic tree, but it was clustered with members of the anginosus and sanguinis groups when groEL gene sequences used for analysis. In this study, we sequenced the draft genome of S. sinensis and used a polyphasic approach, including concatenated genes, whole genomes, and matrix-assisted laser desorption ionization-time of flight mass spectrometry to analyze the phylogeny of S. sinensis. The size of the S. sinensis draft genome is 2.06 Mb, with GC content of 42.2%. Phylogenetic analysis using 50 concatenated genes or whole genomes revealed that S. sinensis formed a distinct cluster with Streptococcus oligofermentans and Streptococcus cristatus, and these three streptococci were clustered with the "sanguinis group." As for phylogenetic analysis using hierarchical cluster analysis of the mass spectra of streptococci, S. sinensis also formed a distinct cluster with S. oligofermentans and S. cristatus, but these three streptococci were clustered with the "mitis group." On the basis of the findings, we propose a novel group, named "sinensis group," to include S. sinensis, S. oligofermentans, and S. cristatus, in the Streptococcus genus. Our study also illustrates the power of phylogenomic analyses for resolving ambiguities in bacterial taxonomy.
Zavalin, Andre; Yang, Junhai; Hayden, Kevin; Vestal, Marvin; Caprioli, Richard M.
We have achieved protein imaging mass spectrometry capabilities at sub-cellular spatial resolution and at high acquisition speed by integrating a transmission geometry ion source with time of flight mass spectrometry. The transmission geometry principle allowed us to achieve a 1 μm laser spot diameter on target. A minimal raster step size of the instrument was 2.5 μm. Use of 2,5-dihydroxyacetophenone robotically sprayed on top of a tissue sample as a matrix together with additional sample preparation steps resulted in single pixel mass spectra from mouse cerebellum tissue sections having more than 20 peaks in a range 3–22 kDa. Mass spectrometry images were acquired in a standard step raster microprobe mode at 5 pixels/s and in a continuous raster mode at 40 pixels/s. PMID:25673247
Kang, Jeong Han; Park, Kwan-Kyu; Lee, In-Seon; Magae, Junji; Ando, Kunio; Kim, Cheorl-Ho; Chang, Young-Chae
Ascochlorin is a prenyl-phenol compound that was isolated from the fungus Ascochyta viciae. Ascochlorin reduces serum cholesterol and triglyceride levels, suppresses hypertension and tumor development, and ameliorates type I and II diabetes. Here, to better understand the mechanisms by which ascochlorin regulates physiological or pathological events and induces responses in the pharmacological treatment of cancer, we performed differential analysis of the proteome of the human osteosarcoma cells U2OS in response to ascochlorin. In addition, we established the first two-dimensional map of the U2OS proteome. The U2OS cell proteomes with and without treatment with ascochlorin were compared using two-dimensional electrophoresis, matrix-assisted laser desorption/ionization mass spectrometry and bioinformatics. The largest differences in expression were observed for the epidermal growth factor receptor (4-fold decrease), ribulose-5-phosphate-epimerase (13-fold decrease), ATP-dependent RNA helicase (8-fold decrease), and kelch-like ECH-associated protein 1 (6-fold decrease). The abundance of heterogeneous nuclear ribonucleoprotein L and minichromosome maintenance protein 7 increased 12- and 8.2-fold, respectively. In addition, Erk 2 was increased 3-fold in U2OS cells treated with ascochlorin. The expression of some selected proteins was confirmed by western blotting, zymography and RT-PCR analysis.
Lasch, Peter; Noda, Isao
Hyperspectral imaging (HSI) techniques are useful for obtaining very detailed structural and compositional information from biomedical, pharmaceutical, or clinical samples, among others. The informative value of these methods can be further increased through the application of different HSI techniques and joint analysis of the data. However, interpretation and understanding of multimodal HSI have been impeded by difficulties in registration of the different HSI data sets and by the lack of integrative analysis concepts. Here, we introduce two-dimensional correlation spectroscopy (2DCOS) as a novel technique for jointly analyzing HSI data which allows one to obtain deeper insights into the chemistry of complex samples by decrypting auto- and heterospectral correlations that may exist between features of the different HSI data. The general workflow of 2DCOS analysis is demonstrated by HSI examples acquired from cryo-sections of hamster brain tissue using Fourier-transform infrared (FT-IR) microspectroscopy, confocal Raman microspectroscopy (CRM), and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Multimodal hyperspectral image analysis by 2DCOS opens up new opportunities for spectral band assignments and thus the interpretation of structure-spectra and composition-spectra relationships. We foresee wide application potential for describing complex samples in various fields ranging from biomedicine to industrial applications.
Mauger, Florence; Gelfand, David H; Gupta, Amar; Bodepudi, Veeraiah; Will, Stephen G; Bauer, Keith; Myers, Thomas W; Gut, Ivo G
Here, we describe a high-throughput, single-tube, allele-specific ribonucleotide analog pyrophosphorolysis-activated polymerization (ribo-PAP) PCR multiplex genotyping and resequencing method. An RNA/DNA chimeric PCR product is generated using genomic DNA as starting template, a panel of allele-selective 5'-tagged primers, a reverse primer, one nucleotide in the ribo-form (90-100%), the other nucleotides in the deoxy-form, a DNA polymerase capable of incorporating ribonucleotides, a suitable buffer and thermal cycling. The RNA/DNA chimeric PCR products are fragmented by treatment with alkali and analyzed by mass spectrometry. All allele-selective primers have a 5' repetitive motif where each repeat unit has a unique, distinct mass upon reverse copying and alkali fragmentation. The mass of the complement repeat fragment or flag identifies the primer or primers that were recruited in the ribo-PAP PCR. The method readily identifies homozygous and heterozygous positions in simplex or duplex ribo-PAP PCR. Many different tags can be analyzed simultaneously. The assay can genotype several SNPs in a single tube. It thus constitutes the simplest genotyping protocol with multiplex analysis. This novel genotyping and resequencing protocol was applied to different genomic loci: NOS1 and H19 in 30 individuals in simplex ribo-PAP PCR and at two SLCO1B1 loci in 95 individuals in duplex ribo-PAP PCR.
Chiu, Jasper Z. S.; Tucker, Ian G.; McDowell, Arlene
High sensitivity quantification of the putative cell-penetrating peptide di-arginine-histidine (RRH) associated with poly (ethyl-cyanoacrylate) (PECA) nanoparticles was achieved without analyte separation, using a novel application of isobaric-tagging and high matrix-assisted laser desorption/ionization coupled to time-of-flight (MALDI-TOF) mass spectrometry. Isobaric-tagging reaction equilibrium was reached after 5 min, with 90% or greater RRH peptide successfully isobaric-tagged after 60 min. The accuracy was greater than 90%, which indicates good reliability of using isobaric-tagged RRH as an internal standard for RRH quantification. The sample intra- and inter-spot coefficients of variations were less than 11%, which indicate good repeatability. The majority of RRH peptides in the nanoparticle formulation were physically associated with the nanoparticles (46.6%), whereas only a small fraction remained unassociated (13.7%). The unrecovered RRH peptide (~40%) was assumed to be covalently associated with PECA nanoparticles.
Deak, Eszter; Charlton, Carmen L; Bobenchik, April M; Miller, Shelley A; Pollett, Simon; McHardy, Ian H; Wu, Max T; Garner, Omai B
This study compared the diagnostic performance of Bruker's Microflex LT and bioMérieux's Vitek MS matrix-assisted laser desorption ionization-time of flight mass spectrometry systems. A total of 477 isolates were tested on both instruments. Discrepant results were resolved by sequencing. Overall, there was no statistically significant difference between the proportion of isolates correctly identified, miscalled or not called by each instrument. Although both systems were good at identifying yeast (66/69 to species level), the confidence level was high only to genus level for 30% of the isolates on the Bruker. Both systems performed with high accuracy when evaluated solely on Food and Drug Administration-approved organisms for each database. A user-based assessment of the 2 instruments revealed an overall preference for the Vitek MS instrument.
Calvano, Cosima D; De Ceglie, Cristina; Zambonin, Carlo G
In foodstuffs, one of the main factors inducing modifications in phospholipids (PLs) structure is the heat treatment. Among PLs, only phosphatidylethanolamines and phosphatidylserines, due to their free amino group, can be involved in Maillard reaction and can form adducts with reducing sugars, besides other by-products called advanced glycation end-products. To date, glycated lipid products are less characterized in comparison to proteins. The aim of this work was to develop a novel, rapid and sensitive extraction protocol for the detection and characterization of modified PLs (glycated and oxidized) by means of matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS). At first, to investigate the formation of glycated and/or short chain by-products in different classes of PLs, representative standards were heated with or without sugar (lactose or glucose) and subjected to traditional lipid extraction methods as Bligh and Dyer and to the novel direct in matrix extraction (DIME) using 1,8-bis(dimethylamino)naphthalene as preconcentrating matrix. MALDI-MS analysis in negative ion mode allowed detecting glycation and oxidation products both on fatty acid and glucose moieties. Then, the procedure was successfully applied to different heat-treated and powdered samples (milk powders, pasteurized milk, ultra-high-temperature milk and soy flour) for the detection of modified PLs in complex foods. The currently developed DIME protocol could be a powerful tool for understanding lipid glycation also in biological samples.
Torres, M J; Petroselli, G; Daz, M; Erra-Balsells, R; Audisio, M C
In this work a new Bacillus sp. strain, isolated from honey, was characterized phylogenetically. Its antibacterial activity against three relevant foodborne pathogenic bacteria was studied; the main bioactive metabolites were analyzed using ultraviolet matrix assisted laser desorption-ionization mass spectrometry (UV-MALDI MS). Bacillus CBMDC3f was phylogenetically characterized as Bacillus subtilis subsp. subtilis after rRNA analysis of the 16S subunit and the gyrA gene (access codes Genbank JX120508 and JX120516, respectively). Its antibacterial potential was evaluated against Listeria monocytogenes (9 strains), B. cereus (3 strains) and Staphylococcus aureus ATCC29213. Its cell suspension and cell-free supernatant (CFS) exerted significant anti-Listeria and anti-S. aureus activities, while the lipopeptides fraction (LF) also showed anti-B. cereus effect. The UV-MALDI-MS analysis revealed surfactin, iturin and fengycin in the CFS, whereas surfactin predominated in the LF. The CFS from CBMDC3f contained surfactin, iturin and fengycin with four, two and four homologues per family, respectively, whereas four surfactin, one iturin and one fengycin homologues were identified in the LF. For some surfactin homologues, their UV-MALDI-TOF/TOF (MS/MS; Laser Induced Decomposition method, LID) spectra were also obtained. Mass spectrometry analysis contributed with relevant information about the type of lipopeptides that Bacillus strains can synthesize. From our results, surfactin would be the main metabolite responsible for the antibacterial effect.
De Ceglie, Cristina; Calvano, Cosima Damiana; Zambonin, Carlo Giorgio
Adulteration of extra-virgin olive oil (EVOO) with hazelnut oil (HO) is an illegal practice that could have severe health consequences for consumers due to the possible exposure to hidden hazelnut allergens. Here, matrix-assisted laser-desorption/ionization (MALDI) mass spectrometry (MS) was used as a rapid and sensitive technique for the detection of a low concentration of hazelnut proteins in oil samples. Different protocols were tested for protein extraction, and the most efficient (cold acetone) was applied to HO and EVOO adulterated with HO. The subsequent in-solution tryptic digestion of protein extracts and MALDI-MS analysis, using α-cyano-4-chlorocinnamic acid as matrix, allowed the detection of stable hazelnut peptide markers (i.e., the m/z ions 1002.52, 1356.71, 1394.70, 1440.81, 1453.85, 1555.76, 1629.83, 1363.73, and 1528.67) attributable to the main hazelnut proteins Cor a 9, Cor a 11, and Cor a 1. Thus, the approach might allow the direct detection of specific hazelnut allergens in EVOO at low concentration without time-consuming pretreatments.
Ranucci, Elisabetta; Ferruti, Paolo; Annunziata, Rita; Gerges, Irini; Spinelli, Giuseppe
Ester-functionalised poly(1-vinylpyrrolidin-2-one) (PVP) oligomers obtained by radical polymerisation in methyl propionate, diethyl malonate and diethyl 2-methylmalonate were characterised by NMR spectroscopy, and MALDI-TOF mass spectrometry. The chain-transfer constants were determined as 5.54 x 10(-4), 1.22 x 10(-3) and 1.70 x 10(-2), respectively, by measuring the variation of the number-average molecular weight on conversion. These values were compared with those of methyl isobutyrate (1.65 x 10(-3)) and ethyl lactate (1.03 x 10(-2)), which had been previously determined. A clear dependence was found on the reactivity of the mobile hydrogen atoms alpha with the ester group. All of the macromolecules carried a single ester function. Therefore, the re-initiation step by the CTA-derived radicals overwhelmingly prevailed over initiation by the primary radicals.
Jansen, Bas C.; Bondt, Albert; Reiding, Karli R.; Scherjon, Sicco A.; Vidarsson, Gestur; Wuhrer, Manfred
During pregnancy, the mother provides multiple nutrients and substances to the foetus, with maternal immunoglobulin G (IgG) being actively transported to the foetus. Newborns depend on maternal IgG for immune-protection in their first months. The glycosylation of IgG has been shown to influence its dynamics, e.g. receptor binding. While minor differences in IgG glycosylation have been found between IgG derived from maternal blood and umbilical cord blood (UC) of newborn children, the differential glycosylation of maternal and UC plasma has hitherto not been studied. Here, we studied the N-glycosylation of IgG and total plasma proteome of both maternal and UC plasma of 42 pairs of mothers and newborn children. A total of 37 N-glycans were quantified for IgG and 45 for the total plasma N-glycome (TPNG). The study showed slightly higher levels of galactosylation for UC IgG than maternal IgG, confirming previous results, as well as lower bisection and sialylation. Furthermore, the TPNG results showed lower values for galactosylation and sialylation, and higher values for fucosylation in the UC plasma. In conclusion, this study presents some novel insights into IgG glycosylation differences as well as the first broad overview of the differential plasma glycosylation between mothers and newborns. PMID:27666402
Haigh, J D; Green, I M; Ball, D; Eydmann, M; Millar, M; Wilks, M
Several studies have reported poor results when trying to identify microorganisms directly from the bioMérieux BacT/ALERT blood culture system using matrix-assisted laser desorption/ionisation-time of flight (MALDI-TOF) mass spectrometry. The aim of this study is to evaluate two new methods, Sepsityper and an enrichment method for direct identification of microorganisms from this system. For both methods the samples were processed using the Bruker Microflex LT mass spectrometer (Biotyper) using the Microflex Control software to obtain spectra. The results from direct analysis were compared with those obtained by subculture and subsequent identification. A total of 350 positive blood cultures were processed simultaneously by the two methods. Fifty-three cultures were polymocrobial or failed to grow any organism on subculture, and these results were not included as there was either no subculture result, or for polymicrobial cultures it was known that the Biotyper would not be able to distinguish the constituent organisms correctly. Overall, the results showed that, contrary to previous reports, it is possible to identify bacteria directly from bioMérieux blood culture bottles, as 219/297 (74%) correct identifications were obtained using the Bruker Sepsityper method and 228/297 (77%) were obtained for the enrichment method when there is only one organism was present. Although the enrichment method was simpler, the reagent costs for the Sepsityper method were approximately pound 4.00 per sample compared to pound 0.50. An even simpler and cheaper method, which was less labour-intensive and did not require further reagents, was investigated. Seventy-seven specimens from positive signalled blood cultures were analysed by inoculating prewarmed blood agar plates and analysing any growth after 1-, 2- and 4-h periods of incubation at 37 degrees C, by either direct transfer or alcohol extraction. This method gave the highest number of correct identifications, 66/77 (86%), and was cheaper and less labour-intensive than either of the two above methods.
Meex, Cécile; Neuville, Florence; Descy, Julie; Huynen, Pascale; Hayette, Marie-Pierre; De Mol, Patrick; Melin, Pierrette
In cases of bacteraemia, a rapid species identification of the causal agent directly from positive blood culture broths could assist clinicians in the timely targeting of empirical antimicrobial therapy. For this purpose, we evaluated the direct identification of micro-organisms from BacT/ALERT (bioMérieux) anaerobic positive blood cultures without charcoal using the Microflex matrix-assisted laser desorption/ionization (MALDI) time of flight MS (Bruker), after bacterial extraction by using two different methods: the MALDI Sepsityper kit (Bruker) and an in-house saponin lysis method. Bruker's recommended criteria for identification were expanded in this study, with acceptance of the species identification when the first three results with the best matches with the MALDI Biotyper database were identical, whatever the scores were. In total, 107 monobacterial cultures and six polymicrobial cultures from 77 different patients were included in this study. Among monomicrobial cultures, we identified up to the species level 67 and 66 % of bacteria with the MALDI Sepsityper kit and the saponin method, respectively. There was no significant difference between the two extraction methods. The direct species identification was particularly inconclusive for Gram-positive bacteria, as only 58 and 52 % of them were identified to the species level with the MALDI Sepsityper kit and the saponin method, respectively. Results for Gram-negative bacilli were better, with 82.5 and 90 % of correct identification to the species level with the MALDI Sepsityper kit and the saponin method, respectively. No misidentifications were given by the direct procedures when compared with identifications provided by the conventional method. Concerning the six polymicrobial blood cultures, whatever the extraction method used, a correct direct identification was only provided for one of the isolated bacteria on solid medium in all cases. The analysis of the time-to-result demonstrated a reduction in the turnaround time for identification ranging from 1 h 06 min to 24 h 44 min, when performing the blood culture direct identification in comparison with the conventional method, whatever the extraction method.
Antagonistic effects of Bacillus subtilis subsp. subtilis and B. amyloliquefaciens against Macrophomina phaseolina: SEM study of fungal changes and UV-MALDI-TOF MS analysis of their bioactive compounds.
Torres, M J; Brandan, C Pérez; Petroselli, G; Erra-Balsells, R; Audisio, M C
The antifungal effect of Bacillus subtilis subsp. subtilis PGPMori7 and Bacillus amyloliquefaciens PGPBacCA1 was evaluated against Macrophomina phaseolina (Tassi) Goid. Cell suspension (CS), cell-free supernatant (CFS) and the lipopeptide fraction (LF) of PGPMori7 and PGPBacCA1 were screened against three different M. phaseolina strains. CS exhibited the highest inhibitory effect (around 50%) when compared to those of CFS and LF, regardless of the fungal strain studied. The synthesis of lipopeptides was studied by UV-MALDI TOF. Chemical analysis of Bacillus metabolite synthesis revealed that surfactin and iturin were mainly produced in liquid medium. Potential fengycin was also co-produced when both Bacillus were cultivated in solid medium. In co-culture assays, the bacterial colony-fungal mycelium interface at the inhibition zone was evaluated by both scanning electron microscopy (SEM) and UV-MALDI TOF, the former to determine the structural changes on M. phaseolina cells and the latter to identify the main bioactive molecules involved in the inhibitory effect. PGPBacCA1 produced surfactin, iturin and fengycin in the inhibition zone while PGPMori7 only produced these metabolites within its colony and not in the narrow inhibition zone. Interestingly, SEM revealed that PGPBacCA1 induced damage in M. phaseolina sclerotia, generating a fungicidal effect as no growth was observed when normal growth conditions were reestablished. In turn, PGPMori7 inhibited the growth of the Macrophomina mycelium without fungal injury, resulting only in a fungistatic activity. From these results, it was determined that the two bacilli significantly inhibited the growth of an important phytopathogenic fungus by at least two different mechanisms: lipopeptide synthesis and competition among microorganisms.
Wang, Aili; Liu, Li; Peng, Yanchun; Islam, Shahidul; Applebee, Marie; Appels, Rudi; Yan, Yueming; Ma, Wujun
Low molecular weight glutenin subunits (LMW-GS) play an important role in determining dough properties and breadmaking quality. However, resolution of the currently used methodologies for analyzing LMW-GS is rather low which prevents an efficient use of genetic variations associated with these alleles in wheat breeding. The aim of the current study is to evaluate and develop a rapid, simple, and accurate method to differentiate LMW-GS alleles using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. A set of standard single LMW-GS allele lines as well as a suite of well documented wheat cultivars were collected from France, CIMMYT, and Canada. Method development and optimization were focused on protein extraction procedures and MALDI-TOF instrument settings to generate reproducible diagnostic spectrum peak profiles for each of the known wheat LMW-GS allele. Results revealed a total of 48 unique allele combinations among the studied genotypes. Characteristic MALDI-TOF peak patterns were obtained for 17 common LMW-GS alleles, including 5 (b, a or c, d, e, f), 7 (a, b, c, d or i, f, g, h) and 5 (a, b, c, d, f) patterns or alleles for the Glu-A3, Glu-B3, and Glu-D3 loci, respectively. In addition, some reproducible MALDI-TOF peak patterns were also obtained that did not match with any known alleles. The results demonstrated a high resolution and throughput nature of MALDI-TOF technology in analyzing LMW-GS alleles, which is suitable for application in wheat breeding programs in processing a large number of wheat lines with high accuracy in limited time. It also suggested that the variation of LMW-GS alleles is more abundant than what has been defined by the current nomenclature system that is mainly based on SDS-PAGE system. The MALDI-TOF technology is useful to differentiate these variations. An international joint effort may be needed to assign allele symbols to these newly identified alleles and determine their effects on end-product quality attributes.
Performance of Two Resin-Containing Blood Culture Media in Detection of Bloodstream Infections and in Direct Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry (MALDI-TOF MS) Broth Assays for Isolate Identification: Clinical Comparison of the BacT/Alert Plus and Bactec Plus Systems
Fiori, Barbara; D'Inzeo, Tiziana; Di Florio, Viviana; De Maio, Flavio; De Angelis, Giulia; Giaquinto, Alessia; Campana, Lara; Tanzarella, Eloisa; Tumbarello, Mario; Antonelli, Massimo; Spanu, Teresa
We compared the clinical performances of the BacT/Alert Plus (bioMérieux) and Bactec Plus (Becton Dickinson) aerobic and anaerobic blood culture (BC) media with adsorbent polymeric beads. Patients ≥16 years old with suspected bloodstream infections (BSIs) were enrolled in intensive care units and infectious disease wards. A single 40-ml blood sample was collected from each and used to inoculate (10 ml/bottle) one set of BacT/Alert Plus cultures and one set of Bactec Plus cultures, each set consisting of one aerobic and one anaerobic bottle. Cultures were incubated ≤5 days in the BacT/Alert 3D and Bactec FX instruments, respectively. A total of 128 unique BSI episodes were identified based on the recovery of clinically significant growth in 212 aerobic cultures (106 BacT/Alert and 106 Bactec) and 151 anaerobic cultures (82 BacT/Alert and 69 Bactec). The BacT/Alert aerobic medium had higher recovery rates for Gram-positive cocci (P = 0.024), whereas the Bactec aerobic medium was superior for recovery of Gram-negative bacilli (P = 0.006). BacT/Alert anaerobic medium recovery rates exceeded those of the Bactec anaerobic medium for total organisms (P = 0.003), Gram-positive cocci (P = 0.013), and Escherichia coli (P = 0.030). In terms of capacity for diagnosing the 128 septic episodes, the BacT/Alert and Bactec sets were comparable, although the former sets diagnosed more BSIs caused by Gram-positive cocci (P = 0.008). They also allowed earlier identification of coagulase-negative staphylococcal growth (mean, 2.8 h; P = 0.003) and growth in samples from patients not on antimicrobial therapy that yielded positive results (mean, 1.3 h; P < 0.001). Similarly high percentages of microorganisms in BacT/Alert and Bactec cultures (93.8% and 93.3%, respectively) were identified by direct matrix-assisted laser desorption ionization–time of flight mass spectrometry assay of BC broths. The BacT/Alert Plus media line appears to be a reliable, timesaving tool for routine detection of BSIs in the population we studied, although further studies are needed to evaluate their performance in other settings. PMID:25031441
Gordonia Species as Emerging Causes of Continuous-Ambulatory-Peritoneal-Dialysis-Related Peritonitis Identified by 16S rRNA and secA1 Gene Sequencing and Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry (MALDI-TOF MS)
Lam, Jimmy Y. W.; Leung, Wai-Shing; Cheung, Ingrid; Chan, Jasper F. W.; Tse, Cindy W. S.; Lee, Rodney A.; Lau, Susanna K. P.
We report here four cases of continuous ambulatory peritoneal dialysis-related peritonitis caused by three different species of Gordonia. The portal of entry was likely through Tenckhoff catheters. 16S rRNA and secA1 gene sequencing are so far the most reliable methods for the accurate identification of Gordonia species. PMID:25428146
Almuzara, Marisa; Barberis, Claudia; Traglia, Germán; Famiglietti, Angela; Ramirez, Maria Soledad; Vay, Carlos
Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) to identify 396 Nonfermenting Gram-Negative Bacilli clinical isolates was evaluated in comparison with conventional phenotypic tests and/or molecular methods. MALDI-TOF MS identified to species level 256 isolates and to genus or complex level 112 isolates. It identified 29 genera including uncommon species.
Chen, Jonathan H K; Cheng, Vincent C C; Wong, Oi-Ying; Wong, Sally C Y; So, Simon Y C; Yam, Wing-Cheong; Yuen, Kwok-Yung
The clinical workflow of using chromogenic agar and matrix-assisted laser desorption ionization time-of-fight mass spectrometry (MALDI-TOF MS) for Clostridium difficile identification was evaluated. The addition of MALDI-TOF MS identification after the chromID C. difficile chromogenic agar culture could significantly improve the diagnostic accuracy of C. difficile.
Within the past few years identification of bacteria by MALDI-TOF MS has become a standard technique in bacteriological laboratories for good reasons. MALDI-TOF MS identification is rapid, robust, automatable, and the per-sample costs are low. Yet, the spectra are very informative and the reliable identification of bacterial species is usually possible. Recently, new MS-based approaches for the identification of bacteria are emerging that are based on the detailed analysis of the bacterial proteome by high-resolution MS. These "proteotyping" approaches are highly discriminative and outperform MALDI-TOF MS-based identification in terms of specificity, but require a laborious proteomic workflow and far more expertise and sophisticated instrumentation than identification on basis of MALDI-TOF MS spectra, which can be obtained with relative simple and uncostly linear MALDI-TOF mass spectrometers. Thus MALDI-TOF MS identification of bacteria remains an attractive option for routine diagnostics. Additionally, MALDI-TOF MS identification protocols have been extended and improved in many respects making linear MALDI-TOF MS a versatile tool that can be useful beyond the identification of a bacterial species, e.g. for the characterization of leucocytes and arthropod vectors of infectious diseases. This review focuses on such improvements and extensions of the typical MALDI-TOF MS workflow in the field of infectious diseases.
Loy, John Dustin; Clawson, Michael L
Genotype 2M. haemolytica predominantly associate over genotype 1 with the lungs of cattle with respiratory disease and ICEs containing antimicrobial resistance genes. Distinct protein masses were detected by MALDI-TOF MS between genotype 1 and 2 strains. MALDI-TOF MS could rapidly differentiate genotype 2 strains in veterinary diagnostic laboratories.
Gonzalez, Mark D; Weber, Carol J; Burnham, Carey-Ann D
We performed a retrospective analysis of a simple modification to MALDI-TOF MS for microorganism identification to accurately improve the turnaround time (TAT) for identification of Enterobacteriaceae recovered in blood cultures. Relative to standard MALDI-TOF MS procedures, we reduced TAT from 28.3 (n=90) to 21.2h (n=107).
Steensels, D; Verhaegen, J; Lagrou, K
Recently, MALDI-TOF MS devices designed for use in clinical laboratories have been commercially introduced in various large centres worldwide. All published studies conclude that MALDI-TOF MS can be implemented easily for routine identification of bacteria and yeasts in a clinical microbiological laboratory. Although all data show that MALDI-TOF MS correctly identifies the great majority of isolates processed routinely, it cannot yet identify every such isolate. Until today, MALDI-TOF MS is inappropriate for the identification of Shigella species, pneumococci and viridans streptococci. Database upgrades and sample enrichment are essential elements to refine the MALDI-TOF MS technique, allowing the method to increase its power. For the identification of a significant proportion of yeasts, an extraction method prior to analysis in the mass spectrometer is mandatory to obtain appropriate spectra. Because of the low marginal costs, and the extreme speed of MALDI-TOF MS, the technique can improve laboratory efficiency when used early in identification protocols. Lengthier, more labour-intensive, and costlier techniques can be reserved for the minority of isolates not identified with high confidence by MALDI-TOF MS. MALDI-TOF MS also has the potential to directly identify pathogens in biological fluids, such as urine samples and blood cultures. For this application however, further well-designed prospective studies are warranted. The potential for identification at the serotype or strain level, and antibiotic resistance profiling within minutes make MALDI-TOF mass spectrometry an ongoing revolution in the clinical microbiology laboratory.
Genotype 2 M. haemolytica predominantly associate over genotype 1 with the lungs of cattle with respiratory disease and ICEs containing antimicrobial resistance genes. Distinct protein masses were detected by MALDI-TOF MS between genotype 1 and 2 strains. MALDI-TOF MS could rapidly differentiate ge...
Marín, Santiago; Gil-Humanes, Javier; Hernando, Alberto; Barro, Francisco
In this work, gliadin composition has been analyzed in 33 accessions of durum wheat using MALDI-TOF MS and compared with A-PAGE results. The MALDI-TOF MS spectra were 29,900-42,500 Da, which corresponds to the α/β- and γ-gliadin regions in A-PAGE. The average of gliadin peaks per line was 23 for MALDI-TOF MS and only 14.8 bands for A-PAGE. MALDI-TOF MS identified 33 gliadin peaks in the durum wheat collection, 20 of which were unique peaks present in 7 lines. A-PAGE analysis identified 30 bands, of which only 4 were unique. Thus, the MALDI-TOF MS method was more sensitive than A-PAGE for identifying α/β- and γ-gliadins in the 33 durum wheat lines studied. Phylogenetic analyses performed using MALDI-TOF MS data assigned the durum wheat lines to two groups. The utility of MALDI-TOF MS to determine relationships among genotypes and for identification of durum wheat accessions is discussed.
Determination of molecular mass distribution of silicone oils by supercritical fluid chromatography, matrix-assisted laser desorption ionization time-of-flight mass spectrometry and their off-line combination.
Chmelík, J; Planeta, J; Rehulka, P; Chmelík, J
Silicone oil samples were characterized by supercritical fluid chromatography (SFC), matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI--TOF MS), and their off-line combination. SFC was used to separate samples of silicone oils on micropacked capillary columns. The fractions for the identification studies were obtained from SFC runs at defined time intervals, when the restrictor was pulled out from the chromatographic flame ionization detector (FID) and inserted into a glass vial with acetone. MALDI--TOF MS was used for the identification of individual oligomers in the fractions separated. The molecular mass distributions determined based on SFC and MALDI--TOF MS measurements were compared. From this comparison, it follows that the results are in good agreement. However, certain differences were observed: MALDI--TOF MS was capable of detecting somewhat larger oligomers than the SFC-FID, but the lower molecular mass oligomers were not present in the MALDI spectra. Differences in the region of lower molecular masses can be explained by evaporation of the more volatile low molecular mass oligomers resulting from heating of the sample during the MALDI--TOF MS measurements as a result of the absorption of the laser shot energy. The fact that no high mass discrimination effects of the MALDI--TOF MS measurements, compared with SFC, were observed is very promising for further applications of MALDI--TOF MS in characterizing synthetic polymers of moderate polydispersity.
Buchanan, R; Ball, D; Dolphin, H; Dave, J
Matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS) was compared with the API NH biochemical method for the identification of Neisseria gonorrhoeae in routine clinical samples. A retrospective review of laboratory records for 1090 isolates for which both biochemical and MALDI-TOF MS identifications were available was performed. Cases of discrepant results were examined in detail for evidence supportive of a particular organism identification. Of 1090 isolates, 1082 were identified as N. gonorrhoeae by API NH. MALDI-TOF MS successfully identified 984 (91%) of these after one analysis, rising to 1081 (99.9%) after two analyses, with a positive predictive value of 99.3%. For those isolates requiring a repeat analysis, failure to generate an identifiable proteomic signature was the reason in 76% of cases, with alternative initial identifications accounting for the remaining 24%. MALDI-TOF MS identified eight isolates as N. gonorrhoeae that were not identified as such by API NH-examination of these discrepant results suggested that the MALDI-TOF MS identification may be the more reliable. MALDI-TOF MS is at least as accurate and reliable a method of identifying N. gonorrhoeae as API NH. We propose that MALDI-TOF MS could potentially be used as a single method for N. gonorrhoeae identification in routine cases by laboratories with access to this technology.
Kumsa, Bersissa; Laroche, Maureen; Almeras, Lionel; Mediannikov, Oleg; Raoult, Didier; Parola, Philippe
Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) technology has recently been reported as a promising method for arthropods identification. More recently, our laboratory reported the correct identification of tick species via the MALDI-TOF MS protein spectra profiling of legs from fresh specimens. The aim of the present study was to assess the use of MALDI-TOF MS for correct identification of ixodid tick species preserved in 70 % ethanol during field collection in Ethiopia. Following morphological identification of 12 tick species, the legs from 85 tick specimens were subjected to MALDI-TOF MS. Spectral analysis revealed an intra-species reproducibility and inter-species specificity that were consistent with the morphological classification. To support the results of the MALDI-TOF MS tick species identification, 41 tick specimens comprising 3 to 5 specimens per tick species were used to create a reference spectra database, which was evaluated using the spectra of the 44 remaining tick specimens. The blind tests revealed that 100 % of the tick specimens studied by MALDI-TOF MS were correctly identified. A relevant Log score value (LSV) of >1.8 was recorded for all of the tick species studied by MALDI-TOF MS, except for Rhipicephalus praetextatus. The morphological and MALDI-TOF MS identifications were confirmed by sequencing the 12S ribosomal RNA (rRNA) gene of 40 tick specimens belonging to 11 ixodid species. Taken together, the results of the present study indicate that MALDI-TOF MS is a reliable tool for tick species identification, even after preservation in ethanol, provided that a reference spectra database is built from specimens that represent the respective species stored under the same conditions.
van Veen, S. Q.; Claas, E. C. J.; Kuijper, Ed J.
Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is suitable for high-throughput and rapid diagnostics at low costs and can be considered an alternative for conventional biochemical and molecular identification systems in a conventional microbiological laboratory. First, we evaluated MALDI-TOF MS using 327 clinical isolates previously cultured from patient materials and identified by conventional techniques (Vitek-II, API, and biochemical tests). Discrepancies were analyzed by molecular analysis of the 16S genes. Of 327 isolates, 95.1% were identified correctly to genus level, and 85.6% were identified to species level by MALDI-TOF MS. Second, we performed a prospective validation study, including 980 clinical isolates of bacteria and yeasts. Overall performance of MALDI-TOF MS was significantly better than conventional biochemical systems for correct species identification (92.2% and 83.1%, respectively) and produced fewer incorrect genus identifications (0.1% and 1.6%, respectively). Correct species identification by MALDI-TOF MS was observed in 97.7% of Enterobacteriaceae, 92% of nonfermentative Gram-negative bacteria, 94.3% of staphylococci, 84.8% of streptococci, 84% of a miscellaneous group (mainly Haemophilus, Actinobacillus, Cardiobacterium, Eikenella, and Kingella [HACEK]), and 85.2% of yeasts. MALDI-TOF MS had significantly better performance than conventional methods for species identification of staphylococci and genus identification of bacteria belonging to HACEK group. Misidentifications by MALDI-TOF MS were clearly associated with an absence of sufficient spectra from suitable reference strains in the MALDI-TOF MS database. We conclude that MALDI-TOF MS can be implemented easily for routine identification of bacteria (except for pneumococci and viridans streptococci) and yeasts in a medical microbiological laboratory. PMID:20053859
Woods, Katherine; Beighton, David; Klein, John L
Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) provides rapid, accurate and cost-effective identification of a range of bacteria and is rapidly changing the face of routine diagnostic microbiology. However, certain groups of bacteria, for example streptococci (in particular viridans or non-haemolytic streptococci), are less reliably identified by this method. We studied the performance of MALDI-TOF MS for identification of the 'Streptococcus anginosus group' (SAG) to species level. In total, 116 stored bacteraemia isolates identified by conventional methods as belonging to the SAG were analysed by MALDI-TOF MS. Partial 16S rRNA gene sequencing, supplemented with sialidase activity testing, was performed on all isolates to provide 'gold standard' identification against which to compare MALDI-TOF MS performance. Overall, 100 % of isolates were correctly identified to the genus level and 93.1 % to the species level by MALDI-TOF MS. However, only 77.6 % were correctly identified to the genus level and 59.5 % to the species level by a MALDI-TOF MS direct transfer method alone. Use of a rapid in situ extraction method significantly improved identification rates when compared with the direct transfer method (P<0.001). We recommend routine use of this method to reduce the number of time-consuming full extractions required for identification of this group of bacteria by MALDI-TOF MS in the routine diagnostic laboratory. Only 22 % (1/9) of Streptococcus intermedius isolates were reliably identified by MALDI-TOF MS to the species level, even after full extraction. MALDI-TOF MS reliably identifies S. anginosus and Streptococcus constellatus to the species level but does not reliably identify S. intermedius.
van Veen, S Q; Claas, E C J; Kuijper, Ed J
Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is suitable for high-throughput and rapid diagnostics at low costs and can be considered an alternative for conventional biochemical and molecular identification systems in a conventional microbiological laboratory. First, we evaluated MALDI-TOF MS using 327 clinical isolates previously cultured from patient materials and identified by conventional techniques (Vitek-II, API, and biochemical tests). Discrepancies were analyzed by molecular analysis of the 16S genes. Of 327 isolates, 95.1% were identified correctly to genus level, and 85.6% were identified to species level by MALDI-TOF MS. Second, we performed a prospective validation study, including 980 clinical isolates of bacteria and yeasts. Overall performance of MALDI-TOF MS was significantly better than conventional biochemical systems for correct species identification (92.2% and 83.1%, respectively) and produced fewer incorrect genus identifications (0.1% and 1.6%, respectively). Correct species identification by MALDI-TOF MS was observed in 97.7% of Enterobacteriaceae, 92% of nonfermentative Gram-negative bacteria, 94.3% of staphylococci, 84.8% of streptococci, 84% of a miscellaneous group (mainly Haemophilus, Actinobacillus, Cardiobacterium, Eikenella, and Kingella [HACEK]), and 85.2% of yeasts. MALDI-TOF MS had significantly better performance than conventional methods for species identification of staphylococci and genus identification of bacteria belonging to HACEK group. Misidentifications by MALDI-TOF MS were clearly associated with an absence of sufficient spectra from suitable reference strains in the MALDI-TOF MS database. We conclude that MALDI-TOF MS can be implemented easily for routine identification of bacteria (except for pneumococci and viridans streptococci) and yeasts in a medical microbiological laboratory.
Westblade, Lars F; Garner, Omai B; MacDonald, Karen; Bradford, Constance; Pincus, David H; Mochon, A Brian; Jennemann, Rebecca; Manji, Ryhana; Bythrow, Maureen; Lewinski, Michael A; Burnham, Carey-Ann D; Ginocchio, Christine C
Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) has revolutionized the identification of clinical bacterial and yeast isolates. However, data describing the reproducibility of MALDI-TOF MS for microbial identification are scarce. In this study, we show that MALDI-TOF MS-based microbial identification is highly reproducible and can tolerate numerous variables, including differences in testing environments, instruments, operators, reagent lots, and sample positioning patterns. Finally, we reveal that samples of bacterial and yeast isolates prepared for MALDI-TOF MS identification can be repeatedly analyzed without compromising organism identification.
Kok, Jen; Chen, Sharon C A; Dwyer, Dominic E; Iredell, Jonathan R
The integration of matrix-assisted laser desorption ionisation-time of flight mass spectrometry (MALDI-TOF MS) into many clinical microbiology laboratories has revolutionised routine pathogen identification. MALDI-TOF MS complements and has good potential to replace existing phenotypic identification methods. Results are available in a more clinically relevant timeframe, particularly in bacteraemic septic shock. Novel applications include strain typing and the detection of antimicrobial resistance, but these are not widely used. This review discusses the technical aspects, current applications, and limitations of MALDI-TOF MS.
Direct detection of the plant pathogens Burkholderia glumae, Burkholderia gladioli pv. gladioli, and Erwinia chrysanthemi pv. zeae in infected rice seedlings using matrix assisted laser desorption/ionization time-of-flight mass spectrometry.
The plant pathogens Burkholderia glumae, Burkholderia gladioli pv. gladioli, and Erwinia chrysanthemi pv. zeae were directly detected in extracts from infected rice seedlings by matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). This method did not require culturing of the pathogens on artificial medium. In the MALDI-TOF MS analysis, peaks originating from bacteria were found in extracts from infected rice seedlings. The spectral peaks showed significantly high scores, in spite of minor differences in spectra. The spectral peaks originating from host plant tissues did not affect this direct MALDI-TOF MS analysis for the rapid identification of plant pathogens.
Na, Dong Hee; DeLuca, Patrick P; Lee, Kang Choon
A quantitative determination of peptides incorporated into poly(d,l-lactide-co-glycolide) microspheres by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was accomplished in a single step without pretreatment for extracting the peptide from the microsphere. The conventional extraction methods often underestimate the actual amount of peptide because of incomplete extraction from the microspheres or loss during the procedures. In this study, the microspheres dissolved in acetonitrile containing 0.1% trifluoroacetic acid were mixed with matrix solution containing the internal standard, and the peptide content was directly determined by MALDI-TOF MS. The drug content values determined by MALDI-TOF MS in both the leuprolide- and salmon calcitonin-incorporated microspheres were closer to the theoretical contents than those determined by the conventional extraction method. This method using MALDI-TOF MS could be a good alternative to time-consuming and less-accurate conventional methods.
Hettick, Justin M; Green, Brett J; Buskirk, Amanda D; Kashon, Michael L; Slaven, James E; Janotka, Erika; Blachere, Francoise M; Schmechel, Detlef; Beezhold, Donald H
Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was used to generate highly reproducible mass spectral 'fingerprints' for twelve Penicillium species. Prior to MALDI-TOF MS analysis, eight replicate cultures of each Penicillium species were subjected to three one-minute bead-beating cycles in an acetonitrile/trifluoroacetic acid solvent. The mass spectra contained abundant peaks in the range of m/z 5000-20 000, and allowed unambiguous discrimination between species. In addition, a biomarker common to all Penicillium mass spectra was observed at m/z 13 900. Discriminant analysis using the MALDI-TOF MS data yielded classification error rates of 0% (i.e. 100% correct identification), indicating that MALDI-TOF MS data may be a useful diagnostic tool for the objective identification of Penicillium species of environmental and clinical importance.
microcolumns of C18 (Millipore). Peptide mass fingerprints were determined by matrix assisted laser desorption ionization ( MALDI )- time- of-flight ( TOF )-MS on...24 Table 2. List ofproteins that were found to be HD-exposure related spots. Proteins were identified by MALDI / TOF /MS analysis. Letters correspond to...were identified by MALDI - TOF /MS analysis. Numbers correspond to spots indicated in Figure 10. Values for MW and pI are according to the SWISSPROT
Chen, Hui; He, Meiyu
MALDI-TOF MS is utilized to perform quantitative analysis on synthetic polymers. Despite the inherent limitations of MALDI, good quantitative results have been obtained in the three sets of experiments described here. An internal standard with similar molecular properties as the analytes is introduced. Plots of relative integrated intensity ratios as a function of theoretical ratios of stoichiometry are drawn based on the results. The satisfactory slopes and correlation coefficients illustrated the practicality of quantitative measurement by MALDI-TOF MS.
Carbonnelle, Etienne; Grohs, Patrick; Jacquier, Hervé; Day, Nesrine; Tenza, Sylvie; Dewailly, Alexandra; Vissouarn, Odile; Rottman, Martin; Herrmann, Jean-Louis; Podglajen, Isabelle; Raskine, Laurent
MALDI-TOF-MS systems (Microflex-Bruker Daltonics/BioTyper™ and Axima-Assurance-Shimadzu/SARAMIS-AnagnosTec) were assessed for bacterial identification. Focusing on bacteria difficult to identify routinely, 296 strains were identified by molecular biology techniques as gold standard. MALDI-TOF-MS identification provided correct results at genus and species level for 94.9%, 83.4% and 83.8%, 65.9% with Biotyper and Saramis respectively.
Thomaz, Danilo Y; Grenfell, Rafaella C; Vidal, Monica S M; Giudice, Mauro C; Del Negro, Gilda M B; Juliano, Luiz; Benard, Gil; de Almeida Júnior, João N
We described the impact of the capsule size for Cryptococcus neoformans and Cryptococcus gattii identification at the species level by Bruker matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). After experimental capsule size modulation, we observed that reducing the capsule size resulted in improved identification by Bruker MALDI-TOF MS across all of the reference strains analyzed.
Roberts, Amity L; Alelew, Aqilah; Iwen, Peter C
Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF-MS) analysis in conjunction with the direct formic acid (FA) sample processing method was evaluated for the ability to differentiate the closely related species of Candida albicans and Candida dubliniensis. The results showed that MALDI-TOF-MS, using the direct FA method, was reliable to differentiate between these species.
van Eck, Kim; Faro, Dirk; Wattenberg, Melanie; de Jong, Arjan; Kuipers, Saskia
We have assessed matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) identification (Bruker) of nontuberculous mycobacteria from newly positive liquid cultures of respiratory samples. Twelve (22%) of 54 isolates were identified directly from liquid medium. After subculture and with manual laser operation, this rose to 49/54 isolates (91%). MALDI-TOF MS is less promising than previously suggested. PMID:27147723
Buckwalter, S. P.; Olson, S. L.; Connelly, B. J.; Lucas, B. C.; Rodning, A. A.; Walchak, R. C.; Deml, S. M.; Wohlfiel, S. L.
The value of matrix-assisted laser desorption ionization−time of flight mass spectrometry (MALDI-TOF MS) for the identification of bacteria and yeasts is well documented in the literature. Its utility for the identification of mycobacteria and Nocardia spp. has also been reported in a limited scope. In this work, we report the specificity of MALDI-TOF MS for the identification of 162 Mycobacterium species and subspecies, 53 Nocardia species, and 13 genera (totaling 43 species) of other aerobic actinomycetes using both the MALDI-TOF MS manufacturer's supplied database(s) and a custom database generated in our laboratory. The performance of a simplified processing and extraction procedure was also evaluated, and, similar to the results in an earlier literature report, our viability studies confirmed the ability of this process to inactivate Mycobacterium tuberculosis prior to analysis. Following library construction and the specificity study, the performance of MALDI-TOF MS was directly compared with that of 16S rRNA gene sequencing for the evaluation of 297 mycobacteria isolates, 148 Nocardia species isolates, and 61 other aerobic actinomycetes isolates under routine clinical laboratory working conditions over a 6-month period. MALDI-TOF MS is a valuable tool for the identification of these groups of organisms. Limitations in the databases and in the ability of MALDI-TOF MS to rapidly identify slowly growing mycobacteria are discussed. PMID:26637381
Tang, Feng; Cen, Si-Ying; He, Huan; Liu, Yi; Yuan, Bi-Feng; Feng, Yu-Qi
Determination of low-molecular-weight compounds by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) has been a great challenge in the analytical research field. Here we developed a universal peptide-based derivatization (peptidylation) strategy for the sensitive analysis of low-molecular-weight compounds by MALDI-TOF-MS. Upon peptidylation, the molecular weights of target analytes increase, thus avoiding serious matrix ion interference in the low-molecular-weight region in MALDI-TOF-MS. Since peptides typically exhibit good signal response during MALDI-TOF-MS analysis, peptidylation endows high detection sensitivities of low-molecular-weight analytes. As a proof-of-concept, we analyzed low-molecular-weight compounds of aldehydes and thiols by the developed peptidylation strategy. Our results showed that aldehydes and thiols can be readily determined upon peptidylation, thus realizing the sensitive and efficient determination of low-molecular-weight compounds by MALDI-TOF-MS. Moreover, target analytes also can be unambiguously detected in biological samples using the peptidylation strategy. The established peptidylation strategy is a universal strategy and can be extended to the sensitive analysis of various low-molecular-weight compounds by MALDI-TOF-MS, which may be potentially used in areas such as metabolomics.
Ferreira, Laura; Sánchez-Juanes, Fernando; González-Ávila, Magdalena; Cembrero-Fuciños, David; Herrero-Hernández, Ana; González-Buitrago, José Manuel; Muñoz-Bellido, Juan Luis
Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has been suggested as a reliable method for bacterial identification from cultures. Direct analysis of clinical samples might increase the usefulness of this method, shortening the time for microorganism identification. We compared conventional methods for the diagnosis of urinary tract infections (UTIs) and identification of the urinary tract pathogens (automated screening, plate cultures, and identification based on biochemical characteristics) and a fast method based on conventional screening and MALDI-TOF MS. For this latter method, 4 ml of urine was centrifuged at a low-revolution setting (2,000 × g) to remove leukocytes and then at high revolutions (15,500 × g) to collect bacteria. The pellet was washed and then applied directly to the MALDI-TOF MS plate. Two hundred sixty urine samples, detected as positive by the screening device (UF-1000i), were processed by culture and MALDI-TOF MS. Twenty samples were positive in the screening device but negative in culture, and all of them were also negative by MALDI-TOF MS. Two-hundred thirty-five samples displayed significant growth of a single morphological type in culture. Two-hundred twenty of them showed bacterial growth of >105 CFU/ml. Microorganism identifications in this group were coincident at the species level in 202 cases (91.8%) and at the genus level in 204 cases (92.7%). The most frequent microorganism was Escherichia coli (173 isolates). MALDI-TOF MS identified this microorganism directly from the urine sample in 163 cases (94.2%). Our results show that MALDI-TOF MS allows bacterial identification directly from infected urine in a short time, with high accuracy, and especially when Gram-negative bacteria with high bacterial counts are involved. PMID:20392910
Development and Validation of an In-House Database for Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry-Based Yeast Identification Using a Fast Protein Extraction Procedure
De Carolis, Elena; Vella, Antonietta; Vaccaro, Luisa; Torelli, Riccardo; Posteraro, Patrizia; Ricciardi, Walter; Posteraro, Brunella
In recent studies evaluating the usefulness of the matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS)-based identification of yeasts for the routine diagnosis of fungal infections, preanalytical sample processing has emerged as a critical step for reliable MALDI-TOF MS outcomes, especially when the Bruker Daltonics Biotyper software was used. In addition, inadequate results often occurred due to discrepancies between the methods used for clinical testing and database construction. Therefore, we created an in-house MALDI-TOF MS library using the spectra from 156 reference and clinical yeast isolates (48 species in 11 genera), which were generated with a fast sample preparation procedure. After a retrospective validation study, our database was evaluated on 4,232 yeasts routinely isolated during a 6-month period and fast prepared for MALDI-TOF MS analysis. Thus, 4,209 (99.5%) of the isolates were successfully identified to the species level (with scores of ≥2.0), with 1,676 (39.6%) having scores of >2.3. For the remaining 23 (0.5%) isolates, no reliable identification (with scores of <1.7) was obtained. Interestingly, these isolates were almost always from species uniquely represented or not included in the database. As the MALDI-TOF MS results were, except for 23 isolates, validated without additional phenotypic or molecular tests, our proposed strategy can enhance the rapidity and accuracy of MALDI-TOF MS in identifying medically important yeast species. However, while continuous updating of our database will be necessary to enrich it with more strains/species of new and emerging yeasts, the present in-house MALDI-TOF MS library can be made publicly available for future multicenter studies. PMID:24554755
Seng, Piseth; Abat, Cedric; Rolain, Jean Marc; Colson, Philippe; Lagier, Jean-Christophe; Gouriet, Frédérique; Fournier, Pierre Edouard; Drancourt, Michel; La Scola, Bernard; Raoult, Didier
During the past 5 years, matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) has become a powerful tool for routine identification in many clinical laboratories. We analyzed our 11-year experience in routine identification of clinical isolates (40 months using MALDI-TOF MS and 91 months using conventional phenotypic identification [CPI]). Among the 286,842 clonal isolates, 284,899 isolates of 459 species were identified. The remaining 1,951 isolates were misidentified and required confirmation using a second phenotypic identification for 670 isolates and using a molecular technique for 1,273 isolates of 339 species. MALDI-TOF MS annually identified 112 species, i.e., 36 species/10,000 isolates, compared to 44 species, i.e., 19 species/10,000 isolates, for CPI. Only 50 isolates required second phenotypic identifications during the MALDI-TOF MS period (i.e., 4.5 reidentifications/10,000 isolates) compared with 620 isolates during the CPI period (i.e., 35.2/10,000 isolates). We identified 128 bacterial species rarely reported as human pathogens, including 48 using phenotypic techniques (22 using CPI and 37 using MALDI-TOF MS). Another 75 rare species were identified using molecular methods. MALDI-TOF MS reduced the time required for identification by 55-fold and 169-fold and the cost by 5-fold and 96-fold compared with CPI and gene sequencing, respectively. MALDI-TOF MS was a powerful tool not only for routine bacterial identification but also for identification of rare bacterial species implicated in human infectious diseases. The ability to rapidly identify bacterial species rarely described as pathogens in specific clinical specimens will help us to study the clinical burden resulting from the emergence of these species as human pathogens, and MALDI-TOF MS may be considered an alternative to molecular methods in clinical laboratories.
Alibi, S; Ferjani, A; Gaillot, O; Marzouk, M; Courcol, R; Boukadida, J
We evaluated the Bruker Biotyper matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) for the identification of 97 Corynebacterium clinical in comparison to identification strains by Api Coryne and MALDI-TOF-MS using 16S rRNA gene and hypervariable region of rpoB genes sequencing as a reference method. C. striatum was the predominant species isolated followed by C. amycolatum. There was an agreement between Api Coryne strips and MALDI-TOF-MS identification in 88.65% of cases. MALDI-TOF-MS was unable to differentiate C. aurimucosum from C. minutissimum and C. minutissimum from C. singulare but reliably identify 92 of 97 (94.84%) strains. Two strains remained incompletely identified to the species level by MALDI-TOF-MS and molecular approaches. They belonged to Cellulomonas and Pseudoclavibacter genus. In conclusion, MALDI-TOF-MS is a rapid and reliable method for the identification of Corynebacterium species. However, some limits have been noted and have to be resolved by the application of molecular methods.
Clark, Andrew E; Kaleta, Erin J; Arora, Amit; Wolk, Donna M
Within the past decade, clinical microbiology laboratories experienced revolutionary changes in the way in which microorganisms are identified, moving away from slow, traditional microbial identification algorithms toward rapid molecular methods and mass spectrometry (MS). Historically, MS was clinically utilized as a high-complexity method adapted for protein-centered analysis of samples in chemistry and hematology laboratories. Today, matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) MS is adapted for use in microbiology laboratories, where it serves as a paradigm-shifting, rapid, and robust method for accurate microbial identification. Multiple instrument platforms, marketed by well-established manufacturers, are beginning to displace automated phenotypic identification instruments and in some cases genetic sequence-based identification practices. This review summarizes the current position of MALDI-TOF MS in clinical research and in diagnostic clinical microbiology laboratories and serves as a primer to examine the "nuts and bolts" of MALDI-TOF MS, highlighting research associated with sample preparation, spectral analysis, and accuracy. Currently available MALDI-TOF MS hardware and software platforms that support the use of MALDI-TOF with direct and precultured specimens and integration of the technology into the laboratory workflow are also discussed. Finally, this review closes with a prospective view of the future of MALDI-TOF MS in the clinical microbiology laboratory to accelerate diagnosis and microbial identification to improve patient care.
Clark, Andrew E.; Kaleta, Erin J.; Arora, Amit
SUMMARY Within the past decade, clinical microbiology laboratories experienced revolutionary changes in the way in which microorganisms are identified, moving away from slow, traditional microbial identification algorithms toward rapid molecular methods and mass spectrometry (MS). Historically, MS was clinically utilized as a high-complexity method adapted for protein-centered analysis of samples in chemistry and hematology laboratories. Today, matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) MS is adapted for use in microbiology laboratories, where it serves as a paradigm-shifting, rapid, and robust method for accurate microbial identification. Multiple instrument platforms, marketed by well-established manufacturers, are beginning to displace automated phenotypic identification instruments and in some cases genetic sequence-based identification practices. This review summarizes the current position of MALDI-TOF MS in clinical research and in diagnostic clinical microbiology laboratories and serves as a primer to examine the “nuts and bolts” of MALDI-TOF MS, highlighting research associated with sample preparation, spectral analysis, and accuracy. Currently available MALDI-TOF MS hardware and software platforms that support the use of MALDI-TOF with direct and precultured specimens and integration of the technology into the laboratory workflow are also discussed. Finally, this review closes with a prospective view of the future of MALDI-TOF MS in the clinical microbiology laboratory to accelerate diagnosis and microbial identification to improve patient care. PMID:23824373
Vlek, Anne L M; Bonten, Marc J M; Boel, C H Edwin
Matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) allows the identification of microorganisms directly from positive blood culture broths. Use of the MALDI-TOF MS for rapid identification of microorganisms from blood culture broths can reduce the turnaround time to identification and may lead to earlier appropriate treatment of bacteremia. During February and April 2010, direct MALDI-TOF MS was routinely performed on all positive blood cultures. During December 2009 and March 2010 no direct MALDI-TOF MS was used. Information on antibiotic therapy was collected from the hospital and intensive care units' information systems from all positive blood cultures during the study period. In total, 253 episodes of bacteremia were included of which 89 during the intervention period and 164 during the control period. Direct performance of MALDI-TOF MS on positive blood culture broths reduced the time till species identification by 28.8-h and was associated with an 11.3% increase in the proportion of patients receiving appropriate antibiotic treatment 24 hours after blood culture positivity (64.0% in the control period versus 75.3% in the intervention period (p0.01)). Routine implementation of this technique increased the proportion of patients on adequate antimicrobial treatment within 24 hours.
Utilization of matrix-assisted laser desorption and ionization time-of-flight mass spectrometry for identification of infantile seborrheic dermatitis-causing Malassezia and incidence of culture-based cutaneous Malassezia microbiota of 1-month-old infants.
Yamamoto, Mikachi; Umeda, Yoshiko; Yo, Ayaka; Yamaura, Mariko; Makimura, Koichi
Matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF-MS) has been utilized for identification of various microorganisms. Malassezia species, including Malassezia restricta, which is associated with seborrheic dermatitis, has been difficult to identify by traditional means. This study was performed to develop a system for identification of Malassezia species with MALDI-TOF-MS and to investigate the incidence and variety of cutaneous Malassezia microbiota of 1-month-old infants using this technique. A Malassezia species-specific MALDI-TOF-MS database was developed from eight standard strains, and the availability of this system was assessed using 54 clinical strains isolated from the skin of 1-month-old infants. Clinical isolates were cultured initially on CHROMagar Malassezia growth medium, and the 28S ribosomal DNA (D1/D2) sequence was analyzed for confirmatory identification. Using this database, we detected and analyzed Malassezia species in 68% and 44% of infants with and without infantile seborrheic dermatitis, respectively. The results of MALDI-TOF-MS analysis were consistent with those of rDNA sequencing identification (100% accuracy rate). To our knowledge, this is the first report of a MALDI-TOF-MS database for major skin pathogenic Malassezia species. This system is an easy, rapid and reliable method for identification of Malassezia.
Bruin, J P; Kostrzewa, M; van der Ende, A; Badoux, P; Jansen, R; Boers, S A; Diederen, B M W
Generally accepted laboratory methods that have been used for decades do not reliably distinguish between H. influenzae and H. haemolyticus isolates. H. haemolyticus strains are often incorrectly identified as nontypeable Haemophilus influenzae (NTHi). To distinguish H. influenzae from H. haemolyticus we have created a new database on the matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) bio-typer 2 and compared the results with routine determination of Haemophilus (growth requirement for X and V factor), and multilocus sequence typing (MLST). In total we have tested 277 isolates, 244 H. influenzae and 33 H. haemolyticus. Using MLST as the gold standard, the agreement of MALDI-TOF MS was 99.6 %. MALDI-TOF MS allows reliable and rapid discrimination between H. influenzae and H. haemolyticus.
Majcherczyk, Paul A; McKenna, Therese; Moreillon, Philippe; Vaudaux, Pierre
To explore the discriminatory power of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for detecting subtle differences in isogenic isolates, we tested isogenic strains of Staphylococcus aureus differing in their expression of resistance to methicillin or teicoplanin. More important changes in MALDI-TOF MS spectra were found with strains differing in methicillin than in teicoplanin resistance. In comparison, very minor or no changes were recorded in pulsed-field gel electrophoresis profiles or peptidoglycan muropeptide digest patterns of these strains, respectively. MALDI-TOF MS might be useful to detect subtle strain-specific differences in ionizable components released from bacterial surfaces and not from their peptidoglycan network.
Sato, Yasuto; Fukuyama, Yuko; Nonami, Hiroshi; Erra-Balsells, Rosa; Stortz, Carlos A; Cerezo, Alberto S; Matulewicz, María C
Matrix-assisted ultraviolet laser desorption/ionization time-of-flight mass spectrometry (UV-MALDI-TOF-MS) has shown to be a very useful technique for the study of the non-volatile and thermally non-stable N-acylated glycopyranosyl- and glycofuranosyl-amines. Of the several matrices tested, 2,5-dihydroxybenzoic acid (DHB) was the most effective giving good spectra in the positive-ion mode. In the linear and reflectron modes, the [M+Na](+) ions appeared with high intensity. Their fragmentation patterns were investigated by post-source decay (PSD) UV-MALDI-TOF-MS showing mainly cross-ring cleavages. In addition, N,O-acylated glycopyranosyl- and glycofuranosyl-amines were also analyzed by this technique. PSD UV-MALDI-TOF-MS gave significant signals for several primary fragment ions, which were proposed but not detected, or observed with very low abundance, in electron ionization mass spectrometry (EI-MS) experiments.
Emami, Kaveh; Nelson, Andrew; Hack, Ethan; Zhang, Jinwei; Green, David H; Caldwell, Gary S; Mesbahi, Ehsan
The genus Pseudoalteromonas constitutes an ecologically significant group of marine Gammaproteobacteria with potential biotechnological value as producers of bioactive compounds and of enzymes. Understanding their roles in the environment and bioprospecting for novel products depend on efficient ways of identifying environmental isolates. Matrix Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) biotyping has promise as a rapid and reliable method of identifying and distinguishing between different types of bacteria, but has had relatively limited application to marine bacteria and has not been applied systematically to Pseudoalteromonas. Therefore, we constructed a MALDI-TOF MS database of 31 known Pseudoalteromonas species, to which new isolates can be compared by MALDI-TOF biotyping. The ability of MALDI-TOF MS to distinguish between species was scrutinized by comparison with 16S rRNA gene sequencing. The patterns of similarity given by the two approaches were broadly but not completely consistent. In general, the resolution of MALDI-TOF MS was greater than that of 16S rRNA gene sequencing. The database was tested with 13 environmental Pseudoalteromonas isolates from UK waters. All of the test strains could be identified to genus level by MALDI-TOF MS biotyping, but most could not be definitely identified to species level. We conclude that several of these isolates, and possibly most, represent new species. Thus, further taxonomic investigation of Pseudoalteromonas is needed before MALDI-TOF MS biotyping can be used reliably for species identification. It is, however, a powerful tool for characterizing and distinguishing among environmental isolates and can make an important contribution to taxonomic studies.
Coello, Andreu; Fernández-Rivas, Gema; Rivaya, Belén; Hidalgo, Jessica; Quesada, María Dolores; Ausina, Vicente
Early diagnosis of urinary tract infections (UTIs) is essential to avoid inadequate or unnecessary empirical antibiotic therapy. Microbiological confirmation takes 24 to 48 h. The use of screening methods, such as cytometry and automated microscopic analysis of urine sediment, allows the rapid prediction of negative samples. In addition, matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) is a widely established technique in clinical microbiology laboratories used to identify microorganisms. We evaluated the ability of MALDI-TOF MS to identify microorganisms from direct urine samples and the predictive value of automated analyzers for the identification of microorganisms in urine by MALDI-TOF MS. A total of 451 urine samples from patients with suspected UTIs were first analyzed using the Sysmex UF-1000i flow cytometer, an automatic sediment analyzer with microscopy (SediMax), culture, and then processed by MALDI-TOF MS with a simple triple-centrifuged procedure to obtain a pellet that was washed and centrifuged and finally applied directly to the MALDI-TOF MS plate. The organisms in 336 samples were correctly identified, mainly those with Gram-negative bacteria (86.10%). No microorganisms were misidentified, and no Candida spp. were correctly identified. Regarding the data from autoanalyzers, the best bacteriuria cutoffs were 1,000 and 200 U/μl for UF-1000i and SediMax, respectively. It was concluded that the combination of a urine screening method and MALDI-TOF MS provided a reliable identification from urine samples, especially in those containing Gram-negative bacteria. PMID:26818668
Renaudin, H.; Cauvin, E.; Del Prá Netto Machado, L.; Tricot, A.; Benoit, F.; Treilles, M.; Bébéar, C.
Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) recently emerged as a technology for the identification of bacteria. In this study, we aimed to evaluate its applicability to human and ruminant mycoplasmal identification, which can be demanding and time-consuming when using phenotypic or molecular methods. In addition, MALDI-TOF MS was tested as a subtyping tool for certain species. A total of 29 main spectra (MSP) from 10 human and 13 ruminant mycoplasma (sub)species were included in a mycoplasma MSP database to complete the Bruker MALDI Biotyper database. After broth culture and protein extraction, MALDI-TOF MS was applied for the identification of 119 human and 143 ruminant clinical isolates that were previously identified by antigenic or molecular methods and for subcultures of 73 ruminant clinical specimens that potentially contained several mycoplasma species. MALDI-TOF MS resulted in accurate (sub)species-level identification with a score of ≥1.700 for 96% (251/262) of the isolates. The phylogenetically closest (sub)species were unequivocally distinguished. Although mixtures of the strains were reliably detected up to a certain cellular ratio, only the predominant species was identified from the cultures of polymicrobial clinical specimens. For typing purposes, MALDI-TOF MS proved to cluster Mycoplasma bovis and Mycoplasma agalactiae isolates by their year of isolation and genome profiles, respectively, and Mycoplasma pneumoniae isolates by their adhesin P1 type. In conclusion, MALDI-TOF MS is a rapid, reliable, and cost-effective method for the routine identification of high-density growing mycoplasmal species and shows promising prospects for its capacity for strain typing. PMID:23903545
Prospective evaluation of a matrix-assisted laser desorption ionization-time of flight mass spectrometry system in a hospital clinical microbiology laboratory for identification of bacteria and yeasts: a bench-by-bench study for assessing the impact on time to identification and cost-effectiveness.
Tan, K E; Ellis, B C; Lee, R; Stamper, P D; Zhang, S X; Carroll, K C
Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has been found to be an accurate, rapid, and inexpensive method for the identification of bacteria and yeasts. Previous evaluations have compared the accuracy, time to identification, and costs of the MALDI-TOF MS method against standard identification systems or commercial panels. In this prospective study, we compared a protocol incorporating MALDI-TOF MS (MALDI protocol) with the current standard identification protocols (standard protocol) to determine the performance in actual practice using a specimen-based, bench-by-bench approach. The potential impact on time to identification (TTI) and costs had MALDI-TOF MS been the first-line identification method was quantitated. The MALDI protocol includes supplementary tests, notably for Streptococcus pneumoniae and Shigella, and indications for repeat MALDI-TOF MS attempts, often not measured in previous studies. A total of 952 isolates (824 bacterial isolates and 128 yeast isolates) recovered from 2,214 specimens were assessed using the MALDI protocol. Compared with standard protocols, the MALDI protocol provided identifications 1.45 days earlier on average (P < 0.001). In our laboratory, we anticipate that the incorporation of the MALDI protocol can reduce reagent and labor costs of identification by $102,424 or 56.9% within 12 months. The model included the fixed annual costs of the MALDI-TOF MS, such as the cost of protein standards and instrument maintenance, and the annual prevalence of organisms encountered in our laboratory. This comprehensive cost analysis model can be generalized to other moderate- to high-volume laboratories.
Íñigo, Melania; Coello, Andreu; Fernández-Rivas, Gema; Rivaya, Belén; Hidalgo, Jessica; Quesada, María Dolores; Ausina, Vicente
Early diagnosis of urinary tract infections (UTIs) is essential to avoid inadequate or unnecessary empirical antibiotic therapy. Microbiological confirmation takes 24 to 48 h. The use of screening methods, such as cytometry and automated microscopic analysis of urine sediment, allows the rapid prediction of negative samples. In addition, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is a widely established technique in clinical microbiology laboratories used to identify microorganisms. We evaluated the ability of MALDI-TOF MS to identify microorganisms from direct urine samples and the predictive value of automated analyzers for the identification of microorganisms in urine by MALDI-TOF MS. A total of 451 urine samples from patients with suspected UTIs were first analyzed using the Sysmex UF-1000iflow cytometer, an automatic sediment analyzer with microscopy (SediMax), culture, and then processed by MALDI-TOF MS with a simple triple-centrifuged procedure to obtain a pellet that was washed and centrifuged and finally applied directly to the MALDI-TOF MS plate. The organisms in 336 samples were correctly identified, mainly those with Gram-negative bacteria (86.10%). No microorganisms were misidentified, and noCandidaspp. were correctly identified. Regarding the data from autoanalyzers, the best bacteriuria cutoffs were 1,000 and 200 U/μl for UF-1000iand SediMax, respectively. It was concluded that the combination of a urine screening method and MALDI-TOF MS provided a reliable identification from urine samples, especially in those containing Gram-negative bacteria.
Quiles Melero, Inmaculada; Peláez, Teresa; Rezusta López, Antonio; Garcia-Rodríguez, Julio
MALDI-TOF (matrix-assisted laser desorption ionization time-of-flight) mass spectrometry (MS) is becoming an essential tool in most microbiology laboratories. At present, by using a characteristic fungal profile obtained from whole cells or through simple extraction protocols, MALDI-TOF MS allows the identification of pathogenic fungi with a high performance potential. This methodology decreases the laboratory turnaround time, optimizing the detection of mycoses. This article describes the state-of-the-art of the use of MALDI-TOF MS for the detection of human clinical fungal pathogens in the laboratory and discusses the future applications of this technology, which will further improve routine mycological diagnosis.
Cassagne, Carole; Ranque, Stéphane; Normand, Anne-Cécile; Fourquet, Patrick; Thiebault, Sandrine; Planard, Chantal; Hendrickx, Marijke; Piarroux, Renaud
Background MALDI-TOF MS recently emerged as a valuable identification tool for bacteria and yeasts and revolutionized the daily clinical laboratory routine. But it has not been established for routine mould identification. This study aimed to validate a standardized procedure for MALDI-TOF MS-based mould identification in clinical laboratory. Materials and Methods First, pre-extraction and extraction procedures were optimized. With this standardized procedure, a 143 mould strains reference spectra library was built. Then, the mould isolates cultured from sequential clinical samples were prospectively subjected to this MALDI-TOF MS based-identification assay. MALDI-TOF MS-based identification was considered correct if it was concordant with the phenotypic identification; otherwise, the gold standard was DNA sequence comparison-based identification. Results The optimized procedure comprised a culture on sabouraud-gentamicin-chloramphenicol agar followed by a chemical extraction of the fungal colonies with formic acid and acetonitril. The identification was done using a reference database built with references from at least four culture replicates. For five months, 197 clinical isolates were analyzed; 20 were excluded because they were not identified at the species level. MALDI-TOF MS-based approach correctly identified 87% (154/177) of the isolates analyzed in a routine clinical laboratory activity. It failed in 12% (21/177), whose species were not represented in the reference library. MALDI-TOF MS-based identification was correct in 154 out of the remaining 156 isolates. One Beauveria bassiana was not identified and one Rhizopus oryzae was misidentified as Mucor circinelloides. Conclusions This work's seminal finding is that a standardized procedure can also be used for MALDI-TOF MS-based identification of a wide array of clinically relevant mould species. It thus makes it possible to identify moulds in the routine clinical laboratory setting and opens new avenues
Lin, Wei-Hung; Hwang, Jyh-Chang; Tseng, Chin-Chung; Chang, Yu-Tzu; Wu, An-Bang; Yan, Jing-Jou
Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) and conventional standard methods were compared for time to pathogen identification and impact on clinical outcomes in peritoneal dialysis-related peritonitis patients. The MALDI-TOF MS method identified the causative microorganisms earlier (average time saved, 64 h for all pathogens), and patients had a shorter hospital stay (mean ± standard deviation, 5.2 ± 4.8 days versus 8.2 ± 4.5 days, P = 0.001). PMID:26912750
Girard, Victoria; Mailler, Sandrine; Polsinelli, Sophie; Jacob, Delphine; Saccomani, Marie Christine; Celliere, Beatrice; Monnin, Valerie; van Belkum, Alex; Hagen, Ferry; Meis, Jacques F; Durand, Géraldine
We here show adequate species identification for bacterial isolates of the genus Nocardia spp. through VITEK mass spectrometry. Application of a specific sample preparation method in combination with a robust matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) database leads to 94% accurate identification to the species level on a set of 164 isolates. The possibility to identify Nocardia spp. using MALDI-TOF MS will be available in the next release of VITEK MS update (IVD Version 3.0).
Purvis, Tanya J; Krouse, Donna; Miller, Dawn; Livengood, Julia; Thirumalapura, Nagaraja R; Tewari, Deepanker
Brucella canis was recovered from dogs that were canine brucellosis suspect by blood culture using a modified lysis method. Organism identity was established by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The instrument-provided security library identified the isolates as Brucella species. The isolates were further identified as B. canis with the help of phenotypic and genotypic characteristics. The mass spectral profiles from characterized B. canis isolates, when added to the MALDI-TOF MS standard reference library, allowed successful presumptive identification of B. canis.
In this study, we describe and evaluate the performance of a simple and rapid mass spectral method for screening fish plasma for estrogen-responsive biomarkers using matrix assisted laster desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) couopled with a short...
Nebbak, Amira; Willcox, Alexandra C; Bitam, Idir; Raoult, Didier; Parola, Philippe; Almeras, Lionel
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.
Mass spectrometric cleaved amplified polymorphic sequence (MS-CAPS) is a method for detecting genes using a combination of short PCR and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). MS-CAPS can identify a single nucleotide polymorphism (SNP) in less than one hour and is suitable for plants, animals, bacteria, and food.
Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been developed for high-throughput screening of naturally-occurring mixtures of rhamnolipids from Pseudomonas spp. Mono- and di-rhamnolipids are readily distinguished by characteristic molecular adduct i...
Kolecka, Anna; Khayhan, Kantarawee; Groenewald, Marizeth; Theelen, Bart; Arabatzis, Michael; Velegraki, Aristea; Kostrzewa, Markus; Mares, Mihai; Taj-Aldeen, Saad J; Boekhout, Teun
Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) was used for an extensive identification study of arthroconidial yeasts, using 85 reference strains from the CBS-KNAW yeast collection and 134 clinical isolates collected from medical centers in Qatar, Greece, and Romania. The test set included 72 strains of ascomycetous yeasts (Galactomyces, Geotrichum, Saprochaete, and Magnusiomyces spp.) and 147 strains of basidiomycetous yeasts (Trichosporon and Guehomyces spp.). With minimal preparation time, MALDI-TOF MS proved to be an excellent diagnostic tool that provided reliable identification of most (98%) of the tested strains to the species level, with good discriminatory power. The majority of strains were correctly identified at the species level with good scores (>2.0) and seven of the tested strains with log score values between 1.7 and 2.0. The MALDI-TOF MS results obtained were consistent with validated internal transcribed spacer (ITS) and/or large subunit (LSU) ribosomal DNA sequencing results. Expanding the mass spectrum database by increasing the number of reference strains for closely related species, including those of nonclinical origin, should enhance the usefulness of MALDI-TOF MS-based diagnostic analysis of these arthroconidial fungi in medical and other laboratories.
Stępień-Pyśniak, Dagmara; Hauschild, Tomasz; Różański, Paweł; Marek, Agnieszka
The aim of this study was to explore the accuracy and feasibility of Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) in identifying bacteria from environmental sources, as compared to rpoA gene sequencing, and to evaluate the occurrence of bacteria of the genus Enterococcus in wild birds. In addition, a phyloproteomic analysis of certain Enterococcus species with spectral relationships was performed. The enterococci were isolated from 25 species of wild birds in central Europe (Poland). Proteomic (MALDI-TOF MS) and genomic (rpoA gene sequencing) methods were used to identify isolates. Using MALDI-TOF MS, all 54 (100%) isolates were identified as Enterococcus spp. Among these, 51 (94.4%) isolates were identified to the species level - log(score) > or =2.0, and three isolates (5.6%) were identified at a level of probable genus identification - log(score) 1.88-1.927. Phylogenetic analysis based on rpoA sequences confirmed that all enterococci had been correctly identified. Enterococcus faecalis was the most prevalent enterococcal species (50%) and Enterococcus faecium (33.3%) the second most frequent species, followed by Enterococcus hirae (9.3%), Enterococcus durans (3.7%) and Enterococcus casseliflavus (3.7%). The phyloproteomic analysis of the spectral profiles of the isolates showed that MALDI-TOF MS is able to differentiate among similar species of the genus Enterococcus.
Abreu, Lucas M; Moreira, Gláucia M; Ferreira, Douglas; Rodrigues-Filho, Edson; Pfenning, Ludwig H
We assessed the species diversity among 45 strains of Clonostachys from different substrates and localities in Brazil using molecular phylogenetics, and compared the results with the phenotypic classification of strains obtained from matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Phylogenetic analyses were based on beta tubulin (Tub), ITS-LSU rDNA, and a combined Tub-ITS DNA dataset. MALDI-TOF MS analyses were performed using intact conidia and conidiophores of strains cultivated on oatmeal agar and 4% malt extract agar. Six known species were identified: Clonostachys byssicola, Clonostachys candelabrum, Clonostachys pseudochroleuca, Clonostachys rhizophaga, Clonostachys rogersoniana, and Clonostachys rosea. Two clades and two singleton lineages did not correspond to known species represented in the reference DNA dataset and were identified as Clonostachys sp. 1-4. Multivariate cluster analyses of MALDI-TOF MS data classified the strains into eight clusters and three singletons, corresponding to the ten identified species plus one additional cluster containing two strains of C. rogersoniana that split from the other co-specific strains. The consistent results of MALDI-TOF MS supported the identification of strains assigned to C. byssicola and C. pseudochroleuca, which did not form well supported clades in all phylogenetic analyses, but formed distinct clusters in the MALDI-TOF dendrograms.
Development of methods for rapid identification of foodborne bacteria is an important area of analytical science and food safety. MALDI-TOF-MS has been utilized to rapidly identify pathogens including foodborne bacteria. Identification typically involves detection of high copy cytosolic proteins i...
Elamin, Wael F; Ball, David; Millar, Michael
Unbiased species-level identification of coagulase-negative staphylococci (CoNS) using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) identified Staphylococcus lugdunensis to be a more commonly isolated CoNS in our laboratory than previously observed. It has also highlighted the possibility of vertical transmission.
Evidence of genotypic diversity among Candida auris isolates by multilocus sequence typing, matrix-assisted laser desorption ionization time-of-flight mass spectrometry and amplified fragment length polymorphism.
Prakash, A; Sharma, C; Singh, A; Kumar Singh, P; Kumar, A; Hagen, F; Govender, N P; Colombo, A L; Meis, J F; Chowdhary, A
Candida auris is a multidrug-resistant nosocomial bloodstream pathogen that has been reported from Asian countries and South Africa. Herein, we studied the population structure and genetic relatedness among 104 global C. auris isolates from India, South Africa and Brazil using multilocus sequence typing (MLST), amplified fragment length polymorphism (AFLP) fingerprinting and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). RPB1, RPB2 and internal transcribed spacer (ITS) and D1/D2 regions of the ribosomal DNA were sequenced for MLST. Further, genetic variation and proteomic assessment was carried out using AFLP and MALDI-TOF MS, respectively. Both MLST and AFLP typing clearly demarcated two major clusters comprising Indian and Brazilian isolates. However, the South African isolates were randomly distributed, suggesting different genotypes. MALDI-TOF MS spectral profiling also revealed evidence of geographical clustering but did not correlate fully with the genotyping methods. Notably, overall the population structure of C. auris showed evidence of geographical clustering by all the three techniques analysed. Antifungal susceptibility testing by the CLSI microbroth dilution method revealed that fluconazole had limited activity against 87% of isolates (MIC90, 64 mg/L). Also, MIC90 of AMB was 4 mg/L. Candida auris is emerging as an important yeast pathogen globally and requires reproducible laboratory methods for identification and typing. Evaluation of MALDI-TOF MS as a typing method for this yeast is warranted.
Bourafa, N.; Loucif, L.; Boutefnouchet, N.; Rolain, J.-M.
Enterococcus hirae is a zoonotic pathogen rarely isolated from human infections. This case is the first description of E. hirae causing urinary tract infection in a diabetic man with benign prostatic hyperplasia from Algeria. The clinical isolate was identified by MALDI-TOF MS and displayed a multisensitivity antibiotic profile. PMID:26543562
Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has proven to be a powerful tool for taxonomic resolution of microorganisms. In this proof-of-concept study, we assessed the effectiveness of this technique to track the current gene sequence-based phylogenet...
Beauruelle, Clémence; Le Bars, Hervé; Pougnet, Laurence; Lesecq, Ludovic; Buisson, Philippe
Fastidiosipila sanguinis is a Gram-positive anaerobic coccus. We report the first case of osteitis implicating this species. The strain was accurately identified by 16S rRNA sequence analysis, matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) identification having failed. The reservoir remained unclear; an endogenous origin is suspected. PMID:24789188
High-yield, glycolipid-based biosurfactants are of increasing interest for use in environmentally benign cleaning or emulsifying agents. We have developed a MALDI-TOF/MS screen for the rapid analysis of several types of biosurfactants, including various acylated rhamnolipids in Pseudomonas extracts...
High-yield, glycolipid-based biosurfactants are of increasing interest for use in environmentally benign cleaning or emulsifying agents. We have developed a MALDI-TOF/MS screen for the rapid analysis of several types of biosurfactants, including various acylated rhamnolipids in Pseudomonas extracts...
Tsai, Chia-Ju; Chen, Yen-Ling; Feng, Chia-Hsien
This study explored dispersive liquid-liquid microextraction for extraction and concentration of lipoic acid in human urine. To improve the detection of lipoic acid by both capillary liquid chromatography (CapLC) with UV detection and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), microwave-assisted derivatization with 4-bromomethyl-6,7-dimethoxycoumarin was performed to render lipoic acid chromophores for UV detection and also high ionization efficiency in MALDI. All parameters that affected lipoic acid extraction and derivatization from urine were investigated and optimized. In the analyses of human urine samples, the two methods had a linear range of 0.1-20 μM with a correlation coefficient of 0.999. The detection limits of CapLC-UV and MALDI-TOF MS were 0.03 and 0.02 μM (S/N ≧ 3), respectively. The major metabolites of lipoic acid, including 6,8-bismethylthio-octanoic acid, 4,6-bismethylthio-hexanoic acid, and 2,4-bismethylthio-butanoic acid were also extracted by dispersive liquid-liquid microextraction and detected by MALDI-TOF MS. The minor metabolites (undetectable by MALDI-TOF MS), bisnorlipoic acid and tetranorlipoic acid were also extracted by dispersive liquid-liquid microextraction and identified with an LTQ Orbitrap mass spectrometer. After dispersive liquid-liquid microextraction and microwave-assisted derivatization, all lipoic acid derivatizations and metabolites were structurally confirmed by LTQ Orbitrap.
Feng, Chia-Hsien; Huang, Hao-Yi; Lu, Chi-Yu
Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been broadly applied to analyze high-molecular-weight compound (such as polymer or proteomic research) but seldom used for low-molecular-weight compound analysis. The objective of this study is the development of a simple analytical method for the determination of the concentration of tripeptide glutathione (GSH) by MALDI-TOF MS. Unfortunately, GSH could not be detected directly by MALDI-TOF MS. Our method is based on the derivatization of GSH with 4-bromomethyl-6,7-dimethoxycoumarin (BrDMC) in acetonitrile using potassium hydroxide (KOH) as a base catalyst. After simple extraction step, the supernatant is spotted on a target plate, mixed with matrix α-cyano-4-hydroxycinnamic acid (CHCA) and then detected by MALDI-TOF MS. Some parameters affecting the derivatization of GSH were investigated, such as the concentration of BrDMC, KOH, different base catalyst, and reaction time, etc. The regression equations of GSH derivative possessed good linearity (r≧0.995) over the range of 1.0-100.0 μM. The relative standard deviation (R.S.D.) and relative error (R.E.) values in intra- and inter-day assays were below 13%, which showed good precision and accuracy. This proposed method was successfully applied to monitor the concentration of GSH in human blood at micro-scale level.
Cenci, Lucia; Anesi, Andrea; Busato, Mirko; Guella, Graziano; Bossi, Alessandra Maria
Molecularly imprinted polymers (MIPs) were combined to MALDI-TOF-MS to evaluate a selective enrichment (SE) method for the determination of clinically relevant biomarkers from complex biological samples. The concept was proven with the myocardial injury marker Troponin I (cTnI). In a first part, MIP materials entailed for the recognition of cTnI epitopes (three peptides selected) were prepared and characterized in dimensions (0.7-2μm), dissociation constants (58-817 nM), kinetics of binding (5-60 min), binding capacity (ca. 1.5 µg/mg polymer), imprinting factors (3 > IF > 5) and selectivity for the peptide epitope. Then, the MIPs, incubated with cTnI peptides and spotted on the target with the DHB matrix, were assayed for the desorption of the peptides in MALDI-TOF-MS. The measured detection limit was ca. 300 femtomols. Finally, the MIP-SE MALDI-TOF-MS was tested for its ability to enrich in the cTnI peptides from a complex sample, mimic of serum (i.e. 81 peptides of digested albumin). The MIP-SE MALDI-TOF-MS successfully enriched in cTnI peptides from the complex sample proving the technique could offer a flexible platform to prepare entailed materials suitable for diagnostic purposes.
Ramalinga, Uma; Clogston, Jeffrey D; Patri, Anil K; Simpson, John T
Determining the molecular weight of nanoparticles can be challenging. The molecular weight characterization of dendrimers, for example, with varying covalent and noncovalent modifications is critical to their use as therapeutics. As such, we describe in this chapter a protocol for the analysis of these molecules by matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS).
Tracz, Dobryan M; Tyler, Andrea D; Cunningham, Ian; Antonation, Kym S; Corbett, Cindi R
A high-quality custom database of MALDI-TOF mass spectral profiles was developed with the goal of improving clinical diagnostic identification of high-consequence bacterial pathogens. A biomarker discovery method is presented for identifying and evaluating MALDI-TOF MS spectra to potentially differentiate biothreat bacteria from less-pathogenic near-neighbour species.
Kamboj, Kamal; Pancholi, Preeti
Mycobacteria cause significant morbidity in humans. Rapid and accurate mycobacterial identification is important for improvement of patient outcomes. However, identification may be challenging due to the slow and fastidious growth of mycobacteria. Several diagnostic methods, such as biochemical, sequencing, and probe methods, are used for mycobacterial identification. We compared the matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) Biotyper system (Bruker Daltonics) to 16S rRNA/hsp65 sequencing and/or DNA probes (Gen-Probe) for mycobacterial identification. One hundred seventy-eight mycobacterial isolates grown on solid and/or broth medium were included in the study. MALDI-TOF MS identified 93.8% of the mycobacteria isolates accurately to the species level and 98.3% to the genus level, independent of the type of medium used for isolation. The identification of mycobacteria directly from cultures using MALDI-TOF MS allows for precise identification in an hour compared to traditional biochemical and phenotypic methods that can take weeks or probes and sequencing that may take a few hours. Identification by MALDI-TOF MS potentially reduces the turnaround time and cost, thereby saving resources within the health care system. PMID:23804379
Bacteriophage cell lysis of Shiga toxin-producing Escherichia coli for top-down proteomic identification of Shiga toxin 1 & 2 using matrix-assisted laser desorption/ionization tandem time-of-light mass spectrometry
RATIONALE: Analysis of bacteria by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) often relies upon sample preparation methods that result in cell lysis, e.g. bead-beating. However, Shiga toxin-producing Escherichia coli (STEC) can undergo bacteriophage...
Kedney, Mollie G.; Strunk, Kevin B.; Giaquinto, Lisa M.; Wagner, Jennifer A.; Pollack, Sidney; Patton, Walter A.
Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS or simply MALDI) has become ubiquitous in the identification and analysis of biomacromolecules. As a technique that allows for the molecular weight determination of otherwise nonvolatile molecules, MALDI has had a profound impact in the molecular…
Analysis of a world-wide collection of strains of Trichoderma asperellum using multilocus genealogies of four genomic regions (tef1, rbp2, act, ITS1, 2, 5.8s), sequence polymorphism-derived (SPD) markers, matrix-assisted laser desorption/ionisation–time of flight mass spectrometry (MALDI-TOF MS) of ...
Liu, Hai-Long; Chang, Yu-Jie; Fan, Ting; Gu, Zhi-Yuan
Stable 2-D metal-organic framework nanosheets were utilized as a superior clean-background matrix for MALDI-TOF MS analysis of small biomolecules and pollutants in both positive and negative ion modes. The matrix could unusually afford up to 1000 mM of the salt concentrations in the monitoring of the enzymatic hydrolysis of neurotransmitter acetylcholine.
Hrabák, Jaroslav; Chudácková, Eva; Walková, Radka
Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has been successfully applied as an identification procedure in clinical microbiology and has been widely used in routine laboratory practice because of its economical and diagnostic benefits. The range of applications of MALDI-TOF MS has been growing constantly, from rapid species identification to labor-intensive proteomic studies of bacterial physiology. The purpose of this review is to summarize the contribution of the studies already performed with MALDI-TOF MS concerning antibiotic resistance and to analyze future perspectives in this field. We believe that current research should continue in four main directions, including the detection of antibiotic modifications by degrading enzymes, the detection of resistance mechanism determinants through proteomic studies of multiresistant bacteria, and the analysis of modifications of target sites, such as ribosomal methylation. The quantification of antibiotics is suggested as a new approach to study influx and efflux in bacterial cells. The results of the presented studies demonstrate that MALDI-TOF MS is a relevant tool for the detection of antibiotic resistance and opens new avenues for both clinical and experimental microbiology.
Kuckova, S; Nemec, I; Hynek, R; Hradilova, J; Grygar, T
Two methods of analysis of organic components of colour layers of art works have been tested: IR microspectroscopy of indigo, Cu-phthalocyanine, and Prussian blue, and MALDI-TOF-MS of proteinaceous binders and a protein-containing red dye. The IR spectra distortion common for smooth outer surfaces and polished cross sections of colour layer of art works is suppressed by reflectance measurement of microtome slices. The detection limit of the three blue pigments examined is approximately 0.3 wt% in reference colour layers in linseed oil binder with calcite as extender and lead white as a drying agent. The sensitivity has been sufficient to identify Prussian blue in repaints on a Gothic painting. MALDI-TOF-MS has been used to identify proteinaceous binders in two historical paintings, namely isinglass (fish glue) and rabbit glue. MALDI-TOF-MS has also been proposed for identification of an insect red dye, cochineal carmine, according to its specific protein component. The enzymatic cleavage with trypsin before MALDI-TOF-MS seems to be a very gentle and specific way of dissolution of the colour layers highly polymerised due to very long aging of old, e.g. medieval, samples.
This document provides a storage plan for portable exhausters POR-004 SKID B, POR-005 SKID C, AND POR-006 SKID D. The exhausters will be stored until they are needed by the TWRS (Tank Waste Remediation Systems) Saltwell Pumping Program. The storage plan provides criteria for portable exhauster storage, periodic inspections during storage, and retrieval from storage.
Lo, Cheikh I; Fall, Bécaye; Sambe-Ba, Bissoume; Diawara, Silman; Gueye, Mamadou W; Mediannikov, Oleg; Sokhna, Cheikh; Faye, Ngor; Diemé, Yaya; Wade, Boubacar; Raoult, Didier; Fenollar, Florence
Our team in Europe has developed the routine clinical laboratory identification of microorganisms by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS). To evaluate the utility of MALDI-TOF MS in tropical Africa in collaboration with local teams, we installed an apparatus in the Hôpital Principal de Dakar (Senegal), performed routine identification of isolates, and confirmed or completed their identification in France. In the case of discordance or a lack of identification, molecular biology was performed. Overall, 153/191 (80.1%) and 174/191 (91.1%) isolates yielded an accurate and concordant identification for the species and genus, respectively, with the 2 different MALDI-TOF MSs in Dakar and Marseille. The 10 most common bacteria, representing 94.2% of all bacteria routinely identified in the laboratory in Dakar (Escherichia coli, Klebsiella pneumoniae, Streptococcus agalactiae, Acinetobacter baumannii, Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus haemolyticus, Enterobacter cloacae, Enterococcus faecalis, and Staphylococcus epidermidis) were accurately identified with the MALDI-TOF MS in Dakar. The most frequent misidentification in Dakar was at the species level for Achromobacter xylosoxidans, which was inaccurately identified as Achromobacter denitrificans, and the bacteria absent from the database, such as Exiguobacterium aurientacum or Kytococcus schroeteri, could not be identified. A few difficulties were observed with MALDI-TOF MS for Bacillus sp. or oral streptococci. 16S rRNA sequencing identified a novel bacterium, "Necropsobacter massiliensis." The robust identification of microorganisms by MALDI-TOF MS in Dakar and Marseille demonstrates that MALDI-TOF MS can be used as a first-line tool in clinical microbiology laboratories in tropical countries.
Sala-Comorera, Laura; Vilaró, Carles; Galofré, Belén; Blanch, Anicet R; García-Aljaro, Cristina
The study of bacterial communities throughout a drinking water treatment plant could provide a basic understanding of the effects of water processing that could then be used to improve the management of such plants. However, it is necessary to develop new analytical techniques that are sufficiently efficient, robust and fast for their effective and useful application in routine analysis. The aim of this study is therefore to assess the performance of matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS), as compared to the PhenePlate™ system, for routine analysis in a drinking water treatment plant. To this end we studied a total of 277 colonies isolated in different seasons and from different points throughout the water treatment process, including: raw water, sand filtration, ultrafiltration, reverse osmosis and chlorination. The colonies were analysed using MALDI-TOF MS by direct deposition of the cells on the plate. The colonies were also biochemically fingerprinted using the PhenePlate™ system, clustered according to their similarity and a representative strain was selected for 16S rRNA gene sequencing and API(®) gallery-based identification. The use of MALDI-TOF MS was reliable compared to the PhenePlate™ system and has the advantage of being faster and relatively cheap. Bacteria typing by MALDI-TOF MS is therefore a promising method to replace conventional routine phenotypic methods for the identification of bacteria in drinking water laboratories, thanks to its robustness. The major limiting factor for MALDI-TOF MS is the lack of a suitable mass spectra database; although each laboratory can develop its own library. This methodology will provide a tracking tool for companies to use in risk management and the detection of possible failures in both the water treatment processes and the distribution network, as well as offering characterization of the intrinsic microbial populations.
Triest, David; Stubbe, Dirk; De Cremer, Koen; Piérard, Denis; Normand, Anne-Cécile; Piarroux, Renaud; Detandt, Monique; Hendrickx, Marijke
The rates of infection with Fusarium molds are increasing, and a diverse number of Fusarium spp. belonging to different species complexes can cause infection. Conventional species identification in the clinical laboratory is time-consuming and prone to errors. We therefore evaluated whether matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is a useful alternative. The 289 Fusarium strains from the Belgian Coordinated Collections of Microorganisms (BCCM)/Institute of Hygiene and Epidemiology Mycology (IHEM) culture collection with validated sequence-based identities and comprising 40 species were used in this study. An identification strategy was developed, applying a standardized MALDI-TOF MS assay and an in-house reference spectrum database. In vitro antifungal testing was performed to assess important differences in susceptibility between clinically relevant species/species complexes. We observed that no incorrect species complex identifications were made by MALDI-TOF MS, and 82.8% of the identifications were correct to the species level. This success rate was increased to 91% by lowering the cutoff for identification. Although the identification of the correct species complex member was not always guaranteed, antifungal susceptibility testing showed that discriminating between Fusarium species complexes can be important for treatment but is not necessarily required between members of a species complex. With this perspective, some Fusarium species complexes with closely related members can be considered as a whole, increasing the success rate of correct identifications to 97%. The application of our user-friendly MALDI-TOF MS identification approach resulted in a dramatic improvement in both time and accuracy compared to identification with the conventional method. A proof of principle of our MALDI-TOF MS approach in the clinical setting using recently isolated Fusarium strains demonstrated its validity.
Alby, Kevin; Kerr, Alan; Jones, Melissa; Gilligan, Peter H.
Matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry (MS) is an emerging technology for rapid identification of bacterial and fungal isolates. In comparison to conventional methods, this technology is much less labor intensive and can provide accurate and reliable results in minutes from a single isolated colony. We compared the cost of performing the bioMérieux Vitek MALDI-TOF MS with conventional microbiological methods to determine the amount saved by the laboratory by converting to the new technology. Identification costs for 21,930 isolates collected between April 1, 2013, and March 31, 2014, were directly compared for MALDI-TOF MS and conventional methodologies. These isolates were composed of commonly isolated organisms, including commonly encountered aerobic and facultative bacteria and yeast but excluding anaerobes and filamentous fungi. Mycobacterium tuberculosis complex and rapidly growing mycobacteria were also evaluated for a 5-month period during the study. Reagent costs and a total cost analysis that included technologist time in addition to reagent expenses and maintenance service agreement costs were analyzed as part of this study. The use of MALDI-TOF MS equated to a net savings of $69,108.61, or 87.8%, in reagent costs annually compared to traditional methods. When total costs are calculated to include technologist time and maintenance costs, traditional identification would have cost $142,532.69, versus $68,886.51 with the MALDI-TOF MS method, resulting in a laboratory savings of $73,646.18, or 51.7%, annually by adopting the new technology. The initial cost of the instrument at our usage level would be offset in about 3 years. MALDI-TOF MS not only represents an innovative technology for the rapid and accurate identification of bacterial and fungal isolates, it also provides a significant cost savings for the laboratory. PMID:25994167
Hazen, Tracy H.; Martinez, Robert J.; Chen, Yanfeng; Lafon, Patricia C.; Garrett, Nancy M.; Parsons, Michele B.; Bopp, Cheryl A.; Sullards, M. Cameron; Sobecky, Patricia A.
Vibrio parahaemolyticus is a pathogenic marine bacterium that is the main causative agent of bacterial seafood-borne gastroenteritis in the United States. An increase in the frequency of V. parahaemolyticus-related infections during the last decade has been attributed to the emergence of an O3:K6 pandemic clone in 1995. The diversity of the O3:K6 pandemic clone and its serovariants has been examined using multiple molecular techniques including multilocus sequence analysis, pulsed-field gel electrophoresis, and group-specific PCR analysis. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has become a powerful tool for rapidly distinguishing between related bacterial species. In the current study, we demonstrate the development of a whole-cell MALDI-TOF MS method for the distinction of V. parahaemolyticus from other Vibrio spp. We identified 30 peaks that were present only in the spectra of the V. parahaemolyticus strains examined in this study that may be developed as MALDI-TOF MS biomarkers for identification of V. parahaemolyticus. We detected variation in the MALDI-TOF spectra of V. parahaemolyticus strains isolated from different geographical locations and at different times. The MALDI-TOF MS spectra of the V. parahaemolyticus strains examined were distinct from those of the other Vibrio species examined including the closely related V. alginolyticus, V. harveyi, and V. campbellii. The results of this study demonstrate the first use of whole-cell MALDI-TOF MS analysis for the rapid identification of V. parahaemolyticus. PMID:19749061
Implementation of Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry in Routine Clinical Laboratories Improves Identification of Coagulase-Negative Staphylococci and Reveals the Pathogenic Role of Staphylococcus lugdunensis
Riegel, Philippe; Lavigne, Thierry; Lefebvre, Nicolas; Grandpré, Nicolas; Hansmann, Yves; Jaulhac, Benoit; Prévost, Gilles; Schramm, Frédéric
The use of matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) for staphylococcal identification is now considered routine in laboratories compared with the conventional phenotypical methods previously used. We verified its microbiological relevance for identifying the main species of coagulase-negative staphylococci (CoNS) by randomly selecting 50 isolates. From 1 January 2007 to 31 August 2008, 12,479 staphylococci were isolated with phenotypic methods, of which 4,594 were identified as Staphylococcus aureus and 7,885 were coagulase negative staphylococci. Using MALDI-TOF MS from 1 January 2011 to 31 August 2012, 14,913 staphylococci were identified, with 5,066 as S. aureus and 9,847 as CoNS. MALDI-TOF MS allowed the identification of approximately 85% of the CoNS strains, whereas only 14% of the CoNS strains were identified to the species level with phenotypic methods because they were often considered contaminants. Furthermore, the use of MALDI-TOF MS revealed the occurrence of recently characterized Staphylococcus species, such as S. pettenkoferi, S. condimenti, and S. piscifermentans. Microbiological relevance analysis further revealed that some species displayed a high rate of microbiological significance, i.e., 40% of the S. lugdunensis strains included in the analysis were associated with infection risk. This retrospective microbiological study confirms the role of MALDI-TOF MS in clinical settings for the identification of staphylococci with clinical consequences. The species distribution reveals the occurrence of the recently identified species S. pettenkoferi and putative virulent species, including S. lugdunensis. PMID:25878345
Implementation of Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry in Routine Clinical Laboratories Improves Identification of Coagulase-Negative Staphylococci and Reveals the Pathogenic Role of Staphylococcus lugdunensis.
Argemi, Xavier; Riegel, Philippe; Lavigne, Thierry; Lefebvre, Nicolas; Grandpré, Nicolas; Hansmann, Yves; Jaulhac, Benoit; Prévost, Gilles; Schramm, Frédéric
The use of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for staphylococcal identification is now considered routine in laboratories compared with the conventional phenotypical methods previously used. We verified its microbiological relevance for identifying the main species of coagulase-negative staphylococci (CoNS) by randomly selecting 50 isolates. From 1 January 2007 to 31 August 2008, 12,479 staphylococci were isolated with phenotypic methods, of which 4,594 were identified as Staphylococcus aureus and 7,885 were coagulase negative staphylococci. Using MALDI-TOF MS from 1 January 2011 to 31 August 2012, 14,913 staphylococci were identified, with 5,066 as S. aureus and 9,847 as CoNS. MALDI-TOF MS allowed the identification of approximately 85% of the CoNS strains, whereas only 14% of the CoNS strains were identified to the species level with phenotypic methods because they were often considered contaminants. Furthermore, the use of MALDI-TOF MS revealed the occurrence of recently characterized Staphylococcus species, such as S. pettenkoferi, S. condimenti, and S. piscifermentans. Microbiological relevance analysis further revealed that some species displayed a high rate of microbiological significance, i.e., 40% of the S. lugdunensis strains included in the analysis were associated with infection risk. This retrospective microbiological study confirms the role of MALDI-TOF MS in clinical settings for the identification of staphylococci with clinical consequences. The species distribution reveals the occurrence of the recently identified species S. pettenkoferi and putative virulent species, including S. lugdunensis.
Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for the identification of Burkholderia pseudomallei from Asia and Australia and differentiation between Burkholderia species.
Suttisunhakul, Vichaya; Pumpuang, Apinya; Ekchariyawat, Peeraya; Wuthiekanun, Vanaporn; Elrod, Mindy G; Turner, Paul; Currie, Bart J; Phetsouvanh, Rattanaphone; Dance, David A B; Limmathurotsakul, Direk; Peacock, Sharon J; Chantratita, Narisara
Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is increasingly used for rapid bacterial identification. Studies of Burkholderia pseudomallei identification have involved small isolate numbers drawn from a restricted geographic region. There is a need to expand the reference database and evaluate B. pseudomallei from a wider geographic distribution that more fully captures the extensive genetic diversity of this species. Here, we describe the evaluation of over 650 isolates. Main spectral profiles (MSP) for 26 isolates of B. pseudomallei (N = 5) and other Burkholderia species (N = 21) were added to the Biotyper database. MALDI-TOF MS was then performed on 581 B. pseudomallei, 19 B. mallei, 6 B. thailandensis and 23 isolates representing a range of other bacterial species. B. pseudomallei originated from northeast and east Thailand (N = 524), Laos (N = 12), Cambodia (N = 14), Hong Kong (N = 4) and Australia (N = 27). All 581 B. pseudomallei were correctly identified, with 100% sensitivity and specificity. Accurate identification required a minimum inoculum of 5 x 107 CFU/ml, and identification could be performed on spiked blood cultures after 24 hours of incubation. Comparison between a dendrogram constructed from MALDI-TOF MS main spectrum profiles and a phylogenetic tree based on recA gene sequencing demonstrated that MALDI-TOF MS distinguished between B. pseudomallei and B. mallei, while the recA tree did not. MALDI-TOF MS is an accurate method for the identification of B. pseudomallei, and discriminates between this and other related Burkholderia species.
Hazen, Tracy H; Martinez, Robert J; Chen, Yanfeng; Lafon, Patricia C; Garrett, Nancy M; Parsons, Michele B; Bopp, Cheryl A; Sullards, M Cameron; Sobecky, Patricia A
Vibrio parahaemolyticus is a pathogenic marine bacterium that is the main causative agent of bacterial seafood-borne gastroenteritis in the United States. An increase in the frequency of V. parahaemolyticus-related infections during the last decade has been attributed to the emergence of an O3:K6 pandemic clone in 1995. The diversity of the O3:K6 pandemic clone and its serovariants has been examined using multiple molecular techniques including multilocus sequence analysis, pulsed-field gel electrophoresis, and group-specific PCR analysis. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has become a powerful tool for rapidly distinguishing between related bacterial species. In the current study, we demonstrate the development of a whole-cell MALDI-TOF MS method for the distinction of V. parahaemolyticus from other Vibrio spp. We identified 30 peaks that were present only in the spectra of the V. parahaemolyticus strains examined in this study that may be developed as MALDI-TOF MS biomarkers for identification of V. parahaemolyticus. We detected variation in the MALDI-TOF spectra of V. parahaemolyticus strains isolated from different geographical locations and at different times. The MALDI-TOF MS spectra of the V. parahaemolyticus strains examined were distinct from those of the other Vibrio species examined including the closely related V. alginolyticus, V. harveyi, and V. campbellii. The results of this study demonstrate the first use of whole-cell MALDI-TOF MS analysis for the rapid identification of V. parahaemolyticus.
Xu, Jing; Xu, Bin; Tang, Chuanhao; Li, Xiaoyan; Qin, Haifeng; Wang, Weixia; Wang, Hong; Wang, Zhongyuan; Li, Liangliang; Li, Zhihua; Gao, Hongjun
Background. Diagnoses of malignant pleural effusion (MPE) are a crucial problem in clinics. In our study, we compared the peptide profiles of MPE and tuberculosis pleural effusion (TPE) to investigate the value of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) in diagnosis of MPE. Material and Methods. The 46 MPE and 32 TPE were randomly assigned to training set and validation set. Peptides were isolated by weak cation exchange magnetic beads and peaks in the m/z range of 800–10000 Da were analyzed. Comparing the peptide profile between 30 MPE and 22 TPE samples in training set by ClinProTools software, we screened the specific biomarkers and established a MALDI-TOF-MS classification of MPE. Finally, the other 16 MPE and 10 TPE were included to verify the model. We additionally determined carcinoembryonic antigen (CEA) in MPE and TPE samples using electrochemiluminescent immunoassay method. Results. Five peptide peaks (917.37 Da, 4469.39 Da, 1466.5 Da, 4585.21 Da, and 3216.87 Da) were selected to separate MPE and TPE by MALDI-TOF-MS. The sensitivity, specificity, and accuracy of the classification were 93.75%, 100%, and 96.15%, respectively, after blinded test. The sensitivity of CEA was significantly lower than MALDI-TOF-MS classification (P = 0.035). Conclusions. The results indicate MALDI-TOF-MS is a potential method for diagnosing MPE. PMID:28386154
Lo, Cheikh I.; Fall, Bécaye; Sambe-Ba, Bissoume; Diawara, Silman; Gueye, Mamadou W.; Mediannikov, Oleg; Sokhna, Cheikh; Faye, Ngor; Diemé, Yaya; Wade, Boubacar; Raoult, Didier; Fenollar, Florence
Our team in Europe has developed the routine clinical laboratory identification of microorganisms by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS). To evaluate the utility of MALDI-TOF MS in tropical Africa in collaboration with local teams, we installed an apparatus in the Hôpital Principal de Dakar (Senegal), performed routine identification of isolates, and confirmed or completed their identification in France. In the case of discordance or a lack of identification, molecular biology was performed. Overall, 153/191 (80.1%) and 174/191 (91.1%) isolates yielded an accurate and concordant identification for the species and genus, respectively, with the 2 different MALDI-TOF MSs in Dakar and Marseille. The 10 most common bacteria, representing 94.2% of all bacteria routinely identified in the laboratory in Dakar (Escherichia coli, Klebsiella pneumoniae, Streptococcus agalactiae, Acinetobacter baumannii, Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus haemolyticus, Enterobacter cloacae, Enterococcus faecalis, and Staphylococcus epidermidis) were accurately identified with the MALDI-TOF MS in Dakar. The most frequent misidentification in Dakar was at the species level for Achromobacter xylosoxidans, which was inaccurately identified as Achromobacter denitrificans, and the bacteria absent from the database, such as Exiguobacterium aurientacum or Kytococcus schroeteri, could not be identified. A few difficulties were observed with MALDI-TOF MS for Bacillus sp. or oral streptococci. 16S rRNA sequencing identified a novel bacterium, “Necropsobacter massiliensis.” The robust identification of microorganisms by MALDI-TOF MS in Dakar and Marseille demonstrates that MALDI-TOF MS can be used as a first-line tool in clinical microbiology laboratories in tropical countries. PMID:26716681
Jay-Russell, M T; Bates, A; Harden, L; Miller, W G; Mandrell, R E
We report the isolation of Campylobacter species from the same population of feral swine that was investigated in San Benito County, California, during the 2006 spinach-related Escherichia coli O157:H7 outbreak. This is the first survey of Campylobacter in a free-ranging feral swine population in the United States. Campylobacter species were cultured from buccal and rectal-anal swabs, colonic faeces and tonsils using a combination of selective enrichment and antibiotic-free membrane filtration methods. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS, Bruker Daltonics, Inc., Billerica, MA, USA) was used to identify species followed by confirmatory multiplex PCR or 16S rRNA sequencing. Genetic relatedness of Campylobacter jejuni and Campylobacter coli strains was determined by multilocus sequence typing (MLST) and porA allele sequencing. Altogether, 12 (40%) of 30 feral swine gastrointestinal and oral cavity specimens were positive, and six species were isolated: Campylobacter coli, Campylobacter fetus, Campylobacter hyointestinalsis, Campylobacter jejuni, Campylobacter lanienae and Campylobacter sputorum. Campylobacter jejuni subtypes were closely related to MLST sequence type 21 (ST-21) and had identical porA sequences. Campylobacter coli subtypes were unrelated to isolates in the pubMLST/porA database. This feral swine population lived in close association with a 'grassfed' beef cattle herd adjacent to spinach and other leafy green row crop fields. The findings underscore the importance of protecting raw vegetable crops from faecal contamination by wild or feral animals. The study also illustrates a potential risk of Campylobacter exposure for hunters during handling and processing of wild swine meat.
Blosser, Sara J.; Drake, Steven K.; Andrasko, Jennifer L.; Henderson, Christina M.; Kamboj, Kamal; Antonara, Stella; Mijares, Lilia; Conville, Patricia; Frank, Karen M.; Harrington, Susan M.; Balada-Llasat, Joan-Miquel
This multicenter study analyzed Nocardia spp., including extraction, spectral acquisition, Bruker matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) identification, and score interpretation, using three Nocardia libraries, the Bruker, National Institutes of Health (NIH), and The Ohio State University (OSU) libraries, and compared the results obtained by each center. A standardized study protocol, 150 Nocardia isolates, and NIH and OSU Nocardia MALDI-TOF MS libraries were distributed to three centers. Following standardized culture, extraction, and MALDI-TOF MS analysis, isolates were identified using score cutoffs of ≥2.0 for species/species complex-level identification and ≥1.8 for genus-level identification. Isolates yielding a score of <2.0 underwent a single repeat extraction and analysis. The overall score range for all centers was 1.3 to 2.7 (average, 2.2 ± 0.3), with common species generally producing higher average scores than less common ones. Score categorization and isolate identification demonstrated 86% agreement between centers; 118 of 150 isolates were correctly identified to the species/species complex level by all centers. Nine strains (6.0%) were not identified by any center, and six (4.0%) of these were uncommon species with limited library representation. A categorical score discrepancy among centers occurred for 21 isolates (14.0%). There was an overall benefit of 21.2% from repeat extraction of low-scoring isolates and a center-dependent benefit for duplicate spotting (range, 2 to 8.7%). Finally, supplementation of the Bruker Nocardia MALDI-TOF MS library with both the OSU and NIH libraries increased the genus-level and species-level identification by 18.2% and 36.9%, respectively. Overall, this study demonstrates the ability of diverse clinical microbiology laboratories to utilize MALDI-TOF MS for the rapid identification of clinically relevant Nocardia spp. and to implement MALDI-TOF MS
Seng, Piseth; Rolain, Jean-Marc; Fournier, Pierre Edouard; La Scola, Bernard; Drancourt, Michel; Raoult, Didier
MALDI-TOF-mass spectrometry (MS) has been successfully adapted for the routine identification of microorganisms in clinical microbiology laboratories in the past 10 years. This revolutionary technique allows for easier and faster diagnosis of human pathogens than conventional phenotypic and molecular identification methods, with unquestionable reliability and cost-effectiveness. This article will review the application of MALDI-TOF-MS tools in routine clinical diagnosis, including the identification of bacteria at the species, subspecies, strain and lineage levels, and the identification of bacterial toxins and antibiotic-resistance type. We will also discuss the application of MALDI-TOF-MS tools in the identification of Archaea, eukaryotes and viruses. Pathogenic identification from colony-cultured, blood-cultured, urine and environmental samples is also reviewed.
García, Patricia; Allende, Fidel; Legarraga, Paulette; Huilcaman, Marcos; Solari, Sandra
Bacterial identification is important for the proper treatment of infected patients hospitalized with serious infections especially in critical care units. Identification by conventional methods used in microbiology laboratories takes at least 16 hours since a culture is positive. The introduction of mass spectrometry, specifically MALDI-TOF MS (matrix-assisted laser desorption/ ionization time-of-flight mass spectrometer) in the microbiology laboratory could mean a radical change in the identification accuracy, turn around time (6 minutes per bacteria) and cost (about 5 times cheaper than conventional identification). Since its introduction in clinical microbiology laboratories in 2008, many reports about its usefulness in identifying microorganisms from colonies, as well as directly from positive blood cultures and urine samples have been published. This review describes MALDI-TOF MS methodology, its identification performance for bacteria (aerobic and anaerobic), mycobacterium and yeasts, its future applications in microbiology and its main disadvantages.
Cheng, Wern-Cherng; Jan, I-Shiow; Chen, Jong-Min; Teng, Shih-Hua; Teng, Lee-Jene; Sheng, Wang-Huei; Ko, Wen-Chien
Among 56 blood isolates of Vibrio species identified by sequencing analysis of 16S rRNA and rpoB genes, the Bruker Biotyper matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) system correctly identified all isolates of Vibrio vulnificus (n = 20), V. parahaemolyticus (n = 2), and V. fluvialis (n = 1) but none of the isolates of serogroup non-O1/O139 (non-serogroup O1, non-O139) V. cholerae (n = 33) to the species level. All of these serogroup non-O1/O139 V. cholerae isolates were correctly identified using the newly created MALDI-TOF MS database. PMID:25740773
Soro-Yao, Amenan A; Schumann, Peter; Thonart, Philippe; Djè, Koffi M; Pukall, Rüdiger
Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) protein analysis, automated ribotyping, and phenotypic tests (e.g., cell morphology, gas production from glucose, growth and acid production on homofermemtative-heterofermentative differential (HHD) agar medium, sugar fermentation patterns) were used to identify 23 lactic acid bacteria (LAB) isolated from fermented cereal foods available in Abidjan, Côte d’Ivoire. Pediococcus acidilactici (56.5%), Lactobacillus fermentum (30.4%), L. salivarius (4.3%), P. pentosaceus (4.3%) and L. plantarum subsp. plantarum (4.3%) were the species and subspecies identified. Protein based identification was confirmed by automated ribotyping for selected isolates and was similar to that provided by the phenotypic characterization. MALDI-TOF MS protein analysis provided a high level of discrimination among the isolates and could be used for the rapid screening of LAB starter cultures. PMID:25279017
Jaskolla, Thorsten; Fuchs, Beate; Karas, Michael; Schiller, Jürgen
Phosphatidylethanolamines (PEs) are abundant lipid constituents of the cellular membrane. The amino group of PEs exhibits high reactivity with hypochlorous acid that is generated under inflammatory conditions in vivo. The analysis of the resulting PE mono- and dichloramines is of significant interest since these species represent important mediators of lipid peroxidation. We have shown in a previous communication that mass spectrometric detection of PE chloramines is only possible with ESI MS, whereas MALDI-TOF MS fails to detect these products if standard matrices are used. In this work we demonstrate that the detection of PE chloramines is also possible by MALDI-TOF MS if 4-chloro-alpha-cyanocinnamic acid is used as matrix. The underlying processes leading to ionization of these species will be discussed in detail. Both, experimental and theoretical studies taking into account possible intramolecular rearrangements were performed to clarify these aspects.
Gray, Timothy J; Thomas, Lee; Olma, Tom; Iredell, Jonathan R; Chen, Sharon C-A
The application of matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) mass spectrometry (MS) directly to blood culture broth has potential to identify bloodstream infection earlier and facilitate timely management. We prospectively tested a novel, rapid, and inexpensive in-house spin-lysis protocol with formic acid extraction and compared MALDI-TOF MS identification of Gram-negative bacteria with traditional phenotypic methods (Phoenix™) directly from 318 BACTEC™ (Becton Dickinson, Franklin Lakes, USA) blood cultures. The MS score was ≥1.7 in 268 (91.8%) monomicrobial broths, with concordance to genus and species level of 100% and 97.0%, respectively. MALDI-TOF MS still has limited capacity to detect all species in polymicrobial broths.
Chen, Hui; He, Meiyu; Pei, Jian; He, Haifeng
Quantitative analyses of synthetic polymers were accomplished using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS). Many factors have hindered the development of quantitative measurement of polymers via MALDI TOF MS, e.g., laser power, matrix, cation salt, and cocrystallization. By probing the optimal conditions, two sets of polymers were studied. Fair repeatability of the samples ensures acceptable results. In set 1, two poly(ethylene glycols) with different end groups showed equal desorption/ionization efficiencies. Two synthetic polymers in set 2 with different chemical properties resulted in different MALDI responses. Good linearity was achieved by plotting the relationship between the sample concentration ratio and the total signal intensity ratio in both sets.
Kurien, Biji T; Scofield, R Hal
Matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI TOF MS) is extremely sensitive to minor impurities in tryptic peptide digests, resulting in suppression of the signal obtained. Therefore, it becomes necessary to purify the sample, especially those samples that fail to yield good mass spectra. Here, we describe a simple protocol using polyvinylidene difluoride (PVDF) membrane for purifying tryptic peptides prior to mass spectrometric analysis. The tryptic digest is spotted on a PVDF membrane, air-dried, and washed. The membrane is then extracted with trifluoroacetic acid/acetonitrile and the extract is then subjected to MALDI TOF MS. Using this procedure, we were able to identify a cross-reactive D1 autoantigen on the surface of neutrophils that bound antibodies targeting Ro 60 autoantigen in systemic lupus erythematosus.
Veloso, A; Leiza, J R
Cross-contamination from the polymeric material of the vial caps used in size exclusion chromatography (SEC) and high-performance liquid chromatography autosamplers in the low molar mass (LMM) range (<2000 Da) of polymer samples was demonstrated with the help of matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) analysis. It is shown that polyethylene (PE) caps, if exposed to either short or long periods in tetrahydrofuran solvent, might extract oligomers that contaminate the LMM end of the SEC chromatograms. On the other hand, polytetrafluoroethylene (PTFE)/silicone caps have shown to be inert if used properly; namely, the PTFE part in contact with the sample solution. However, care should be taken if accidentally the silicon part is exposed to the solution because silicone oligomer peaks have been detected by MALDI-TOF MS analysis that might mislead the results.
Zhu, Xiaoqing; Wu, Lianglan; Mungra, Divyesh C; Xia, Sijing; Zhu, Jin
In matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS), the analysis capability, especially for small molecules, is often compromised by the addition of organic matrices due to the existence of background signals. Herein we report a new detection method on the utility of core-shell nanoparticles (CSNPs) as energy transfer structure in LDI-TOF-MS. The LDI-TOF-MS based on gold-silica core-shell nanoparticles with ultrathin silica shell of 2-4 nm (Au@utSiO(2) CSNPs) was effectively applied to the analysis of many compounds, especially for small functional molecules and polymers, which was more promising than MALDI-TOF-MS.
Pföhler, Claudia; Hollemeyer, Klaus; Heinzle, Elmar; Altmeyer, Wolfgang; Graeber, Stefan; Müller, Cornelia S L; Stark, Alexandra; Jager, Sven Uwe; Tilgen, Wolfgang
The incidence and prevalence of onychomycosis are rising worldwide. Common diagnostic techniques often lack sensitivity or specificity. Differentiation between non-infectious nail disorders is frequently not possible. The aim of this study was to establish a better diagnostic routine procedure based on modern mass spectrometric peptide analysis techniques. One hundred and fifty-five nail samples from 145 patients with clinically suspected onychomycosis (n = 96, 62%) and without onychomycosis [e.g. nail psoriasis or nail dystrophy resulting from eczema (n = 59, 38%)] were investigated using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) peptide mass fingerprinting in comparison with standard techniques. We demonstrated that MALDI-TOF MS represents a precise, robust and fast tool in diagnostic investigation of nail disorders, which is superior to common standard methods.
Li, Yejia; Hoskins, Jessica N; Sreerama, Subramanya G; Grayson, Michael A; Grayson, Scott M
Recent advances in the resolving power of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) enable the detailed characterization of linear homopolymers, and in particular provide invaluable data for the determination of their end-group functionalities. With the growing importance of macromolecular coupling reactions in building complex polymer architectures, the ability to accurately monitor end-group transformations is becoming increasingly important for synthetic polymer chemists. This tutorial demonstrates the application of MALDI-TOF MS in determining both end-group functionalities and their transformations for linear homopolymers. Examples of both polycaprolactone and polystyrene are examined, and the strengths and weaknesses of various approaches to data analysis are given.
Pusztahelyi, Tünde; Szabó, Judit; Dombrádi, Zsuzsanna; Kovács, Szilvia; Pócsi, István
Listeria monocytogenes is a foodborne pathogen, and the detection and differentiation of this bacterium from the nonpathogenic Listeria species are of great importance to the food industry. Differentiation of Listeria species is very difficult, even with the sophisticated MALDI-TOF MS technique because of the close genetic relationship of the species and the usual gene transfer. The present paper emphasizes the difficulties of the differentiation through the standardized detection and confirmation according to ISO 11290-1:1996 and basic available L. monocytogenes detection methods and tests (such as API Listeria test, MALDI-TOF MS analysis, and hly gene PCR). With the increase of reports on the pathogenesis of atypical Listeria strains in humans, the significance of species level determination has become questionable, especially in food quality control, and the detection of pathogenic characteristics seems to be more relevant.
Amazonian vegetable oils and fats: fast typification and quality control via triacylglycerol (TAG) profiles from dry matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry fingerprinting.
Saraiva, Sérgio A; Cabral, Elaine C; Eberlin, Marcos N; Catharino, Rodrigo R
Amazonian oils and fats display unique triacylglycerol (TAG) profiles and, because of their economic importance as renewable raw materials and use by the cosmetic and food industries, are often subject to adulteration and forgery. Representative samples of these oils (andiroba, Brazil nut, buriti, and passion fruit) and fats (cupuaçu, murumuru, and ucuúba) were characterized without pre-separation or derivatization via dry (solvent-free) matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Characteristic profiles of TAG were obtained for each oil and fat. Dry MALDI-TOF MS provides typification and direct and detailed information, via TAG profiles, of their variable combinations of fatty acids. A database from spectra could be developed and may be used for their fast and reliable typification, application screening, and quality control.
Lu, Y; Qi, Y X; Zhang, H; Zhang, H Q; Pu, J J; Xie, Y X
To establish a proteomic reference map of Musa acuminate Colla (banana) leaf, we separated and identified leaf proteins using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and mass spectrometry (MS). Tryptic digests of 44 spots were subjected to peptide mass fingerprinting (PMF) by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS. Three spots that were not identified by MALDI-TOF MS analysis were identified by searching against the NCBInr, SwissProt, and expressed sequence tag (EST) databases. We identified 41 unique proteins. The majority of the identified leaf proteins were found to be involved in energy metabolism. The results indicate that 2D-PAGE is a sensitive and powerful technique for the separation and identification of Musa leaf proteins. A summary of the identified proteins and their putative functions is discussed.
Wen, Xi; Gu, Yan; Chen, Feng
Detection of pubertal growth peak is vital in orthodontic treatment timing and planning. Gingival crevicular fluid (GCF) contains abundant proteins from different sources and has been proven to be an ideal source of biomarkers. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) is an advanced technique that can detect low-molecular-weight peptides with high sensitivity and resolution. The aim of this research was to identify novel candidate biomarkers in GCF to help the diagnosis of pubertal growth peak by MALDI-TOF/MS. Results showed that the peak intensities of six peptides were significantly different between two groups: 1660.2 Da, 1783.0 Da, 2912.5 Da, 4178.6 Da, 5064.9 Da, and 6108.9 Da and are considered to be potential candidate biomarkers to identify pubertal growth peak. Further studies are needed to identify sequence information of these candidate biomarkers. PMID:27869666
Hattan, Stephen J.; Parker, Kenneth C.; Vestal, Marvin L.; Yang, Jane Y.; Herold, David A.; Duncan, Mark W.
Measurement of glycated hemoglobin is widely used for the diagnosis and monitoring of diabetes mellitus. Matrix assisted laser desorption/ionization (MALDI) time of flight (TOF) mass spectrometry (MS) analysis of patient samples is used to demonstrate a method for quantitation of total glycation on the β-subunit of hemoglobin. The approach is accurate and calibrated with commercially available reference materials. Measurements were linear (R2 > 0.99) across the clinically relevant range of 4% to 20% glycation with coefficients of variation of ≤ 2.5%. Additional and independent measurements of glycation of the α-subunit of hemoglobin are used to validate β-subunit glycation measurements and distinguish hemoglobin variants. Results obtained by MALDI-TOF MS were compared with those obtained in a clinical laboratory using validated HPLC methodology. MALDI-TOF MS sample preparation was minimal and analysis times were rapid making the method an attractive alternative to methodologies currently in practice.
Sammra, Osama; Balbutskaya, Anna; Hijazin, Muaz; Nagib, Samy; Alber, Jörg; Lämmler, Christoph; Abdulmawjood, Amir; Prenger-Berninghoff, Ellen; Timke, Markus; Kostrzewa, Markus; Siebert, Ursula
Arcanobacterium phocisimile, a newly described species with the type strain A. phocisimile 2698T isolated from a vaginal swab of a harbour seal and four additional A. phocisimile strains also isolated from four harbour seals could reliably be identified by phenotypic properties, by matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS), and by sequencing the genomic targets 16S rDNA and 16S-23S rDNA intergenic spacer region and the genes rpoB and gap. The A. phocisimile strains investigated in the present study were isolated together with several other bacterial species indicating that the pathogenic importance of A. phocisimile remains unclear. However, the detection of peptidic spectra by MALDI-TOF MS and the presented phenotypic and genotypic approach might help to identify A. phocisimile in future. PMID:26464945
Peel, Trisha N; Cole, Nicolynn C; Dylla, Brenda L; Patel, Robin
Identification of pathogen(s) associated with prosthetic joint infection (PJI) is critical for patient management. Historically, many laboratories have not routinely identified organisms such as coagulase-negative staphylococci to the species level. The advent of matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) has enhanced clinical laboratory capacity for accurate species-level identification. The aim of this study was to describe the species-level identification of microorganisms isolated from periprosthetic tissue and fluid specimens using MALDI-TOF MS alongside other rapid identification tests in a clinical microbiology laboratory. Results of rapid identification of bacteria isolated from periprosthetic joint fluid and/or tissue specimens were correlated with clinical findings at Mayo Clinic, Rochester, Minnesota, between May 2012 and May 2013. There were 178 PJI and 82 aseptic failure (AF) cases analyzed, yielding 770 organisms (median, 3/subject; range, 1-19/subject). MALDI-TOF MS was employed for the identification of 455 organisms (59%) in 197 subjects (123 PJIs and 74 AFs), with 89% identified to the species level using this technique. Gram-positive bacteria accounted for 68% and 93% of isolates in PJI and AF, respectively. However, the profile of species associated with infection compared to specimen contamination differed. Staphylococcus aureus and Staphylococcus caprae were always associated with infection, Staphylococcus epidermidis and Staphylococcus lugdunensis were equally likely to be a pathogen or a contaminant, whereas the other coagulase-negative staphylococci were more frequently contaminants. Most streptococcal and Corynebacterium isolates were pathogens. The likelihood that an organism was a pathogen or contaminant differed with the prosthetic joint location, particularly in the case of Propionibacterium acnes. MALDI-TOF MS is a valuable tool for the identification of bacteria isolated from patients
Angelakis, Emmanouil; Million, Matthieu; Henry, Mireille; Raoult, Didier
Probiotic food is manufactured by adding probiotic strains simultaneously with starter cultures in fermentation tanks. Here, we investigate the accuracy and feasibility of matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) for bacterial identification at the species level in probiotic food and yoghurts. Probiotic food and yoghurts were cultured in Columbia and Lactobacillus specific agar and tested by quantitative real-time PCR (qPCR) for the detection and quantification of Lactobacillus sp. Bacterial identification was performed by MALDI-TOF analysis and by amplification and sequencing of tuf and 16S rDNA genes. We tested 13 probiotic food and yoghurts and we identified by qPCR that they presented 10(6) to 10(7) copies of Lactobacillus spp. DNA/g. All products contained very large numbers of living bacteria varying from 10(6) to 10(9) colony forming units/g. These bacteria were identified as Lactobacillus casei, Lactococcus lactis, Bifidobacterium animalis, Lactobacillus delbrueckii, and Streptococcus thermophilus. MALDI-TOF MS presented 92% specificity compared to the molecular assays. In one product we found L. lactis, instead of Bifidus spp. which was mentioned on the label and for another L. delbrueckii and S. thermophilus instead of Bifidus spp. MALDI-TOF MS allows a rapid and accurate bacterial identification at the species level in probiotic food and yoghurts. Although the safety and functionality of probiotics are species and strain dependent, we found a discrepancy between the bacterial strain announced on the label and the strain identified. Practical Application: MALDI-TOF MS is rapid and specific for the identification of bacteria in probiotic food and yoghurts. Although the safety and functionality of probiotics are species and strain dependent, we found a discrepancy between the bacterial strain announced on the label and the strain identified.
Clinical and microbiological features of a cystic fibrosis patient chronically colonized with Pandoraea sputorum identified by combining 16S rRNA sequencing and matrix-assisted laser desorption ionization-time of flight mass spectrometry.
Fernández-Olmos, A; Morosini, M I; Lamas, A; García-Castillo, M; García-García, L; Cantón, R; Máiz, L
Clonal isolates identified as various nonfermentative Gram-negative bacilli over a 5-year period from sputum cultures of a 30-year-old cystic fibrosis patient were successfully reidentified as Pandoraea sputorum by combining 16S rRNA sequencing and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Decreased lung function improved after 1 year of azithromycin and inhaled 7%-hypertonic saline treatment.
Application of Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry for Identification of the Fastidious Pediatric Pathogens Aggregatibacter, Eikenella, Haemophilus, and Kingella
Powell, Eleanor A.; Blecker-Shelly, Deborah; Montgomery, Sandra
The accuracy of matrix-assisted laser desorption–ionization time of flight mass spectrometry (MALDI-TOF MS) in the identification of Haemophilus, Aggregatibacter, Cardiobacterium, Eikenella, and Kingella (HACEK) species was compared to that of phenotypic methods (Remel RapID and Vitek 2). Overall, Vitek MS correctly identified more isolates, incorrectly identified fewer isolates, and failed to identify fewer isolates than both phenotypic methods. PMID:23966506
Celandroni, Francesco; Salvetti, Sara; Gueye, Sokhna Aissatou; Mazzantini, Diletta; Lupetti, Antonella; Senesi, Sonia; Ghelardi, Emilia
The soil-related Bacillus and Paenibacillus species have increasingly been implicated in various human diseases. Nevertheless, their identification still poses problems in the clinical microbiology laboratory and, with the exception of Bacillus anthracis and Bacillus cereus, little is known on their pathogenicity for humans. In this study, we evaluated the use of matrix-assisted laser desorption-ionization time of flight mass spectrometry (MALDI-TOF MS) in the identification of clinical isolates of these genera and conducted genotypic and phenotypic analyses to highlight specific virulence properties. Seventy-five clinical isolates were subjected to biochemical and MALDI-TOF MS identification. 16S rDNA sequencing and supplemental tests were used to solve any discrepancies or failures in the identification results. MALDI-TOF MS significantly outperformed classical biochemical testing for correct species identification and no misidentification was obtained. One third of the collected strains belonged to the B. cereus species, but also Bacillus pumilus and Bacillus subtilis were isolated at high rate. Antimicrobial susceptibility testing showed that all the B. cereus, B. licheniformis, B. simplex, B. mycoides, Paenibacillus glucanolyticus and Paenibacillus lautus isolates are resistant to penicillin. The evaluation of toxin/enzyme secretion, toxin-encoding genes, motility, and biofilm formation revealed that B. cereus displays the highest virulence potential. However, although generally considered nonpathogenic, most of the other species were shown to swim, swarm, produce biofilms, and secrete proteases that can have a role in bacterial virulence. In conclusion, MALDI-TOF MS appears useful for fast and accurate identification of Bacillus and Paenibacillus strains whose virulence properties make them of increasing clinical relevance.
induced dissociation (CID), Electron transfer dissociation , or post-source decay (PSD) of ionized tryptic peptides derived from bacterial proteins , i.e...fingerprints of the bacterial proteins , i.e. MALDI-TOF-MS technique, or amino acid sequences, from tandem MS/MS analysis, of proteins from collision ...William 2002), present in biological threat microorganisms. The vast amount of protein and peptide data generated from a typical LC- tandem MS
Oviaño, Marina; Barba, Maria José; Fernández, Begoña; Ortega, Adriana; Aracil, Belén; Oteo, Jesús; Campos, José
A rapid and sensitive (100%) matrix-assisted laser desorption ionization−time of flight mass spectrometry (MALDI-TOF MS) assay was developed to detect OXA-48-type producers, using 161 previously characterized clinical isolates. Ertapenem was monitored to detect carbapenem resistance, and temocillin was included in the assay as a marker for OXA-48-producers. Structural analysis of temocillin is described. Data are obtained within 60 min. PMID:26677247
Tomazi, Tiago; Gonçalves, Juliano Leonel; Barreiro, Juliana Regina; de Campos Braga, Patrícia Aparecida; Prada e Silva, Luis Felipe; Eberlin, Marcos Nogueira; dos Santos, Marcos Veiga
Coagulase-negative staphylococci (CoNS) are among the main pathogens causing bovine intramammary infection (IMI) in many countries. However, one of the limitations related to the specific diagnosis of CoNS is the lack of an accurate, rapid, and convenient method that can differentiate the bacterial species comprising this group. The aim of this study was to evaluate the ability of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) to accurately identify CoNS species in dairy cow IMI. In addition, the study aimed to determine the frequency of CoNS species causing bovine IMI. A total of 108 bacterial isolates were diagnosed as CoNS by microbiological cultures from two milk samples collected from 21 dairy herds; the first sample was collected at the cow level (i.e., 1,242 composite samples from all quarters), while the second sample was collected at the mammary quarter level (i.e., 1,140 mammary samples collected from 285 cows). After CoNS isolation was confirmed by microbiological culture for both samples, all CoNS isolates (n=108) were genotypically differentiated by PCR restriction fragment length polymorphism (RFLP) analysis of a partial groEL gene sequence and subjected to the MALDI-TOF MS identification procedure. MALDI-TOF MS correctly identified 103 (95.4%) of the CoNS isolates identified by PCR-RFLP at the species level. Eleven CoNS species isolated from bovine IMI were identified by PCR-RFLP, and the most prevalent species was Staphylococcus chromogenes (n=80; 74.1%). In conclusion, MALDI-TOF MS may be a reliable alternative method for differentiating CoNS species causing bovine IMI.
Amukele, Timothy K; Street, Jeff; Carroll, Karen; Miller, Heather; Zhang, Sean X
Unmanned aerial vehicles (UAVs) could potentially be used to transport microbiological specimens. To examine the impact of UAVs on microbiological specimens, blood and sputum culture specimens were seeded with usual pathogens and flown in a UAV for 30 ± 2 min. Times to recovery, colony counts, morphologies, and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS)-based identifications of the flown and stationary specimens were similar for all microbes studied.
with respect to molecular weight, with the dominant species present being methylated derivatives of the polycyclic aromatic hydrocarbons ( PAH ...carbon products. 3. Synthesize pure alkylated polycyclic aromatic hydrocarbons ( PAHs ) with molecular weights (mol wts) in the range of 300-2000. These...response of matrix-assisted, laser desorption ionization time-of- flight mass spectrometry (MALDI-TOF-MS) to alkylated PAH compounds. 4. With MALDI
Cools, Piet; Haelters, Jan; Lopes dos Santos Santiago, Guido; Claeys, Geert; Boelens, Jerina; Leroux-Roels, Isabel; Vaneechoutte, Mario; Deschaght, Pieter
Whale strandings remain poorly understood, although bacterial infections have been suggested to contribute. We isolated Edwardsiella tarda from the blood of a stranded sperm whale. The pathogen was identified with MALDI-TOF MS, confirmed by 16S rRNA gene sequencing and quantified in blood by qPCR. We report the first case of sepsis in a sperm whale. The zoonotic potential of E. tarda and the possible role of bacterial infections in the enigmatic strandings of cetaceans are discussed.
Bloemberg, Guido V.; Zbinden, Reinhard; Böttger, Erik C.; Hombach, Michael
Reported matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) identification rates of Gram-positive rods (GPR) are low compared to identification rates of Gram-positive cocci. In this study, three sample preparation methods were compared for MALDI-TOF MS identification of 190 well-characterized GPR strains: direct transfer, direct transfer-formic acid preparation, and ethanol-formic acid extraction. Using the interpretation criteria recommended by the manufacturer, identification rates were significantly higher for direct transfer-formic acid preparation and ethanol-formic acid extraction than for direct transfer. Reducing the species cutoff from 2.0 to 1.7 significantly increased species identification rates. In a subsequent prospective study, 215 clinical GPR isolates were analyzed by MALDI-TOF MS, and the results were compared to those for identification using conventional methods, with discrepancies being resolved by 16S rRNA and rpoB gene analysis. Using the direct transfer-formic acid preparation and a species cutoff of 1.7, congruencies on the genus and species levels of 87.4% and 79.1%, respectively, were achieved. In addition, the rate of nonidentified isolates dropped from 12.1% to 5.6% when using an extended database, i.e., the Bruker database amended by reference spectra of the 190 GPR of the retrospective study. Our data demonstrate three ways to improve GPR identification by the Bruker MALDI Biotyper, (i) optimize sample preparation using formic acid, (ii) reduce cutoff scores for species identification, and (iii) expand the database. Based on our results, we suggest an identification algorithm for the clinical laboratory combining MALDI-TOF MS with nucleic acid sequencing. PMID:24452159
Garcia, Estefânia F; Luciano, Winnie A; Xavier, Danilo E; da Costa, Whyara C A; de Sousa Oliveira, Kleber; Franco, Octávio L; de Morais Júnior, Marcos A; Lucena, Brígida T L; Picão, Renata C; Magnani, Marciane; Saarela, Maria; de Souza, Evandro L
This study aimed to identify lactic acid bacteria (LAB) in byproducts of fruit (Malpighia glabra L., Mangifera indica L., Annona muricata L., and Fragaria vesca L.) pulp processing. Fifty strains of LAB were identified using matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) and 16S rRNA gene sequence (16S rRNA) analysis. Species belonging to Lactobacillus genus were the predominant LAB in all fruit pulp processing byproducts. The average congruency between the MALDI-TOF MS and 16S rRNA in LAB species identification reached 86%. Isolates of L. plantarum, L. brevis, L. pentosus, L. lactis and L. mesenteroides were identified with 100% congruency. MALDI-TOF MS and 16S rRNA analysis presented 86 and 100% efficiency of LAB species identification, respectively. Further, five selected Lactobacillus strains (L. brevis 59, L. pentosus 129, L. paracasei 108, L. plantarum 49, and L. fermentum 111) were evaluated for desirable probiotic-related properties and growth behavior on two different cultivation media. The exposure to pH 2.0 sharply decreased the counts of the different Lactobacillus strains after a 1 or 2 h incubation, while varied decreases were noted after 3 h of exposure to pH 3.0. Overall, the exposure to pH 5.0 and to bile salts (0.15, 0.30, and 1.00%) did not decrease the counts of the Lactobacillus strains. All tested Lactobacillus strains presented inhibitory activity against Staphylococcus aureus, Salmonella Typhimurium, Salmonella Enteritidis, Listeria monocytogenes and Escherichia coli, and presented variable susceptibility to different antibiotics. The selected Lactobacillus strains presented satisfactory and reproducible growth behavior. In conclusion, MALDI-TOF MS and 16S rRNA analysis revealed high efficiency and congruency for LAB species identification, and the selected Lactobacillus strains may be candidates for further investigation of novel probiotic strains.
Månsson, Viktor; Resman, Fredrik; Kostrzewa, Markus; Nilson, Bo; Riesbeck, Kristian
Haemophilus influenzae type b (Hib) is, in contrast to non-type b H. influenzae, associated with severe invasive disease, such as meningitis and epiglottitis, in small children. To date, accurate H. influenzae capsule typing requires PCR, a time-consuming and cumbersome method. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) provides rapid bacterial diagnostics and is increasingly used in clinical microbiology laboratories. Here, MALDI-TOF MS was evaluated as a novel approach to separate Hib from other H. influenzae. PCR-verified Hib and non-Hib reference isolates were selected based on genetic and spectral characteristics. Mass spectra of reference isolates were acquired and used to generate different classification algorithms for Hib/non-Hib differentiation using both ClinProTools and the MALDI Biotyper software. A test series of mass spectra from 33 Hib and 77 non-Hib isolates, all characterized by PCR, was used to evaluate the algorithms. Several algorithms yielded good results, but the two best were a ClinProTools model based on 22 separating peaks and subtyping main spectra (MSPs) using MALDI Biotyper. The ClinProTools model had a sensitivity of 100% and a specificity of 99%, and the results were 98% reproducible using a different MALDI-TOF MS instrument. The Biotyper subtyping MSPs had a sensitivity of 97%, a specificity of 100%, and 93% reproducibility. Our results suggest that it is possible to use MALDI-TOF MS to differentiate Hib from other H. influenzae. This is a promising method for rapidly identifying Hib in unvaccinated populations and for the screening and surveillance of Hib carriage in vaccinated populations.
Manono, Janet; Dougherty, Casey A.; Jones, Kirsten; DeMuth, Joshua; Holl, Mark M. Banaszak; DiMaggio, Stassi
The synthesis, isolation, and characterization of generation 3 poly(amidoamine) (G3 PAMAM) dendrimer containing precise ratios of 5-carboxytetramethylrhodamine succinimidyl ester (TAMRA) dye (n = 1–3) per polymer particle are reported. Stochastic conjugation of TAMRA dye to the dendrimer was followed by separation into precise dye-polymer ratios using rp-HPLC. The isolated materials were characterized by rp-UPLC, MALDI-TOF-MS, and 1H NMR spectroscopy, UV–vis, and fluorescence spectroscopies. PMID:26549978
Hsueh, Po-Ren; Kuo, Lu-Cheng; Chang, Tsung-Chain; Lee, Tai-Fen; Teng, Shih-Hua; Chuang, Yu-Chung; Teng, Lee-Jene
Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) (Bruker Biotyper) was able to accurately identify 98.6% (142/144) of Acinetobacter baumannii isolates, 72.4% (63/87) of A. nosocomialis isolates, and 97.6% (41/42) of A. pittii isolates. All Acinetobacter junii, A. ursingii, A. johnsonii, and A. radioresistens isolates (n = 28) could also be identified correctly by Bruker Biotyper. PMID:24899038
Schott, Ann-Sophie; Behr, Jürgen; Quinn, Jennifer; Vogel, Rudi F.
Lactic acid bacteria (LAB) are widely used as starter cultures in the manufacture of foods. Upon preparation, these cultures undergo various stresses resulting in losses of survival and fitness. In order to find conditions for the subsequent identification of proteomic biomarkers and their exploitation for preconditioning of strains, we subjected Lactobacillus (Lb.) paracasei subsp. paracasei TMW 1.1434 (F19) to different stress qualities (osmotic stress, oxidative stress, temperature stress, pH stress and starvation stress). We analysed the dynamics of its stress responses based on the expression of stress proteins using MALDI-TOF mass spectrometry (MS), which has so far been used for species identification. Exploiting the methodology of accumulating protein expression profiles by MALDI-TOF MS followed by the statistical evaluation with cluster analysis and discriminant analysis of principle components (DAPC), it was possible to monitor the expression of low molecular weight stress proteins, identify a specific time point when the expression of stress proteins reached its maximum, and statistically differentiate types of adaptive responses into groups. Above the specific result for F19 and its stress response, these results demonstrate the discriminatory power of MALDI-TOF MS to characterize even dynamics of stress responses of bacteria and enable a knowledge-based focus on the laborious identification of biomarkers and stress proteins. To our knowledge, the implementation of MALDI-TOF MS protein profiling for the fast and comprehensive analysis of various stress responses is new to the field of bacterial stress responses. Consequently, we generally propose MALDI-TOF MS as an easy and quick method to characterize responses of microbes to different environmental conditions, to focus efforts of more elaborate approaches on time points and dynamics of stress responses. PMID:27783652
Garcia, Estefânia F.; Luciano, Winnie A.; Xavier, Danilo E.; da Costa, Whyara C. A.; de Sousa Oliveira, Kleber; Franco, Octávio L.; de Morais Júnior, Marcos A.; Lucena, Brígida T. L.; Picão, Renata C.; Magnani, Marciane; Saarela, Maria; de Souza, Evandro L.
This study aimed to identify lactic acid bacteria (LAB) in byproducts of fruit (Malpighia glabra L., Mangifera indica L., Annona muricata L., and Fragaria vesca L.) pulp processing. Fifty strains of LAB were identified using matrix-assisted laser desorption/ionization–time of flight mass spectrometry (MALDI-TOF MS) and 16S rRNA gene sequence (16S rRNA) analysis. Species belonging to Lactobacillus genus were the predominant LAB in all fruit pulp processing byproducts. The average congruency between the MALDI-TOF MS and 16S rRNA in LAB species identification reached 86%. Isolates of L. plantarum, L. brevis, L. pentosus, L. lactis and L. mesenteroides were identified with 100% congruency. MALDI-TOF MS and 16S rRNA analysis presented 86 and 100% efficiency of LAB species identification, respectively. Further, five selected Lactobacillus strains (L. brevis 59, L. pentosus 129, L. paracasei 108, L. plantarum 49, and L. fermentum 111) were evaluated for desirable probiotic-related properties and growth behavior on two different cultivation media. The exposure to pH 2.0 sharply decreased the counts of the different Lactobacillus strains after a 1 or 2 h incubation, while varied decreases were noted after 3 h of exposure to pH 3.0. Overall, the exposure to pH 5.0 and to bile salts (0.15, 0.30, and 1.00%) did not decrease the counts of the Lactobacillus strains. All tested Lactobacillus strains presented inhibitory activity against Staphylococcus aureus, Salmonella Typhimurium, Salmonella Enteritidis, Listeria monocytogenes and Escherichia coli, and presented variable susceptibility to different antibiotics. The selected Lactobacillus strains presented satisfactory and reproducible growth behavior. In conclusion, MALDI-TOF MS and 16S rRNA analysis revealed high efficiency and congruency for LAB species identification, and the selected Lactobacillus strains may be candidates for further investigation of novel probiotic strains. PMID:27625647
The data was of insufficient quality to obtain definitive biomarkers. Trips were also made to AFRL/HEDR at Brooks City Base to assist with their...sample analysis using the Finnigan LTQ located there. Mr. Mullens and Ms. Nagore assisted with training personnel at AFRL/HEDR and when necessary...techniques with saliva samples and matrix- assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS), we have been able to
Comparison of the accuracy of matrix-assisted laser desorption ionization-time of flight mass spectrometry with that of other commercial identification systems for identifying Staphylococcus saprophyticus in urine.
Lee, Tai-Fen; Lee, Hao; Chen, Chung-Ming; Du, Shin-Hei; Cheng, Ya-Chih; Hsu, Chen-Ching; Chung, Meng-Yu; Teng, Shih-Hua; Teng, Lee-Jene; Hsueh, Po-Ren
Among 30 urinary isolates of Staphylococcus saprophyticus identified by sequencing methods, the rate of accurate identification was 100% for Bruker Biotyper matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), 86.7% for the Phoenix PID and Vitek 2 GP systems, 93.3% for the MicroScan GP33 system, and 46.7% for the BBL CHROMagar Orientation system.
Jiang, Yang; Lu, Yi-Xuan; Cui, Yu-Xin; Zhou, Qi-Feng; Ma, Yuguo; Pei, Jian
A novel family of giant pi-conjugated dendrimers (G0, G1, and G2) solely constructed by 5,5,10,10,15,15-hexahexyltruxene units has been developed in a convergent manner through a Suzuki cross-coupling reaction. The overall yields to such large rigid conjugated dendrimers are quite satisfying. The structures and purity of these nanosize rigid dendrimers are verified by 1H and 13C NMR, MALDI-TOF MS, and elemental analysis.
Liu, Wen-Jun; Zhou, Yan; Zhou, Qi-Feng; Ma, Yuguo; Pei, Jian
Bimolecular coupling/unimolecular cyclization strategies, including McMurry- and Glaser-type homocoupling reactions, were utilized to synthesize two shape-persistent elliptic macrocycles, which consist of polycyclic aromatic hydrocarbon units. The identity and purity of both macrocycles MC1 and MC2 were verified by 1H and 13C NMR, elemental analysis, as well as MALDI-TOF MS. The photophysical properties of MC1 and MC2 in dilute solution were also investigated.
Wang, Jin-Liang; Chan, Yi-Tsu; Moorefield, Charles N; Pei, Jian; Modarelli, David A; Romano, Natalie C; Newkome, George R
A new series of shape-persistent metallomacromolecules G0 and G1 as well as corresponding ligands, based on thiophene-functionalized bisterpyridine-Ru(II) with truxene moieties, as the nodes, were developed. All new compounds were fully characterized by (1) H, COSY, and (13) C NMR, as well as MALDI-TOF MS. Their photophysical properties revealed that the branched scaffold resulted in high molar absorption coefficients and broad absorption making these materials potential candidates for light-harvesting.
Kaplan-Sandquist, Kimberly; LeBeau, Marc A; Miller, Mark L
Chemical analysis of latent fingermarks, "touch chemistry," has the potential of providing intelligence or forensically relevant information. Matrix-assisted laser desorption ionization/time-of-flight mass spectrometry (MALDI/TOF MS) was used as an analytical platform for obtaining mass spectra and chemical images of target drugs and explosives in fingermark residues following conventional fingerprint development methods and MALDI matrix processing. There were two main purposes of this research: (1) develop effective laboratory methods for detecting drugs and explosives in fingermark residues and (2) determine the feasibility of detecting drugs and explosives after casual contact with pills, powders, and residues. Further, synthetic latent print reference pads were evaluated as mimics of natural fingermark residue to determine if the pads could be used for method development and quality control. The results suggest that artificial amino acid and sebaceous oil residue pads are not suitable to adequately simulate natural fingermark chemistry for MALDI/TOF MS analysis. However, the pads were useful for designing experiments and setting instrumental parameters. Based on the natural fingermark residue experiments, handling whole or broken pills did not transfer sufficient quantities of drugs to allow for definitive detection. Transferring drugs or explosives in the form of powders and residues was successful for preparing analytes for detection after contact with fingers and deposition of fingermark residue. One downfall to handling powders was that the analyte particles were easily spread beyond the original fingermark during development. Analyte particles were confined in the original fingermark when using transfer residues. The MALDI/TOF MS was able to detect procaine, pseudoephedrine, TNT, and RDX from contact residue under laboratory conditions with the integration of conventional fingerprint development methods and MALDI matrix. MALDI/TOF MS is a nondestructive
Emerging characterization tools entailing elements of both microscopy and spectroscopy (nanoIR, X-ray microtomography, MALDI TOF -MS, TOF -SIMS...technologies. • Development of mechanical models of soft materials ( polymers ) will be required to allow their greater incorporation into tribological...systems; viscoelastic models are needed to describe the deformation of polymers from initiation to fracture; • Expansion of mechanical models to
Street, Jeff; Carroll, Karen; Miller, Heather; Zhang, Sean X.
Unmanned aerial vehicles (UAVs) could potentially be used to transport microbiological specimens. To examine the impact of UAVs on microbiological specimens, blood and sputum culture specimens were seeded with usual pathogens and flown in a UAV for 30 ± 2 min. Times to recovery, colony counts, morphologies, and matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS)-based identifications of the flown and stationary specimens were similar for all microbes studied. PMID:27535683
Rapid Identification of Bacteria Directly from Positive Blood Cultures by Use of a Serum Separator Tube, Smudge Plate Preparation, and Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry.
Chen, Yan; Porter, Vanessa; Mubareka, Samira; Kotowich, Leona; Simor, Andrew E
We analyzed the matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) of smudge plate growth for bacterial identification from 400 blood cultures. Ninety-seven percent of Gram-negative bacilli and 85% of Gram-positive organisms were correctly identified within 4 h; only eight isolates (2.0%) were misidentified. This method provided rapid and accurate microbial identification from positive blood cultures.
Calderaro, Adriana; Piergianni, Maddalena; Buttrini, Mirko; Montecchini, Sara; Piccolo, Giovanna; Gorrini, Chiara; Rossi, Sabina; Chezzi, Carlo; Arcangeletti, Maria Cristina; Medici, Maria Cristina; De Conto, Flora
Detection of Entamoeba histolytica and its differentiation from Entamoeba dispar is an important goal of the clinical parasitology laboratory. The aim of this study was the identification and differentiation of E. histolytica and E. dispar by MALDI-TOF MS, in order to evaluate the application of this technique in routine diagnostic practice. MALDI-TOF MS was applied to 3 amebic reference strains and to 14 strains isolated from feces that had been differentiated by molecular methods in our laboratory. Protein extracts from cultures of these strains (axenic cultures for the 3 reference strains and monoxenic cultures for the 14 field isolates) were analyzed by MALDI-TOF MS and the spectra obtained were analyzed by statistical software. Five peaks discriminating between E. histolytica and E. dispar reference strains were found by protein profile analysis: 2 peaks (8,246 and 8,303 Da) specific for E. histolytica and 3 (4,714; 5,541; 8,207 Da) for E. dispar. All clinical isolates except one showed the discriminating peaks expected for the appropriate species. For 2 fecal samples from which 2 strains (1 E. histolytica and 1 E. dispar) out of the 14 included in this study were isolated, the same discriminating peaks found in the corresponding isolated amebic strains were detected after only 12h (E. histolytica) and 24h (E. dispar) of incubation of the fecal samples in Robinson's medium without serum. Our study shows that MALDI-TOF MS can be used to discriminate between E. histolytica and E. dispar using in vitro xenic cultures and it also could have potential for the detection of these species in clinical samples.
Paauw, Armand; Jonker, Debby; Roeselers, Guus; Heng, Jonathan M E; Mars-Groenendijk, Roos H; Trip, Hein; Molhoek, E Margo; Jansen, Hugo-Jan; van der Plas, Jan; de Jong, Ad L; Majchrzykiewicz-Koehorst, Joanna A; Speksnijder, Arjen G C L
E. coli-Shigella species are a cryptic group of bacteria in which the Shigella species are distributed within the phylogenetic tree of E. coli. The nomenclature is historically based and the discrimination of these genera developed as a result of the epidemiological need to identify the cause of shigellosis, a severe disease caused by Shigella species. For these reasons, this incorrect classification of shigellae persists to date, and the ability to rapidly characterize E. coli and Shigella species remains highly desirable. Until recently, existing matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) assays used to identify bacteria could not discriminate between E. coli and Shigella species. Here we present a rapid classification method for the E. coli-Shigella phylogroup based on MALDI-TOF MS which is supported by genetic analysis. E. coli and Shigella isolates were collected and genetically characterized by MLVA. A custom reference library for MALDI-TOF MS that represents the genetic diversity of E. coli and Shigella strains was developed. Characterization of E. coli and Shigella species is based on an approach with Biotyper software. Using this reference library it was possible to distinguish between Shigella species and E. coli. Of the 180 isolates tested, 94.4% were correctly classified as E. coli or shigellae. The results of four (2.2%) isolates could not be interpreted and six (3.3%) isolates were classified incorrectly. The custom library extends the existing MALDI-TOF MS method for species determination by enabling rapid and accurate discrimination between Shigella species and E. coli.
Lee, Meng-Rui; Tsai, Chia-Jung; Liang, Sheng-Kai; Lin, Ching-Kai; Huang, Yu-Tsung; Hsueh, Po-Ren
The clinical characteristics of 89 patients with Lactobacillus bacteraemia treated at a university-affiliated hospital in northern Taiwan during 2000-2014 were retrospectively evaluated. Lactobacillus spp. were identified by 16S rRNA sequencing analysis and matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF/MS). Antimicrobial susceptibilities of the isolates were determined by broth microdilution. The most commonly isolated species was Lactobacillus salivarius (n = 21), followed by Lactobacillus paracasei (n = 16) and Lactobacillus fermentum (n = 13). Excluding three isolates with lower 16S rRNA sequence similarity, MALDI-TOF/MS provided correct identification for 84.9% (73/86) of Lactobacillus isolates. Concordant identification was lowest for Lactobacillus casei (11%). The main infection foci were intra-abdominal infection (49%) and catheter-related bloodstream infection (17%). Only one-half of the patients received adequate antibiotic treatment during the bacteraemic episode. The majority of patients with Lactobacillus bacteraemia were immunocompromised. The 7-day and in-hospital mortality rates were 21% and 62%, respectively, and underlying malignancy was associated with a higher in-hospital mortality rate (odds ratio = 2.666). There were no significant differences in mortality (7-day, 14-day, 30-day and in-hospital) among patients with bacteraemia due to different Lactobacillus spp. Minimum inhibitory concentrations were highest for glycopeptides, cephalosporins and fluoroquinolones and were lowest for carbapenems and aminopenicillins. Lactobacillus bacteraemia was associated with a high mortality rate, and patient outcome was associated with underlying malignancy. MALDI-TOF/MS was able to accurately identify 84.9% of the Lactobacillus isolates, and L. salivarius was the predominant pathogen. The accuracy rate for identification of Lactobacillus spp. by MALDI-TOF/MS was lowest for L. casei.
Evaluation of the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry Bruker Biotyper for identification of Penicillium marneffei, Paecilomyces species, Fusarium solani, Rhizopus species, and Pseudallescheria boydii.
Chen, Ying-Sheng; Liu, Yen-Hung; Teng, Shih-Hua; Liao, Chun-Hsing; Hung, Chien-Ching; Sheng, Wang-Huei; Teng, Lee-Jene; Hsueh, Po-Ren
We evaluated the performance of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), the MALDI Bruker Biotyper system (microflex LT; Bruker Daltonik GmbH, Bremen, Germany), on the identification of 50 isolates of clinically encountered molds, including Penicillium marneffei (n = 28), Paecilomyces species (n = 12), Fusarium solani (n = 6), Rhizopus species (n = 3), and Pseudallescheria boydii (n = 1). The isolates were identified to species levels by sequence analysis of the internal transcribed spacer (ITS) regions using primers ITS1 and ITS4. None of the 28 genetically well characterized isolates of P. marneffei were identified as P. marneffei by MALDI-TOF MS, because P. marneffei was not present in either Bruker general library (DB 5627) or Bruker filamentous fungi library V1.0. However, the rate of accurate identification as P. marneffei (score value ≥ 2.000) was 85.7% based on newly created database from one P. marneffei strain (NTUH-3370) by MALDI Biotyper system. Sequencing analysis of these 22 non-P. marneffei isolates of molds revealed seven Paecilomyces variotii, six F. solani, four Paecilomyces lilacinus, and one each of Paecilomyces sinensis, Rhizopus arrhizus, R. oryzae, R. microspores, and P. boydii. Although all the seven P. variotii isolates, four of the six F. solani, two of the four P. lilacinus, and two of the three isolates of Rhizopus species, and the P. boydii isolate had concordant identification results between MALDI-TOF MS and sequencing analysis, the score values of these isolates were all of <1.700. This study indicated that the MALDI Bruker Biotyper is ineffective for identifying P. marneffei and other unusual molds because of the current database limitations. Therefore, it is necessary to continuously update the MALDI-TOF MS databases.
Wieme, Anneleen D; Spitaels, Freek; Aerts, Maarten; De Bruyne, Katrien; Van Landschoot, Anita; Vandamme, Peter
Applicability of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for identification of beer-spoilage bacteria was examined. To achieve this, an extensive identification database was constructed comprising more than 4200 mass spectra, including biological and technical replicates derived from 273 acetic acid bacteria (AAB) and lactic acid bacteria (LAB), covering a total of 52 species, grown on at least three growth media. Sequence analysis of protein coding genes was used to verify aberrant MALDI-TOF MS identification results and confirmed the earlier misidentification of 34 AAB and LAB strains. In total, 348 isolates were collected from culture media inoculated with 14 spoiled beer and brewery samples. Peak-based numerical analysis of MALDI-TOF MS spectra allowed a straightforward species identification of 327 (94.0%) isolates. The remaining isolates clustered separately and were assigned through sequence analysis of protein coding genes either to species not known as beer-spoilage bacteria, and thus not present in the database, or to novel AAB species. An alternative, classifier-based approach for the identification of spoilage bacteria was evaluated by combining the identification results obtained through peak-based cluster analysis and sequence analysis of protein coding genes as a standard. In total, 263 out of 348 isolates (75.6%) were correctly identified at species level and 24 isolates (6.9%) were misidentified. In addition, the identification results of 50 isolates (14.4%) were considered unreliable, and 11 isolates (3.2%) could not be identified. The present study demonstrated that MALDI-TOF MS is well-suited for the rapid, high-throughput and accurate identification of bacteria isolated from spoiled beer and brewery samples, which makes the technique appropriate for routine microbial quality control in the brewing industry.
Celandroni, Francesco; Salvetti, Sara; Gueye, Sokhna Aissatou; Mazzantini, Diletta; Lupetti, Antonella; Senesi, Sonia; Ghelardi, Emilia
The soil-related Bacillus and Paenibacillus species have increasingly been implicated in various human diseases. Nevertheless, their identification still poses problems in the clinical microbiology laboratory and, with the exception of Bacillus anthracis and Bacillus cereus, little is known on their pathogenicity for humans. In this study, we evaluated the use of matrix-assisted laser desorption—ionization time of flight mass spectrometry (MALDI-TOF MS) in the identification of clinical isolates of these genera and conducted genotypic and phenotypic analyses to highlight specific virulence properties. Seventy-five clinical isolates were subjected to biochemical and MALDI-TOF MS identification. 16S rDNA sequencing and supplemental tests were used to solve any discrepancies or failures in the identification results. MALDI-TOF MS significantly outperformed classical biochemical testing for correct species identification and no misidentification was obtained. One third of the collected strains belonged to the B. cereus species, but also Bacillus pumilus and Bacillus subtilis were isolated at high rate. Antimicrobial susceptibility testing showed that all the B. cereus, B. licheniformis, B. simplex, B. mycoides, Paenibacillus glucanolyticus and Paenibacillus lautus isolates are resistant to penicillin. The evaluation of toxin/enzyme secretion, toxin-encoding genes, motility, and biofilm formation revealed that B. cereus displays the highest virulence potential. However, although generally considered nonpathogenic, most of the other species were shown to swim, swarm, produce biofilms, and secrete proteases that can have a role in bacterial virulence. In conclusion, MALDI-TOF MS appears useful for fast and accurate identification of Bacillus and Paenibacillus strains whose virulence properties make them of increasing clinical relevance. PMID:27031639
Clinical and Microbiological Features of a Cystic Fibrosis Patient Chronically Colonized with Pandoraea sputorum Identified by Combining 16S rRNA Sequencing and Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry
Fernández-Olmos, A.; Morosini, M. I.; Lamas, A.; García-Castillo, M.; García-García, L.; Máiz, L.
Clonal isolates identified as various nonfermentative Gram-negative bacilli over a 5-year period from sputum cultures of a 30-year-old cystic fibrosis patient were successfully reidentified as Pandoraea sputorum by combining 16S rRNA sequencing and matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS). Decreased lung function improved after 1 year of azithromycin and inhaled 7%-hypertonic saline treatment. PMID:22170922
Zhao, Ying; Chan, Jasper Fuk-Woo; Wang, He; Guo, Dawen; Pan, Yuhong; Xiao, Yuling; Yue, Na; Lau, Susanna Kar-Pui
Our case series showed that uncomplicated Yarrowia lipolytica fungemia might be treated with catheter removal alone. The Vitek 2 YST identification (ID) card system, matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS), and internal transcribed spacer and 25S nuclear ribosomal DNA (nrDNA) gene sequencing provided reliable identification. All isolates had low MICs to voriconazole, echinocandins, and amphotericin B. PMID:26311865
Wenning, Mareike; Breitenwieser, Franziska; Konrad, Regina; Huber, Ingrid; Busch, Ulrich; Scherer, Siegfried
The food industry requires easy, accurate, and cost-effective techniques for microbial identification to ensure safe products and identify microbial contaminations. In this work, FTIR spectroscopy and MALDI-TOF mass spectrometry were assessed for their suitability and applicability for routine microbial diagnostics of food-related microorganisms by analyzing their robustness according to changes in incubation time and medium, identification accuracy and their ability to differentiate isolates down to the strain level. Changes in the protocol lead to a significantly impaired performance of FTIR spectroscopy, whereas they had only little effects on MALDI-TOF MS. Identification accuracy was tested using 174 food-related bacteria (93 species) from an in-house strain collection and 40 fresh isolates from routine food analyses. For MALDI-TOF MS, weaknesses in the identification of bacilli and pseudomonads were observed; FTIR spectroscopy had most difficulties in identifying pseudomonads and enterobacteria. In general, MALDI-TOF MS obtained better results (52-85% correct at species level), since the analysis of mainly ribosomal proteins is more robust and seems to be more reliable. FTIR spectroscopy suffers from the fact that it generates a whole-cell fingerprint and intraspecies diversity may lead to overlapping species borders which complicates identification. In the present study values between 56% and 67% correct species identification were obtained. On the opposite, this high sensitivity offers the opportunity of typing below the species level which was not possible using MALDI-TOF MS. Using fresh isolates from routine diagnostics, both techniques performed well with 88% (MALDI-TOF) and 75% (FTIR) correct identifications at species level, respectively.
Camafeita, E; Solís, J; Alfonso, P; López, J A; Sorell, L; Méndez, E
The gluten toxic fractions responsible for the mucosal damage in coeliac disease (CD), so-called gliadins, hordeins, secalins and avenins from a large number (30-40) of wheat, barley, rye and oats cultivars respectively, have been mass analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Gliadin, secalin and avenin characteristic mass profiles are nearly identical amongst distinct cultivars from the corresponding cereal, while hordeins profiles show more variability depending on the particular barley cultivar. On the basis of these four distinguishable characteristic mass patterns spreading within the 20,000-40,000 Da range, MALDI-TOF-MS has permitted the direct and simultaneous visualization of gliadins, hordeins, secalins and avenins in foods elaborated with cereal mixtures of wheat, barley, rye and oats. This capacity has been demonstrated by mass analyzing foods made with these four cereals in varying ratios. Thus MALDI-TOF-MS can be preliminarily established as a unique system with the ability to discriminate the specific type of gluten toxic fractions present in food samples.
Pailhoriès, Hélène; Daure, Sophie; Eveillard, Matthieu; Joly-Guillou, Marie-Laure; Kempf, Marie
Acinetobacter baumannii belongs to the Acinetobacter calcoaceticus-baumannii complex (Acb) containing 2 other pathogenic species: Acinetobacter pittii and Acinetobacter nosocomialis. Identification of these bacteria remains problematic despite the use of matrix-assisted laser ionization time-of-flight mass spectrometry (MALDI-TOF MS). Here, we enriched the SARAMIS™ database of the Vitek MS® plus mass spectrometer to improve the identification of species of the Acb complex. For each species, we incremented reference spectra. Then, a SuperSpectrum was created based on the selection of 40 specific masses. In a second step, we validated reference spectra and SuperSpectra with 100 isolates identified by rpoB gene sequencing. All the isolates were correctly identified by MALDI-TOF MS with the database we created as compared to the identifications obtained by rpoB sequencing. Our database enabled rapid and reliable identification of the pathogen species belonging to the Acb complex. Identification by MALDI-TOF MS with our database is a good alternative to molecular biology.
Camafeita, E; Alfonso, P; Mothes, T; Méndez, E
The first epitope-independent procedure for rapidly quantifying gluten gliadins in food by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI/TOF-MS) based on the direct observation of the characteristic gliadin mass pattern is presented. This pattern was identified in both processed and unprocessed gluten-containing food samples. The procedure allows the micro-quantification of gluten in food samples below levels toxic for coeliac patients, with a linear response in the 0.4-10 mg per 100 g range and a high detection sensitivity similar to that of enzyme-linked immunosorbent assay (ELISA) systems. Food samples simultaneously analyzed by MALDI/TOF-MS and a highly sensitive laboratory-made sandwich ELISA revealed a good correlation between the two techniques. In addition, MALDI/TOF-MS provides a rapid screening system to determine the presence of gliadins in food samples by directly monitoring the occurrence of the protonated gliadin mass pattern. The procedure also permits the study of the alteration of gliadins in food during the baking process, providing data on the heart effect by changes in protein mass signals.
Welker, Martin; Moore, Edward R B
In the last few years matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been increasingly studied and applied for the identification and typing of microorganisms. Very recently, MALDI-TOF MS has been introduced in clinical routine microbiological diagnostics with marked success, which is remarkable considering that not long ago the technology was generally seen as being far from practical application. The identification of microbial isolates by whole-cell mass spectrometry (WC-MS) is being recognized as one of the latest tools forging a revolution in microbial diagnostics, with the potential of bringing to an end many of the time-consuming and man-power-intensive identification procedures that have been used for decades. Apart from applications of WC-MS in clinical diagnostics, other fields of microbiology also have adopted the technology with success. In this article, an over-view of the principles of MALDI-TOF MS and WC-MS is presented, highlighting the characteristics of the technology that allow its utilization for systematic microbiology.
Fernández-No, I C; Böhme, K; Calo-Mata, P; Cañas, B; Gallardo, J M; Barros-Velázquez, J
Streptococcus parauberis is known as an etiological agent of mastitis in cows and for producing streptococcosis in farmed fish, although its presence in foods has seldom been reported. In this work, two bacterial isolates were recovered from a spoiled vacuum-packaged refrigerated seafood product. Both isolates were identified by 16S rRNA gene sequencing, exhibiting 99% homology with respect to S. parauberis. Both isolates were also characterized by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS). Genetic analysis revealed the clonal homogeneity of the isolates and their grouping together with other S. parauberis strains in a different cluster with respect to Streptococcus uberis strains. Proteomic analysis by MALDI-TOF MS allowed for the identification of five mass peaks in the range of 2200-6000 m/z that resulted to be specific to the species S. parauberis and allowed its rapid and direct identification with respect to other pathogenic and spoilage bacteria potentially present in seafood and other food products. This study represents, to our knowledge, the first report of S. parauberis in seafood in general and in vacuum-packed food products in particular. Moreover, it provides a rapid method based on MALDI-TOF MS for the identification of S. parauberis.
Emami, Kaveh; Askari, Vahid; Ullrich, Matthias; Mohinudeen, Khwajah; Anil, Arga Chandrashekar; Khandeparker, Lidita; Burgess, J Grant; Mesbahi, Ehsan
To evaluate a rapid and cost-effective method for monitoring bacteria in ballast water, several marine bacterial isolates were characterized by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Since International Maritime Organization (IMO) regulations are concerned with the unintended transportation of pathogenic bacteria through ballast water, emphasis was placed on detecting species of Vibrio, enterococci and coliforms. Seawater samples collected from the North Sea were incubated in steel ballast tanks and the presence of potentially harmful species of Pseudomonas was also investigated. At the genus-level, the identification of thirty six isolates using MALDI-TOF MS produced similar results to those obtained by 16S rRNA gene sequencing. No pathogenic species were detected either by 16S rRNA gene analysis or by MALDI-TOF MS except for the opportunistically pathogenic bacterium Pseudomonas aeruginosa. In addition, in house software that calculated the correlation coefficient values (CCV) of the mass spectral raw data and their variation was developed and used to allow the rapid and efficient identification of marine bacteria in ballast water for the first time.
Profiling N-glycans of the egg jelly coat of the sea urchin Paracentrotus lividus by MALDI-TOF mass spectrometry and capillary liquid chromatography electrospray ionization-ion trap tandem mass spectrometry systems.
Şahar, Umut; Deveci, Remziye
Sea urchin eggs are surrounded by a carbohydrate-rich layer, termed the jelly coat, that consists of polysaccharides and glycoproteins. In the present study, we describe two mass spectrometric strategies to characterize the N-glycosylation of the Paracentrotus lividus egg jelly coat, which has an alecithal-type extracellular matrix like mammalian eggs. Egg jelly was isolated, lyophilized, and dialysed, followed by peptide N-glycosidase F (PNGase-F) treatment to release N-glycans from their protein chain. These N-glycans were then derivatized by permethylation reaction, and analysed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and capillary liquid chromatography electrospray ionization-ion trap tandem mass spectroscopy (CapLC ESI-Ion trap-MS/MS). N-glycans in the egg jelly coat glycoproteins were indicated by sodiated molecules at m/z 1579.8, 1783.9, 1988.0, 2192.0, and 2397.1 for permethylated oligosaccharides on MALDI-TOF MS. Fragmentation and structural characterization of these oligosaccharides were performed by ESI-Ion trap MS/MS. Then, MALDI-TOF-MS and ESI-Ion trap-MS/MS spectra were interpreted using the GlycoWorkbench software suite, a tool for building, displaying, and profiling glycan masses to identify the original oligosaccharide structures. The oligosaccharides of the isolated egg jelly coat were mainly of the high mannose type. This article is protected by copyright. All rights reserved.
Arinto-Garcia, Raquel; Pinho, Marcos Daniel; Carriço, João André; Melo-Cristino, José
The heterogeneity of members of the Streptococcus anginosus group (SAG) has traditionally hampered their correct identification. Recently, the group was subdivided into 6 taxa whose prevalence among human infections is poorly described. We evaluated the accuracy of the Rapid ID32 Strep test, matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS), and a PCR multiplex method to identify 212 SAG isolates recovered from human infections to the species and subspecies level by using multilocus sequence analysis (MLSA) as the gold standard. We also determined the antimicrobial susceptibilities of the isolates. Representatives of all SAG taxa were found among our collection. MALDI-TOF MS and the Rapid ID32 Strep test correctly identified 92% and 68% of the isolates to the species level, respectively, but showed poor performance at the subspecies level, and the latter was responsible for major identification errors. The multiplex PCR method results were in complete agreement with the MLSA identifications but failed to distinguish the subspecies Streptococcus constellatus subsp. pharyngis and S. constellatus subsp. viborgensis. A total of 145 MLSA sequence types were present in our collection, indicating that within each taxon a number of different lineages are capable of causing infection. Significant antibiotic resistance was observed only to tetracycline, erythromycin, and clindamycin and was present in most taxa. MALDI-TOF MS is a reliable method for routine SAG species identification, while the need for identification to the subspecies level is not clearly established. PMID:26354817
Fall, Bécaye; Lo, Cheikh Ibrahima; Samb-Ba, Bissoume; Perrot, Nadine; Diawara, Silman; Gueye, Mamadou Wague; Sow, Kowry; Aubadie-Ladrix, Maxence; Mediannikov, Oleg; Sokhna, Cheikh; Diemé, Yaya; Chatellier, Sonia; Wade, Boubacar; Raoult, Didier; Fenollar, Florence
Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) represents a revolution in routine pathogen identification in clinical microbiology laboratories. A MALDI-TOF MS was introduced to tropical Africa in the clinical microbiology laboratory of the Hôpital Principal de Dakar (Senegal) and used for routine pathogen identification. Using MS, 2,429 bacteria and fungi isolated from patients were directly assayed, leading to the identification of 2,082 bacteria (85.7%) and 206 fungi (8.5%) at the species level, 109 bacteria (4.5%) at the genus level, and 16 bacteria (0.75%) at the family level. Sixteen isolates remained unidentified (0.75%). Escherichia coli was the most prevalent species (25.8%) followed by Klebsiella pneumoniae (14.8%), Streptococcus agalactiae (6.2%), Acinetobacter baumannii (6.1%), Pseudomonas aeruginosa (5.9%), and Staphylococcus aureus (5.9%). MALDI-TOF MS has also enabled the detection of rare bacteria and fungi. MALDI-TOF MS is a powerful tool for the identification of bacterial and fungal species involved in infectious diseases in tropical Africa. PMID:25601995
Kolínská, Renáta; Spanělová, Petra; Dřevínek, Michal; Hrabák, Jaroslav; Zemličková, Helena
Strains of genus Citrobacter (152 isolates from 1950 to 1988 deposited in the Czech National Collection of Type Cultures, Prague) were re-classified using biological and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) methods. One-hundred thirty-six strains (ca. 90 %) were identified to the species level using the biological method with evaluation by Farmer matrix. MALDI-TOF MS exhibited better identification capability, the data being more compact; the method was unambiguously successful in typing 145 (95 %) strains. Comparison of the results of identification by the two methods revealed differences (for 12 samples) in identified species which, considering all biochemical and/or MS characteristics, could be attributed to the natural variability of strains and close relation of the misidentified species (all of them belonged to the Citrobacter freundii complex). Taking into account all the above data, both methods can be considered reliable; however, the MALDI-TOF MS exhibits higher accuracy, efficiency, and rapidity.
Kern, Carola C; Vogel, Rudi F; Behr, Jürgen
Lactobacillus (L.) brevis is one of the most frequently encountered bacteria in beer-spoilage incidents. As the species Lactobacillus brevis comprises strains showing varying ability to grow in beer, ranging from growth in low hopped wheat to highly hopped pilsner beer, differentiation and classification of L. brevis with regard to their beer-spoiling ability is of vital interest for the brewing industry. Matrix-Assisted-Laser-Desorption-Ionization-Time-Of-Flight Mass Spectrometry (MALDI-TOF MS) has been shown as a powerful tool for species and sub-species differentiation of bacterial isolates and is increasingly used for strain-level differentiation. Seventeen L. brevis strains, representative of different spoilage types, were characterized according to their tolerance to iso-alpha-acids and their growth in wheat-, lager- and pilsner beer. MALDI-TOF MS spectra were acquired to perform strain-level identification, cluster analysis and biomarker detection. Strain-level identification was achieved in 90% out of 204 spectra. Misidentification occurred nearly exclusively among strains belonging to the same spoilage type. Though spectra of strongly beer-spoiling strains showed remarkable similarity, no decisive single markers were detected to be present in all strains of one group. However, MALDI-TOF MS spectra can be reliably assigned to the corresponding strain and thus allow to track single strains and connect them to their physiological properties.
Emami, Kaveh; Askari, Vahid; Ullrich, Matthias; Mohinudeen, Khwajah; Anil, Arga Chandrashekar; Khandeparker, Lidita; Burgess, J. Grant; Mesbahi, Ehsan
To evaluate a rapid and cost-effective method for monitoring bacteria in ballast water, several marine bacterial isolates were characterized by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Since International Maritime Organization (IMO) regulations are concerned with the unintended transportation of pathogenic bacteria through ballast water, emphasis was placed on detecting species of Vibrio, enterococci and coliforms. Seawater samples collected from the North Sea were incubated in steel ballast tanks and the presence of potentially harmful species of Pseudomonas was also investigated. At the genus-level, the identification of thirty six isolates using MALDI-TOF MS produced similar results to those obtained by 16S rRNA gene sequencing. No pathogenic species were detected either by 16S rRNA gene analysis or by MALDI-TOF MS except for the opportunistically pathogenic bacterium Pseudomonas aeruginosa. In addition, in house software that calculated the correlation coefficient values (CCV) of the mass spectral raw data and their variation was developed and used to allow the rapid and efficient identification of marine bacteria in ballast water for the first time. PMID:22685576
Chen, Hui; He, Meiyu; Pei, Jian; Liu, Bin
An analytical method based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been applied to provide information on the structure of a copolymer, e.g., repeat unit and end group. Seven conjugated polymers, which have been demonstrated as the active component in blue light-emitting diodes, were synthesized through Suzuki polycondensation reaction in the presence of Pd(PPh3)4 catalyst. Their molecular weights were obtained using gel permeation chromatography analysis. MALDI-TOF MS was used to investigate the structure information in detail. The proposed end-group structures were confirmed by the identity between the observed and the simulated isotopic distribution of each polymer. The results demonstrate that these synthetic polymers possess various end groups and even contain macrocycles. The catalyst Pd(PPh3)4 was found to introduce phenyl end groups via aryl-aryl exchange between the catalytic palladium intermediate and the triphenylphosphine ligand. All these results are based on the analysis of the mass spectrum data, which suggests that MALDI-TOF MS is an extraordinarily strong tool in synthetic polymer structure analysis.
Structural characterization of native high-methoxylated pectin using nuclear magnetic resonance spectroscopy and ultraviolet matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Comparative use of 2,5-dihydroxybenzoic acid and nor-harmane as UV-MALDI matrices.
Monge, María Eugenia; Negri, R Martín; Kolender, Adriana A; Erra-Balsells, Rosa
The successful analysis by ultraviolet matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (UV-MALDI-TOF MS) of native and hydrolyzed high-methoxylated pectin samples is described. In order to find the optimal conditions for UV-MALDI-TOF MS analysis several experimental variables were studied such as: different UV-MALDI matrices (nor-harmane, 2,5-dihydroxybenzoic acid), sample preparation methods (mixture, sandwich), inorganic salt addition (doping salts, NaCl, KCl, NH(4)Cl), ion mode (positive, negative), linear and reflectron mode, etc. nor-Harmane has never been used as a UV-MALDI matrix for the analysis of pectins but its use avoids pre-treatment of the sample, such as an enzymatic digestion or an acid hydrolysis, and there is no need to add salts, making the analysis easier and faster. This study suggested an alternative way of analyzing native high-methoxylated pectins, with UV-MALDI-TOF MS, by using nor-harmane as the matrix in negative ion mode. The analysis by (1)H and (13)C nuclear magnetic resonance (NMR) spectroscopy of the native and hydrolyzed pectin is also briefly described.
Picariello, Gianluca; Romano, Raffaele; Addeo, Francesco
The potential of matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) for the analysis of intact triacylglycerols (TAGs) is generally limited by the extensive in-source prompt fragmentation. The sequential deposition of matrix and TAGs over the stainless steel target precoated with a thin layer of nitrocellulose (NC) drastically reduced fragmentation in the MALDI-TOF MS profiling of oils and fats. The NC MALDI-TOF MS profiles of native and thermally stressed virgin olive oil and butter are reported as case studies, along with test analyses of a standard mixture of mono-, di-, and triacylglycerols. Mass spectra were almost completely devoid of both fragment and matrix ion signals, thus disclosing relevant information, especially in the low molecular mass range. The detection of several partial acylglycerols of low abundance and minor TAGs that are barely observed with other techniques also provided evidence for an increased dynamic range of NC MALDI-TOF MS that was due to the minimization of suppressive effects. The NC film substrate also improved the shot-to-shot and sample-to-sample reproducibility of the ion production through the exhibition of a more homogeneous matrix/analyte cocrystallization, thus enabling MALDI-based measurements to a consistent quantification of TAGs.
Ludwig, Katrin; Habbach, Schähdi; Krieglstein, Josef; Klumpp, Susanne; König, Simone
Background Protein histidine phosphatase (PHP) is an enzyme which removes phosphate groups from histidine residues. It was described for vertebrates in the year 2002. The recombinant human 16 kDa protein forms multimeric complexes in physiological buffer and in the gas phase. High-mass calibration in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has remained a problem due to the lack of suitable standards. Large proteins can hardly be freed of their substructural microheterogeneity by classical purification procedures so that their use as calibrants is limited. A small adduct-forming protein of validated quality is a valuable alternative for that purpose. Methodology/Principal Findings Three major PHP clusters of ∼113, 209 and >600 kDa were observed in gel filtration analysis. Re-chromatography of the monomer peak showed the same cluster distribution. The tendency to associate was detected also in MALDI-TOF MS measuring regular adducts up to 200 kDa. Conclusions/Significance PHP forms multimers consisting of up to more than 35 protein molecules. In MALDI-TOF MS it generates adduct ions every 16 kDa. The protein can be produced with high quality so that its use as calibration compound for high mass ranges above 100 kDa, where standards are difficult to obtain, is feasible. PMID:21876758
Tadros, Manal; Petrich, Astrid
Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) can be used to identify bacteria directly from positive blood and sterile fluid cultures. The authors evaluated a commercially available kit - the Sepsityper Kit (Bruker Daltonik, Germany) - and MALDI-TOF MS for the rapid identification of organisms from 80 flagged positive blood culture broths, of which 73 (91.2%) were blood culture specimens and seven (8.7%) were cerebrospinal fluid specimens, in comparison with conventional identification methods. Correct identification to the genus and species levels was obtained in 75 of 80 (93.8%) and 39 of 50 (78%) blood culture broths, respectively. Applying the blood culture analysis module, a newly developed software tool, improved the species identification of Gram-negative organisms from 94.7% to 100% and of Gram-positive organisms from 66.7% to 70%. MALDI-TOF MS is a promising tool for the direct identification of organisms cultured from sterile sites.
Schuster, Dominik; Rickmeyer, Jasmin; Gajdiss, Mike; Thye, Thorsten; Lorenzen, Stephan; Reif, Marion; Josten, Michaele; Szekat, Christiane; Melo, Luís D R; Schmithausen, Ricarda M; Liégeois, Florian; Sahl, Hans-Georg; Gonzalez, Jean-Paul J; Nagel, Michael; Bierbaum, Gabriele
The species Staphylococcus argenteus was separated recently from Staphylococcus aureus (Tong S.Y., F. Schaumburg, M.J. Ellington, J. Corander, B. Pichon, F. Leendertz, S.D. Bentley, J. Parkhill, D.C. Holt, G. Peters, and P.M. Giffard, 2015). The objective of this work was to characterise the genome of a non-human S. argenteus strain, which had been isolated from the faeces of a wild-living western lowland gorilla in Gabon, and analyse the spectrum of this species in matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). The full genome sequence revealed a scarcity of virulence genes and absence of resistance genes, indicating a decreased virulence potential compared to S. aureus and the human methicillin-resistant S. argenteus isolate MSHR1132(T). Spectra obtained by MALDI-TOF MS and the analysis of available sequences in the genome databases identified several MALDI-TOF MS signals that clearly differentiate S. argenteus, the closely related Staphylococcus schweitzeri and S. aureus. In conclusion, in the absence of biochemical tests that identify the three species, mass spectrometry should be employed as method of choice.
Khot, Prasanna D; Fisher, Mark A
Shigella species are so closely related to Escherichia coli that routine matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) cannot reliably differentiate them. Biochemical and serological methods are typically used to distinguish these species; however, "inactive" isolates of E. coli are biochemically very similar to Shigella species and thus pose a greater diagnostic challenge. We used ClinProTools (Bruker Daltonics) software to discover MALDI-TOF MS biomarker peaks and to generate classification models based on the genetic algorithm to differentiate between Shigella species and E. coli. Sixty-six Shigella spp. and 72 E. coli isolates were used to generate and test classification models, and the optimal models contained 15 biomarker peaks for genus-level classification and 12 peaks for species-level classification. We were able to identify 90% of E. coli and Shigella clinical isolates correctly to the species level. Only 3% of tested isolates were misidentified. This novel MALDI-TOF MS approach allows laboratories to streamline the identification of E. coli and Shigella species.
Fangous, Marie-Sarah; Mougari, Faiza; Gouriou, Stéphanie; Calvez, Elodie; Raskine, Laurent; Cambau, Emmanuelle; Payan, Christopher
Mycobacterium abscessus, as a species, has been increasingly implicated in respiratory infections, notably in cystic fibrosis patients. The species comprises 3 subspecies, which can be difficult to identify. Since they differ in antibiotic susceptibility and clinical relevance, developing a routine diagnostic tool discriminating Mycobacterium abscessus at the subspecies level is a real challenge. Forty-three Mycobacterium abscessus species isolates, previously identified by multilocus sequence typing, were analyzed by matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS). A subspecies identification algorithm, based on five discriminating peaks, was drawn up and validated by blind identification of a further 49 strains, 94% of which (n = 46) were correctly identified. Two M. abscessus subsp. massiliense strains were misidentified as M. abscessus subsp. abscessus, and for 1 other strain identification failed. Inter- and intralaboratory reproducibility tests were conclusive. This study presents, for the first time, a classification algorithm for MALDI-TOF MS identification of the 3 M. abscessus subspecies. MALDI-TOF MS proved effective in discriminating within the M. abscessus species and might be easily integrated into the workflow of microbiology labs. PMID:25009048
Moura, Hercules; Izquierdo, Fernando; Woolfitt, Adrian R; Wagner, Glauber; Pinto, Tatiana; del Aguila, Carmen; Barr, John R
Emerging methods based on mass spectrometry (MS) can be used in the rapid identification of microorganisms. Thus far, these practical and rapidly evolving methods have mainly been applied to characterize prokaryotes. We applied matrix-assisted laser-desorption-ionization-time-of-flight mass spectrometry MALDI-TOF MS in the analysis of whole cells of 18 N. fowleri isolates belonging to three genotypes. Fourteen originated from the cerebrospinal fluid or brain tissue of primary amoebic meningoencephalitis patients and four originated from water samples of hot springs, rivers, lakes or municipal water supplies. Whole Naegleria trophozoites grown in axenic cultures were washed and mixed with MALDI matrix. Mass spectra were acquired with a 4700 TOF-TOF instrument. MALDI-TOF MS yielded consistent patterns for all isolates examined. Using a combination of novel data processing methods for visual peak comparison, statistical analysis and proteomics database searching we were able to detect several biomarkers that can differentiate all species and isolates studied, along with common biomarkers for all N. fowleri isolates. Naegleria fowleri could be easily separated from other species within the genus Naegleria. A number of peaks detected were tentatively identified. MALDI-TOF MS fingerprinting is a rapid, reproducible, high-throughput alternative method for identifying Naegleria isolates. This method has potential for studying eukaryotic agents.
Nhu, Truong Quynh; Park, Seong Bin; Kim, Si Won; Lee, Jung Seok; Im, Se Pyeong; Lazarte, Jassy Mary S.; Seo, Jong Pyo; Lee, Woo-Jai; Kim, Jae Sung
Edwardsiella (E.) ictaluri is a major bacterial pathogen that affects commercially farmed striped catfish (Pangasius hypothalamus) in Vietnam. In a previous study, 19 strains of E. ictaluri collected from striped catfish were biochemically identified with an API-20E system. Here, the same 19 strains were used to assess the ability of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS; applied using a MALDI Biotyper) to conduct rapid, easy and accurate identification of E. ictaluri. MALDI-TOF MS could directly detect the specific peptide patterns of cultured E. ictaluri colonies with high (> 2.0, indicating species-level identification) scores. MALDI Biotyper 3.0 software revealed that all of the strains examined in this study possessed highly similar peptide peak patterns. In addition, electrophoresis (SDS-PAGE) and subsequent immuno-blotting using a specific chicken antibody (IgY) against E. ictaluri revealed that the isolates had highly similar protein profiles and antigenic banding profiles. The results of this study suggest that E. ictaluri isolated from striped catfish in Vietnam have homologous protein compositions. This is important, because it indicates that MALDI-TOF MS analysis could potentially outperform the conventional methods of identifying E. ictaluri. PMID:26726022
Wang, Ting Yu; Zhou, Hua; Wong, Yuen Fan; Wu, Pui Kei; Hsiao, Wen-Luan Wendy; Leung, Elaine Lai-Han; Liu, Liang
Qingfu Guanjieshu (QFGJS) is an herbal preparation for treating rheumatoid arthritis (RA). Previous studies revealed that QFGJS significantly inhibited experimental arthritis and acute inflammation, accompanied by reduction of proinflammatory cytokines and elevation of anti-inflammatory cytokines. This study aims to identify the targeted proteins and predict the proteomic network associated with the drug action of QFGJS by using 2D gel and MALDI-TOF-MS/MS techniques. Thirty female Wistar rats were evenly grouped as normal and vehicle- and QFGJS-treated CIA rats. The antiarthritic effect of QFGJS was examined with a 19-day treatment course, and the knee synovial tissues of animals from each group were obtained for 2D gel and MALDI-TOF-MS/MS analysis. Results showed that QFGJS significantly ameliorated collagen II-induced arthritis when administrated at 2.8 g/kg body weight for 19 days. 2D gel image analysis revealed 89 differentially expressed proteins in the synovial tissues among the normal and vehicle- and QFGJS-treated CIA rats from over 1000 proteins of which 63 proteins were identified by MALDI-TOF-MS/MS analysis, and 32 proteins were included for classification of functions using Gene Ontology (GO) method. Finally, 14 proteins were analyzed using bioinformatics, and a predicted proteomic network related to the anti-arthritic effect of QFGJS was established, and Pgk1 plays a central role. PMID:23781264
Plenz, Bastian; Schmidt, Volker; Grosse-Herrenthey, Anke; Krüger, Monika; Pees, Michael
The aim of this study was to identify aerobic bacterial isolates from the respiratory tract of boids with matrix-assisted laser desorption ionisation-time-of-flight mass spectrometry (MALDI-TOF MS). From 47 boid snakes, swabs from the oral cavity, tracheal wash samples and, in cases in which postmortem examination was performed, pulmonary tissue samples were taken. Each snake was classified as having inflammation of the respiratory tract and/or oral cavity, or without evidence of inflammation based on combination of clinical, cytological and histopathological findings. Samples collected from the respiratory tract and oral cavity were inoculated onto routine media and bacteria were cultured aerobically. All morphologically distinct individual colonies obtained were analysed using MALDI-TOF MS. Unidentified isolates detected in more than three snakes were selected for further 16S rDNA PCR and sequencing. Among all examined isolates (n=243), 49 per cent (n=119) could be sufficiently speciated using MALDI-TOF MS. Molecular biology revealed several bacterial species that have not been previously described in reptiles. With an average of 6.3 different isolates from the respiratory tract and/or oral cavity, boids with inflammatory disease harboured significantly more bacterial species than boids without inflammatory disease (average 2.8 isolates).
Petkovic, Marijana; Petrovic, Biljana; Savic, Jasmina; Bugarcic, Zivadin D.; Dimitric-Markovic, Jasmina; Momic, Tatjana; Vasic, Vesna
Matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a suitable method for the analysis of inorganic and organic compounds and biomolecules. This makes MALDI-TOF MS convenient for monitoring the interaction of metallo-drugs with biomolecules. Results presented in this manuscript demonstrate that flavonoids such as apigenin, kaempferol and luteolin are suitable for MALDI-TOF MS analysis of Pt(II), Pd(II), Pt(IV) and Ru(III) complexes, giving different signal-to-noise ratios of the analyte peak. The MALDI-TOF mass spectra of inorganic complexes acquired with these flavonoid matrices are easy to interpret and have some advantages over the application of other commonly used matrices: a low number of matrix peaks are detectable and the coordinative metal-ligand bond is, in most cases, preserved. On the other hand, flavonoids do not act as typical matrices, as their excess is not required for the acquisition of MALDI-TOF mass spectra of inorganic complexes.
Yaman, Gorkem; Ciftci, Ugur; Laleli, Yahya Rauf
In clinical microbiology laboratories, routine microbial identification is mostly performed using culture based methodologies requiring 24 to 72 hours from culturing to identification. Matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) technology has been established as a cost effective, reliable, and faster alternative identification platform. In this study, we evaluated the reliability of the two available MALDI-TOF MS systems for their routine clinical level identification accuracy and efficiency in a clinical microbiology laboratory setting. A total of 1,341 routine phenotypically identified clinical bacterial and fungal isolates were selected and simultaneously analyzed using VITEK MS (bioMérieux, France) and Microflex LT (Bruker Diagnostics, Germany) MALDI-TOF MS systems. For any isolate that could not be identified with either of the systems and for any discordant result, 16S rDNA gene or ITS1/ITS2 sequencing was used. VITEK MS and Microflex LT correctly identified 1,303 (97.17%) and 1,298 (96.79%) isolates to the species level, respectively. In 114 (8.50%) isolates initial phenotypic identification was inaccurate. Both systems showed a similar identification efficiency and workflow robustness, and they were twice as more accurate compared to routine phenotypic identification in our sample pool. MALDITOF systems with their accuracy and robustness offer a good identification platform for routine clinical microbiology laboratories. PMID:26793718
Fall, Bécaye; Lo, Cheikh Ibrahima; Samb-Ba, Bissoume; Perrot, Nadine; Diawara, Silman; Gueye, Mamadou Wague; Sow, Kowry; Aubadie-Ladrix, Maxence; Mediannikov, Oleg; Sokhna, Cheikh; Diemé, Yaya; Chatellier, Sonia; Wade, Boubacar; Raoult, Didier; Fenollar, Florence
Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) represents a revolution in routine pathogen identification in clinical microbiology laboratories. A MALDI-TOF MS was introduced to tropical Africa in the clinical microbiology laboratory of the Hôpital Principal de Dakar (Senegal) and used for routine pathogen identification. Using MS, 2,429 bacteria and fungi isolated from patients were directly assayed, leading to the identification of 2,082 bacteria (85.7%) and 206 fungi (8.5%) at the species level, 109 bacteria (4.5%) at the genus level, and 16 bacteria (0.75%) at the family level. Sixteen isolates remained unidentified (0.75%). Escherichia coli was the most prevalent species (25.8%) followed by Klebsiella pneumoniae (14.8%), Streptococcus agalactiae (6.2%), Acinetobacter baumannii (6.1%), Pseudomonas aeruginosa (5.9%), and Staphylococcus aureus (5.9%). MALDI-TOF MS has also enabled the detection of rare bacteria and fungi. MALDI-TOF MS is a powerful tool for the identification of bacterial and fungal species involved in infectious diseases in tropical Africa.
Zhang, Ying-Tao; Geng, Yi-Ping; Zhou, Le; Lai, Bao-Chang; Si, Lv-Sheng; Wang, Yi-Li
AIM: To conduct the proteomic analysis of human colorectal carcinoma cell line, SW480 by using two-dimensional electrophoresis (2-DE) and matrix-assisted laser desorption /ionization-time of flight mass spectrometry (MALDI-TOFMS). METHODS: The total proteins of human colorectal carcinoma cell line, SW480 were separated with 2-DE by using immobilized pH gradient strips and visualized by staining with silver nitrate. The gel images were acquired by scanner and 2-DE analysis software, Image Master 2D Elite. Nineteen distinct protein spots were excised from gel randomly and digested in gel by TPCK-trypsin. Mass analysis of the tryptic digest peptides mixture was performed by using MALDI-TOF MS. Peptide mass fingerprints (PMFs) obtained by the MALDI-TOF analysis were used to search NCBI, SWISS-PROT and MSDB databases by using Mascot software. RESULTS: PMF maps of all spots were obtained by MALDI-TOF MS and thirteen proteins were preliminarily identified. CONCLUSION: The methods of analysis and identification of protein spots of tumor cells in 2-DE gel with silver staining by MALDI-TOF MS derived PMF have been established. Protein expression profile of SW480 has been obtained. It is demonstrated that a combination of proteomics and cell culture is a useful approach to comprehend the process of colon carcinogenesis. PMID:16094709
Singhal, Neelja; Kumar, Manish; Kanaujia, Pawan K.; Virdi, Jugsharan S.
Currently microorganisms are best identified using 16S rRNA and 18S rRNA gene sequencing. However, in recent years matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has emerged as a potential tool for microbial identification and diagnosis. During the MALDI-TOF MS process, microbes are identified using either intact cells or cell extracts. The process is rapid, sensitive, and economical in terms of both labor and costs involved. The technology has been readily imbibed by microbiologists who have reported usage of MALDI-TOF MS for a number of purposes like, microbial identification and strain typing, epidemiological studies, detection of biological warfare agents, detection of water- and food-borne pathogens, detection of antibiotic resistance and detection of blood and urinary tract pathogens etc. The limitation of the technology is that identification of new isolates is possible only if the spectral database contains peptide mass fingerprints of the type strains of specific genera/species/subspecies/strains. This review provides an overview of the status and recent applications of mass spectrometry for microbial identification. It also explores the usefulness of this exciting new technology for diagnosis of diseases caused by bacteria, viruses, and fungi. PMID:26300860
Genetic, phenotypic and matrix-assisted laser desorption ionization time-of-flight mass spectrometry-based identification of anaerobic bacteria and determination of their antimicrobial susceptibility at a University Hospital in Japan.
Yunoki, Tomoyuki; Matsumura, Yasufumi; Nakano, Satoshi; Kato, Karin; Hotta, Go; Noguchi, Taro; Yamamoto, Masaki; Nagao, Miki; Takakura, Shunji; Ichiyama, Satoshi
The accuracies of matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) and the phenotypic method using VITEK 2 were compared to the accuracy of 16S rRNA sequence analysis for the identification of 170 clinically isolated anaerobes. The antimicrobial susceptibility of the isolates was also evaluated. Genetic analysis identified 21 Gram-positive species in 14 genera and 29 Gram-negative species in 11 genera. The most frequently isolated genera were Prevotella spp. (n = 46), Bacteroides spp. (n = 25) and Clostridium spp. (n = 25). MALDI-TOF MS correctly identified more isolates compared with VITEK 2 at the species (80 vs. 58%, respectively; p < 0.01) and genus (85 vs. 71%, respectively; p < 0.01) levels. More than 90% of the isolates of the three major genera identified (Prevotella, Bacteroides, and Clostridium species other than Clostridium difficile) were susceptible to beta-lactam/beta-lactamase inhibitor combinations, carbapenems, metronidazole and chloramphenicol. MALDI-TOF MS provided better identification results than VITEK2. Commonly used anti-anaerobic agents indicated that the isolates of the three most frequently identified anaerobic genera exhibited good antimicrobial susceptibility.
Saleeb, Paul G; Drake, Steven K; Murray, Patrick R; Zelazny, Adrian M
Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has recently been introduced into the clinical microbiology laboratory as a rapid and accurate method to identify bacteria and yeasts. In this paper we describe our work on the use of MALDI-TOF MS for the identification of mycobacterial isolates. We developed a protocol for protein extraction from mycobacteria and utilized it to construct a database containing 42 clinically relevant type and reference strains of mycobacteria. The database was used to identify 104 clinical isolates of mycobacteria. All members of the Mycobacterium tuberculosis complex were identified accurately at the complex level but could not be separated at the species level. All other organisms were identified at the species level, with the exception of one strain of M. kansasii (accurately identified but with a low spectral score) and three pairs of closely related strains: M. abscessus and M. massiliense, M. mucogenicum and M. phocaicum, and M. chimaera and M. intracellulare. These pairs of organisms can currently be identified only by multilocus gene sequence analysis. We conclude that MALDI-TOF MS analysis can be incorporated into the work flow of the microbiology laboratory for rapid and accurate identification of most strains of mycobacteria isolated from solid growth media.
Huang, Bin; Zhang, Lei; Zhang, Weizheng; Liao, Kang; Zhang, Shihong; Zhang, Zhiquan; Ma, Xingyan; Chen, Jialong; Zhang, Xiuhong; Qu, Pinghua; Wu, Shangwei; Chen, Cha; Tang, Yi-Wei
Rapid and accurate detection and identification of microbial pathogens causing urinary tract infections allows prompt and specific treatment. We optimized the specimen processing to maximize the limit of detection (LOD) by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) and evaluated the capacity of combination of MALDI-TOF MS and urine analysis (UA) for direct detection and identification of bacterial pathogens from urine samples. The optimal volumes of processed urine, formic acid/acetonitrile and supernatant spotted onto the target plate were 15 mL, 3 μL and 3 μL, respectively, yielding a LOD of 1.0×10(5) colony-forming units/mL. Among the total of 1,167 urine specimens collected from three hospital centers, 612 (52.4%) and 351 (30.1%) were respectively positive by UA and urine culture. Compared with a reference method comprised of urine culture and 16S rRNA gene sequencing, the sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of MALDI-TOF MS alone and MALDI-TOF MS coupled with UA were 86.6% vs 93.4% (χ(2)=8.93, P<0.01), 91.5% vs 96.3% (χ(2)=7.06, P<0.01), 81.5% vs 96.4% (χ(2)=37.32, P<0.01) and 94.1% vs 93.1% (χ(2)=0.40, P>0.05), respectively. No significant performance differences were revealed among the three sites while the specificity and NPV of MALDI-TOF MS for males were significantly higher than for females (specificity: 94.3% vs 77.3%, χ(2)=44.90, P<0.01; NPV: 95.5% vs 86.1%, χ(2)=18.85, P<0.01). Our results indicated that optimization of specimen processing significantly enhanced analytical sensitivity, and the combination of UA and MALDI-TOF MS provided an accurate and rapid detection and identification of bacterial pathogens directly from urine.
Vithanage, Nuwan R; Yeager, Thomas R; Jadhav, Snehal R; Palombo, Enzo A; Datta, Nivedita
Psychrotrophic bacteria in raw milk produce heat-resistant extracellular proteases, resulting in spoilage and shelf-life reduction of ultrahigh temperature treated milk and milk products. Controlling of these spoilage microbes requires rapid and reliable identification systems for screening of raw milk. This study aimed to compare commercial bacterial identification systems with a genetic method (considered as the 'gold standard' method) for the identification of heat-resistant protease producing bacteria in raw milk. Five bacterial identification systems were compared based on typability, discrimination power (i.e. Simpson's Index of Diversity), reproducibility and speed of analysis. The accuracy of 16S rRNA gene sequencing, Biolog, MALDI-TOF MS, API, and Microbact for the identification of Gram negative bacilli at the species level was 100.0%, 86.8%, 63.2%, 60.5% and 57.9%, respectively. The Gram positive bacilli were identified by 16S rRNA gene sequencing, Biolog, MALDI-TOF MS, and API with accuracies at the species level of 100.0%, 85.0%, 95.0% and 90.0%, respectively. The 16S rRNA gene sequencing and phylogenetic analysis discriminated Pseudomonas fluorescens, Pseudomonas syringae, Hafnia alvei, Bacillus cereus, Bacillus pumilus and Bacillus licheniformis to the subspecies level. The Simpson's Index of Diversity scores were 0.966, 0.711, 0.496, 0.472, and 0.140, for 16S rRNA gene sequencing, Biolog, MALDI-TOF MS, API and Microbact, respectively. Limited reference profiles in the databases of Biolog, MALDI-TOF MS, API and Microbact systems reduced their accuracy in bacterial identification, compared to 16S rRNA gene sequencing. The rapidity of each assay is in the following order; MALDI-TOF MS>16S rRNA gene sequencing>Biolog>Microbact>API. The reproducibility of the assays is in the order of 16S rRNA gene sequencing>API>Microbact>MALDI-TOF MS>Biolog. Thus, 16S rRNA gene sequencing appears to be the most reliable and robust system for the identification of dairy
Gorasia, Dhana G.; Veith, Paul D.; Hanssen, Eric G.; Glew, Michelle D.; Sato, Keiko; Yukitake, Hideharu; Nakayama, Koji; Reynolds, Eric C.
The type IX secretion system (T9SS) has been recently discovered and is specific to Bacteroidetes species. Porphyromonas gingivalis, a keystone pathogen for periodontitis, utilizes the T9SS to transport many proteins including the gingipain virulence factors across the outer membrane and attach them to the cell surface via a sortase-like mechanism. At least 11 proteins have been identified as components of the T9SS including PorK, PorL, PorM, PorN and PorP, however the precise roles of most of these proteins have not been elucidated and the structural organization of these components is unknown. In this study, we purified PorK and PorN complexes from P. gingivalis and using electron microscopy we have shown that PorN and the PorK lipoprotein interact to form a 50 nm diameter ring-shaped structure containing approximately 32–36 subunits of each protein. The formation of these rings was dependent on both PorK and PorN, but was independent of PorL, PorM and PorP. PorL and PorM were found to form a separate stable complex. PorK and PorN were protected from proteinase K cleavage when present in undisrupted cells, but were rapidly degraded when the cells were lysed, which together with bioinformatic analyses suggests that these proteins are exposed in the periplasm and anchored to the outer membrane via the PorK lipid. Chemical cross-linking and mass spectrometry analyses confirmed the interaction between PorK and PorN and further revealed that they interact with the PG0189 outer membrane protein. Furthermore, we established that PorN was required for the stable expression of PorK, PorL and PorM. Collectively, these results suggest that the ring-shaped PorK/N complex may form part of the secretion channel of the T9SS. This is the first report showing the structural organization of any T9SS component. PMID:27509186
Gorasia, Dhana G; Veith, Paul D; Hanssen, Eric G; Glew, Michelle D; Sato, Keiko; Yukitake, Hideharu; Nakayama, Koji; Reynolds, Eric C
The type IX secretion system (T9SS) has been recently discovered and is specific to Bacteroidetes species. Porphyromonas gingivalis, a keystone pathogen for periodontitis, utilizes the T9SS to transport many proteins including the gingipain virulence factors across the outer membrane and attach them to the cell surface via a sortase-like mechanism. At least 11 proteins have been identified as components of the T9SS including PorK, PorL, PorM, PorN and PorP, however the precise roles of most of these proteins have not been elucidated and the structural organization of these components is unknown. In this study, we purified PorK and PorN complexes from P. gingivalis and using electron microscopy we have shown that PorN and the PorK lipoprotein interact to form a 50 nm diameter ring-shaped structure containing approximately 32-36 subunits of each protein. The formation of these rings was dependent on both PorK and PorN, but was independent of PorL, PorM and PorP. PorL and PorM were found to form a separate stable complex. PorK and PorN were protected from proteinase K cleavage when present in undisrupted cells, but were rapidly degraded when the cells were lysed, which together with bioinformatic analyses suggests that these proteins are exposed in the periplasm and anchored to the outer membrane via the PorK lipid. Chemical cross-linking and mass spectrometry analyses confirmed the interaction between PorK and PorN and further revealed that they interact with the PG0189 outer membrane protein. Furthermore, we established that PorN was required for the stable expression of PorK, PorL and PorM. Collectively, these results suggest that the ring-shaped PorK/N complex may form part of the secretion channel of the T9SS. This is the first report showing the structural organization of any T9SS component.
Verroken, A.; Janssens, M.; Berhin, C.; Bogaerts, P.; Huang, T.-D.; Wauters, G.; Glupczynski, Y.
The identification of Nocardia species, usually based on biochemical tests together with phenotypic in vitro susceptibility and resistance patterns, is a difficult and lengthy process owing to the slow growth and limited reactivity of these bacteria. In this study, a panel of 153 clinical and reference strains of Nocardia spp., altogether representing 19 different species, were characterized by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). As reference methods for species identification, full-length 16S rRNA gene sequencing and phenotypical biochemical and enzymatic tests were used. In a first step, a complementary homemade reference database was established by the analysis of 110 Nocardia isolates (pretreated with 30 min of boiling and extraction) in the MALDI BioTyper software according to the manufacturer's recommendations for microflex measurement (Bruker Daltonik GmbH, Leipzig, Germany), generating a dendrogram with species-specific cluster patterns. In a second step, the MALDI BioTyper database and the generated database were challenged with 43 blind-coded clinical isolates of Nocardia spp. Following addition of the homemade database in the BioTyper software, MALDI-TOF MS provided reliable identification to the species level for five species of which more than a single isolate was analyzed. Correct identification was achieved for 38 of the 43 isolates (88%), including 34 strains identified to the species level and 4 strains identified to the genus level according to the manufacturer's log score specifications. These data suggest that MALDI-TOF MS has potential for use as a rapid (<1 h) and reliable method for the identification of Nocardia species without any substantial costs for consumables. PMID:20861335
Perreault, H.; Hronowski, X. L.; Koul, O.; Street, J.; McCluer, R. H.; Costello, C. E.
Stage-specific embryonic antigen (SSEA) glycosphingolipids (GSLs) found in the central nervous system are implicated in regulating cell-cell recognition, targeting and migration of cells during development. Through the action of fucosyltransferase enzymes, SSEA-1 (Lewisx) glycolipids are biosynthesized in the brain by fucosylation of lipid substrates with the neolacto series glycolipid core structure [Gal[beta]1 --> 4GlcNAc[beta]1 --> 3Gal[beta]1 --> 4Glc[beta]1 --> 1'Cer] (originally termed paragloboside) or its higher analogs. In order to optimize methodology for high sensitivity structural determinations of SSEA-1 type glycolipids from fetal calf brain, potential precursors and SSEA-1 glycolipids of previously established structure were first isolated from bovine erythrocytes and beige mutant mouse kidney, purified by column chromatography and characterized by matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) MS, liquid secondary ionization mass spectrometry (LSIMS), and tandem mass spectrometry (MS/MS), among other techniques. Peracetylated derivatives were detected at the low femtomole level by MALDI-TOF MS and the subnanomole level by LSIMS. MALDI-TOF MS produced mainly [M + Na] + and [M + K]+ species. On the basis of the direct and tandem mass spectral analyses of peracetylated and permethylated derivatives, the carbohydrate sequences in the selected bovine erythrocyte and mouse kidney GSL fractions were found to be consistent with those of glycolipids previously-reported from larger-scale studies of these sources. Their heterogeneous ceramide moieties were characterized by collision induced decomposition (CID) MS/MS of abundant Z0-type ions in the LSI mass spectra of the permethylated GSLs. MALDI-PSD-TOF mass spectral analyses of low and subpicomole amounts of derivatized GSL fractions from fetal calf brain provided carbohydrate sequence information that indicates the presence of mono- and difucosylated SSEA-1 neolacto series
Zboromyrska, Y; Rubio, E; Alejo, I; Vergara, A; Mons, A; Campo, I; Bosch, J; Marco, F; Vila, J
The current gold standard method for the diagnosis of urinary tract infections (UTI) is urine culture that requires 18-48 h for the identification of the causative microorganisms and an additional 24 h until the results of antimicrobial susceptibility testing (AST) are available. The aim of this study was to shorten the time of urine sample processing by a combination of flow cytometry for screening and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) for bacterial identification followed by AST directly from urine. The study was divided into two parts. During the first part, 675 urine samples were processed by a flow cytometry device and a cut-off value of bacterial count was determined to select samples for direct identification by MALDI-TOF-MS at ≥5 × 10(6) bacteria/mL. During the second part, 163 of 1029 processed samples reached the cut-off value. The sample preparation protocol for direct identification included two centrifugation and two washing steps. Direct AST was performed by the disc diffusion method if a reliable direct identification was obtained. Direct MALDI-TOF-MS identification was performed in 140 urine samples; 125 of the samples were positive by urine culture, 12 were contaminated and 3 were negative. Reliable direct identification was obtained in 108 (86.4%) of the 125 positive samples. AST was performed in 102 identified samples, and the results were fully concordant with the routine method among 83 monomicrobial infections. In conclusion, the turnaround time of the protocol described to diagnose UTI was about 1 h for microbial identification and 18-24 h for AST.
Comparison of matrix-assisted laser desorption ionization-time of flight mass spectrometry and molecular biology techniques for identification of Culicoides (Diptera: ceratopogonidae) biting midges in senegal.
Sambou, Masse; Aubadie-Ladrix, Maxence; Fenollar, Florence; Fall, Becaye; Bassene, Hubert; Almeras, Lionel; Sambe-Ba, Bissoume; Perrot, Nadine; Chatellier, Sonia; Faye, Ngor; Parola, Philippe; Wade, Boubacar; Raoult, Didier; Mediannikov, Oleg
Biting midges of the genus Culicoides are implicated as vectors for a wide variety of pathogens. The morphological identification of these arthropods may be difficult because of a lack of detailed investigation of taxonomy for this species in Africa. However, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) profiling is efficient for arthropod identification at the species level. This study established a spectrum database of Culicoides spp. from Senegal using MALDI-TOF. Identification of Culicoides insects to the species level before mass spectrometry was performed on the basis of morphological characters. MALDI-TOF MS reference spectra were determined for 437 field-caught Culicoides of 10 species. The protein profiles of all tested Culicoides revealed several peaks with mass ranges of 2 to 20 kDa. In a validation study, 72 Culicoides specimens in the target species were correctly identified at the species level with a similarity of 95 to 99.9%. Four Culicoides protein profiles were misidentified. Nevertheless, six SuperSpectra (C. imicola, C. enderleini, C. oxystoma, C. kingi, C. magnus, and C. fulvithorax) were created. Abdomens of midges were used to amplify and sequence a portion of the mitochondrial cytochrome oxidase I gene (COI). The results obtained using the MALDI-TOF MS method were consistent with the morphological identification and similar to the genetic identification. Protein profiling using MALDI-TOF is an efficient approach for the identification of Culicoides spp., and it is economically advantageous for approaches that require detailed and quantitative information of vector species that are collected in field. The database of African Culicoides MS spectra created is the first database in Africa. The COI sequences of five Culicoides species that were previously noncharacterized using molecular methods were deposited in GenBank.
Agustini, Bruna Carla; Silva, Luciano Paulino; Bloch, Carlos; Bonfim, Tania M B; da Silva, Gildo Almeida
Yeast identification using traditional methods which employ morphological, physiological, and biochemical characteristics can be considered a hard task as it requires experienced microbiologists and a rigorous control in culture conditions that could implicate in different outcomes. Considering clinical or industrial applications, the fast and accurate identification of microorganisms is a crescent demand. Hence, molecular biology approaches has been extensively used and, more recently, protein profiling using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has proved to be an even more efficient tool for taxonomic purposes. Nonetheless, concerning to mass spectrometry, data available for the differentiation of yeast species for industrial purpose is limited and reference databases commercially available comprise almost exclusively clinical microorganisms. In this context, studies focusing on environmental isolates are required to extend the existing databases. The development of a supplementary database and the assessment of a commercial database for taxonomic identifications of environmental yeast are the aims of this study. We challenge MALDI-TOF MS to create protein profiles for 845 yeast strains isolated from grape must and 67.7 % of the strains were successfully identified according to previously available manufacturer database. The remaining 32.3 % strains were not identified due to the absence of a reference spectrum. After matching the correct taxon for these strains by using molecular biology approaches, the spectra concerning the missing species were added in a supplementary database. This new library was able to accurately predict unidentified species at first instance by MALDI-TOF MS, proving it is a powerful tool for the identification of environmental yeasts.
Mino, S.; Nakagawa, S.; Sawabe, T.; Miyazaki, J.; Makita, H.; Nunoura, T.; Yamamoto, M.; Takai, K.
Deep-sea hydrothermal fields are areas on the seafloor of high biological productivity fueled primarily by microbial chemosynthesis. Chemolithoautotrophic Epsilonproteobacteria and Persephonella with an ability to utilize inorganic substrates such as elemental sulfur and hydrogen are important members in wide range of temperature conditions in deep-sea hydrothermal vents. However, little is known about their population genetic structure such as intraspecific genetic diversity, distribution pattern, and phenotypic characteristics. Previously, using genetic approach based on multi-locus sequence analysis (MLSA), we clarified that Epsilonproteobacteria Group A, B, F, and Persephonella populations were geographically separated, and Epsilonproteobacteria appeared to diverge by mutation rather than recombination. Contrary to genetic evidence for allopatric segregation in deep-sea chemoautotrophs, however, phenotypic evidence has never been found. In addition, analyzing such a phenotypic characteristic may lead to a better understanding of the interactions microbes have with their environment. In this study, we present a metabolomic approach based on matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) to reveal phenotypic biogeographical discrimination. We demonstrated the whole-cell MALDI-TOF MS method on Epsilonproteobacteria and Persephonella populations. These chemoautotrophic strains used in this study were isolated from chimney structures, vent fluids, and hydrothermal sediments. These hydrothermal samples were collected from geographically separated hydrothermal areas of the South Mariana Trough, Okinawa Trough and Central Indian Ridge. Based on mass peaks (signal/noise >10) within the m/z range of 2000-14000, phenotypic analysis was carried out by cluster analysis. The result of phenotypic analysis was compared with the genotypic clusters. The whole-cell MALDI-TOF MS revealed that Persephonella population was identified to
Mather, Cheryl A.; Werth, Brian J.; Sivagnanam, Shobini; SenGupta, Dhruba J.
Vancomycin is the standard of care for the treatment of invasive methicillin-resistant Staphylococcus aureus (MRSA) infections. Infections with vancomycin-nonsusceptible MRSA, including vancomycin-intermediate S. aureus (VISA) and heterogeneous VISA (hVISA), are clinically challenging and are associated with poor patient outcomes. The identification of VISA in the clinical laboratory depends on standard susceptibility testing, which takes at least 24 h to complete after isolate subculture, whereas hVISA is not routinely detected in clinical labs. We therefore sought to determine whether VISA and hVISA can be differentiated from vancomycin-susceptible S. aureus (VSSA) using the spectra produced by matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS). Strains of MRSA were characterized for vancomycin susceptibility phenotype by broth microdilution and modified population analysis. We tested 21 VISA, 21 hVISA, and 38 VSSA isolates by MALDI-TOF MS. Susceptibility phenotypes were separated by using a support vector machine (SVM) machine learning algorithm. The resulting model was validated by leave-one-out cross validation. Models were developed and validated by using spectral profiles generated under various subculture conditions, as well as with and without hVISA strains. Using SVM, we correctly identified 100% of the VISA and 97% of the VSSA isolates with an overall classification accuracy of 98%. Addition of hVISA to the model resulted in 76% hVISA identification, 100% VISA identification, and 89% VSSA identification, for an overall classification accuracy of 89%. We conclude that VISA/hVISA and VSSA isolates are separable by MALDI-TOF MS with SVM analysis. PMID:26763961
Eriksson, Helena; Bagge, Elisabeth; Båverud, Viveca; Fellström, Claes; Jansson, Désirée S
This study investigated organic laying hen farms for the presence of Erysipelothrix rhusiopathiae in the house environment and from potential carriers (i.e. insects and mice) during ongoing erysipelas outbreaks, and compared the obtained isolates with those from laying hens. The samples were investigated by selective culture followed by species-specific polymerase chain reaction on cultures. E. rhusiopathiae was isolated from the spleen, jejunal contents, manure, dust and swabs from water nipples. Three more samples from the house environment tested positive by polymerase chain reaction compared with selective culture alone. Selected isolates were investigated by pulsed-field gel electrophoresis (PFGE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). One farm was represented by isolates from laying hens only, and one of these isolates differed in one PFGE band from the others. Different banding patterns were observed for isolates from laying hens and manure on one farm. On the remaining two farms, the isolates from the house environment and laying hens were identical but differed between farms. Outbreaks reoccurred in the next flock on two of the farms, and different PFGE types were isolated from consecutive flocks. Our results suggest an external source of infection, which would explain the previously reported increased risk of outbreaks in free-range flocks. Contaminated manure and dust may represent sources of transmission. For the isolates, MALDI-TOF MS and biochemical typing results were in agreement but, since the type strain of Erysipelothrix tonsillarum was typed as E. rhusiopathiae using MALDI-TOF MS, further studies into this method are needed.
Gautier, M; Normand, A-C; Ranque, S
The use of multi-locus DNA sequence analysis has led to the description of previously unknown 'cryptic' Aspergillus species, whereas classical morphology-based identification of Aspergillus remains limited to the section or species-complex level. The current literature highlights two main features concerning these 'cryptic' Aspergillus species. First, the prevalence of such species in clinical samples is relatively high compared with emergent filamentous fungal taxa such as Mucorales, Scedosporium or Fusarium. Second, it is clearly important to identify these species in the clinical laboratory because of the high frequency of antifungal drug-resistant isolates of such Aspergillus species. Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) has recently been shown to enable the identification of filamentous fungi with an accuracy similar to that of DNA sequence-based methods. As MALDI-TOF MS is well suited to the routine clinical laboratory workflow, it facilitates the identification of these 'cryptic' Aspergillus species at the routine mycology bench. The rapid establishment of enhanced filamentous fungi identification facilities will lead to a better understanding of the epidemiology and clinical importance of these emerging Aspergillus species. Based on routine MALDI-TOF MS-based identification results, we provide original insights into the key interpretation issues of a positive Aspergillus culture from a clinical sample. Which ubiquitous species that are frequently isolated from air samples are rarely involved in human invasive disease? Can both the species and the type of biological sample indicate Aspergillus carriage, colonization or infection in a patient? Highly accurate routine filamentous fungi identification is central to enhance the understanding of these previously unknown Aspergillus species, with a vital impact on further improved patient care.
Misidentification of Aspergillus nomius and Aspergillus tamarii as Aspergillus flavus: characterization by internal transcribed spacer, β-Tubulin, and calmodulin gene sequencing, metabolic fingerprinting, and matrix-assisted laser desorption ionization-time of flight mass spectrometry.
Tam, Emily W T; Chen, Jonathan H K; Lau, Eunice C L; Ngan, Antonio H Y; Fung, Kitty S C; Lee, Kim-Chung; Lam, Ching-Wan; Yuen, Kwok-Yung; Lau, Susanna K P; Woo, Patrick C Y
Aspergillus nomius and Aspergillus tamarii are Aspergillus species that phenotypically resemble Aspergillus flavus. In the last decade, a number of case reports have identified A. nomius and A. tamarii as causes of human infections. In this study, using an internal transcribed spacer, β-tubulin, and calmodulin gene sequencing, only 8 of 11 clinical isolates reported as A. flavus in our clinical microbiology laboratory by phenotypic methods were identified as A. flavus. The other three isolates were A. nomius (n = 2) or A. tamarii (n = 1). The results corresponded with those of metabolic fingerprinting, in which the A. flavus, A. nomius, and A. tamarii strains were separated into three clusters based on ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC MS) analysis. The first two patients with A. nomius infections had invasive aspergillosis and chronic cavitary and fibrosing pulmonary and pleural aspergillosis, respectively, whereas the third patient had A. tamarii colonization of the airway. Identification of the 11 clinical isolates and three reference strains by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) showed that only six of the nine strains of A. flavus were identified correctly. None of the strains of A. nomius and A. tamarii was correctly identified. β-Tubulin or the calmodulin gene should be the gene target of choice for identifying A. flavus, A. nomius, and A. tamarii. To improve the usefulness of MALDI-TOF MS, the number of strains for each species in MALDI-TOF MS databases should be expanded to cover intraspecies variability.
The occurrence of Legionella species other than Legionella pneumophila in clinical and environmental samples in Denmark identified by mip gene sequencing and matrix-assisted laser desorption ionization time-of-flight mass spectrometry.
Svarrer, C W; Uldum, S A
In Denmark, several laboratories use PCR as a routine diagnostic method for Legionnaires' disease, and almost all PCR-positive samples are investigated by culture. From 1993 to 2010, isolates of Legionella species other than Legionella pneumophila were obtained from respiratory samples from 33 patients, and from 1997 to 2010, 42 isolates of Legionella non-pneumophila species were obtained and saved from water samples from 39 different sites in Denmark. Macrophage infectivity potentiator gene (mip) sequencing was used as a reference method to identify the Legionella non-pneumophila species. Only one of the 75 isolates did not meet the acceptance criterion of a similarity of ≥98% to sequences in the database. The species distribution between clinical and environmental isolates varied. For the former, four species were detected, with Legionella bozemanae and Legionella micdadei predominating (both 44%). For the latter, eight species were detected, with Legionella anisa predominating (52%). The distribution among the Danish clinical isolates was different from the general distribution both in Europe and outside Europe, where L. bozemanae and Legionella longbeachae are the most commonly found clinical Legionella non-pneumophila species. The 75 isolates were also investigated by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS): 64 were correctly identified, with a score of ≥2.0; eight had a score of <2.0, but only two of these were wrongly identified; and three gave no results with MALDI-TOF MS. Both mip sequencing and MALDI-TOF MS are robust methods for Legionella species identification.
Advantages of Using Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry as a Rapid Diagnostic Tool for Identification of Yeasts and Mycobacteria in the Clinical Microbiological Laboratory
Chen, Jonathan H. K.; Yam, Wing-Cheong; Ngan, Antonio H. Y.; Fung, Ami M. Y.; Woo, Wai-Lan; Yan, Mei-Kum; Choi, Garnet K. Y.; Ho, Pak-Leung; Cheng, Vincent C. C.
Yeast and mycobacteria can cause infections in immunocompromised patients and normal hosts. The rapid identification of these organisms can significantly improve patient care. There has been an increasing number of studies on using matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) for rapid yeast and mycobacterial identifications. However, studies on direct comparisons between the Bruker Biotyper and bioMérieux Vitek MS systems for the identification of yeast and mycobacteria have been limited. This study compared the performance of the two systems in their identification of 98 yeast and 102 mycobacteria isolates. Among the 98 yeast isolates, both systems generated species-level identifications in >70% of the specimens, of which Candida albicans was the most commonly cultured species. At a genus-level identification, the Biotyper system identified more isolates than the Vitek MS system for Candida (75/78 [96.2%]versus 68/78 [87.2%], respectively; P = 0.0426) and non-Candida yeasts (18/20 [90.0%]versus 7/20 [35.0%], respectively; P = 0.0008). For mycobacterial identification, the Biotyper system generated reliable identifications for 89 (87.3%) and 64 (62.8%) clinical isolates at the genus and species levels, respectively, from solid culture media, whereas the Vitek MS system did not generate any reliable identification. The MS method differentiated 12/21 clinical species, despite the fact that no differentiation between Mycobacterium abscessus and Mycobacterium chelonae was found by using 16S rRNA gene sequencing. In summary, the MALDI-TOF MS method provides short turnaround times and a standardized working protocol for the identification of yeast and mycobacteria. Our study demonstrates that MALDI-TOF MS is suitable as a first-line test for the identification of yeast and mycobacteria in clinical laboratories. PMID:24048537
Mayer-Scholl, Anne; Murugaiyan, Jayaseelan; Neumann, Jennifer; Bahn, Peter; Reckinger, Sabine; Nöckler, Karsten
Human trichinellosis occurs through consumption of raw or inadequately processed meat or meat products containing larvae of the parasitic nematodes of the genus Trichinella. Currently, nine species and three genotypes are recognized, of which T. spiralis, T. britovi and T. pseudospiralis have the highest public health relevance. To date, the differentiation of the larvae to the species and genotype level is based primarily on molecular methods, which can be relatively time consuming and labor intensive. Due to its rapidness and ease of use a matrix assisted laser desorption / ionization time of flight mass spectrometry (MALDI-TOF MS) reference spectra database using Trichinella strains of all known species and genotypes was created. A formicacid/acetonitrile protein extraction was carried out after pooling 10 larvae of each Trichinella species and genotype. Each sample was spotted 9 times using α-cyano 4-hydoxy cinnamic acid matrix and a MicroFlex LT mass spectrometer was used to acquire 3 spectra (m/z 2000 to 20000 Da) from each spot resulting in 27 spectra/species or genotype. Following the spectra quality assessment, Biotyper software was used to create a main spectra library (MSP) representing nine species and three genotypes of Trichinella. The evaluation of the spectra generated by MALDI-TOF MS revealed a classification which was comparable to the results obtained by molecular methods. Also, each Trichinella species utilized in this study was distinct and distinguishable with a high confidence level. Further, different conservation methods such as freezing and conservation in alcohol and the host species origin of the isolated larvae did not have a significant influence on the generated spectra. Therefore, the described MALDI-TOF MS can successfully be implemented for both genus and species level identification and represents a major step forward in the use of this technique in foodborne parasitology.
Modified 16S-23S rRNA intergenic region restriction endonuclease analysis for species identification of Enterococcus strains isolated from pigs, compared with identification using classical methods and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.
Nowakiewicz, Aneta; Ziółkowska, Grażyna; Zięba, Przemysław; Trościańczyk, Aleksandra; Banach, Tomasz; Kowalski, Cezary
Fast and reliable identification of bacteria to at least the species level is currently the basis for correct diagnosis and appropriate treatment of infections. This is particularly important in the case of bacteria of the genus Enterococcus, whose resistance profile is often correlated with their species (e.g. resistance to vancomycin). In this study, we evaluated restriction endonuclease analysis of the 16S-23S rRNA gene intergenic transcribed spacer (ITS) region for species identification of Enterococcus. The utility of the method was compared with that of phenotypic methods [biochemical profile evaluation and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS)]. Identification was based on 21 Enterococcus reference strains, of the species E. faecalis, E. faecium, E. hirae, E. durans, E. casseliflavus, E. gallinarum, E. avium, E. cecorum and E. columbae, and 47 Enterococcus field strains isolated from pigs. Restriction endonuclease analysis of the ITS-PCR product using HinfI, RsaI and MboI, in the order specified, enabled species differentiation of the Enterococcus reference and field strains, and in the case of the latter, the results of species identification were identical (47/47) to those obtained by MALDI-TOF MS. Moreover, as a result of digestion with MboI, a unique restriction profile was also obtained for the strains (3/3) identified by MALDI-TOF MS as E. thailandicus. In our opinion, restriction endonuclease analysis of the 16S-23S rRNA gene ITS region of Enterococcus may be a simple and relatively fast (less than 4 h) alternative method for identifying the species occurring most frequently in humans and animals.
In situ analysis of plant tissue underivatized carbohydrates and on-probe enzymatic degraded starch by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry by using carbon nanotubes as matrix.
Gholipour, Yousef; Nonami, Hiroshi; Erra-Balsells, Rosa
Underivatized carbohydrates of tulip bulb and leaf tissues were characterized in situ by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) by using carbon nanotubes (CNTs) as matrix. Two sample preparation methods--(i) depositing CNTs on the fresh tissue slices placed on the probe and (ii) locating semitransparent tissues on a dried layer of CNTs on the probe--were examined. Furthermore, practicability of in situ starch analysis by MALDI-TOF MS was examined by detection of glucose originated from on-probe amyloglucosidase-catalyzed degradation of starch on the tissue surface. Besides, CNTs could efficiently desorb/ionize natural mono-, di-, and oligosaccharides extracted from tulip bulb tissues as well as glucose resulting from starch enzymatic degradation in vitro. These results were compared with those obtained by in situ MALDI-TOF MS analysis of similar tissues. Positive ion mode showed superior signal reproducibility. CNTs deposited under semitransparent tissue could also desorb/ionize neutral carbohydrates, leading to nearly complete elimination of matrix cluster signals but with an increase in tissue-originated signals. Furthermore, several experiments were carried out to compare the efficiency of 2,5-dihydroxybenzoic acid, nor-harmane, alpha-cyano-4-hydroxycinnamic acid, and CNTs as matrices for MALDI of neutral carbohydrates from the intact plant tissue surface and for enzymatic tissue starch degradation; these results are discussed in brief. Among matrices studied, the lowest laser power was needed to acquire carbohydrate signals with high signal-to-noise ratio and resolution when CNTs were used.
Martinez, Raquel M; Bauerle, Elizabeth R; Fang, Ferric C; Butler-Wu, Susan M
The identification of organisms from positive blood cultures generally takes several days. However, recently developed rapid diagnostic methods offer the potential for organism identification within only a few hours of blood culture positivity. In this study, we evaluated the performance of three commercial methods to rapidly identify organisms directly from positive blood cultures: QuickFISH (AdvanDx, Wolburn, MA), Verigene Gram-Positive Blood Culture (BC-GP; Nanosphere, Northbrook, IL), and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) with Sepsityper processing (Bruker Daltonics, Billerica, MA). A total of 159 blood cultures (VersaTREK Trek Diagnostic Systems, Cleveland, OH) positive for Gram-positive and Gram-negative bacteria as well as yeast were analyzed with QuickFISH and MALDI-TOF MS. In all, 102 blood cultures were analyzed using the BC-GP assay. For monomicrobial cultures, we observed 98.0% concordance with routine methods for both QuickFISH (143/146) and the BC-GP assay (93/95). MALDI-TOF MS demonstrated 80.1% (117/146) and 87.7% (128/146) concordance with routine methods to the genus and species levels, respectively. None of the methods tested were capable of consistently identifying polymicrobial cultures in their entirety or reliably differentiating Streptococcus pneumoniae from viridans streptococci. Nevertheless, the methods evaluated in this study are convenient and accurate for the most commonly encountered pathogens and have the potential to dramatically reduce turnaround time for the provision of results to the treating physician.
da Silva, Kesia Esther; Maciel, Wirlaine Glauce; Sacchi, Flávia Patussi Correia; Carvalhaes, Cecilia Godoy; Rodrigues-Costa, Fernanda; da Silva, Ana Carolina Ramos; Croda, Mariana Garcia; Negrão, Fábio Juliano; Croda, Julio; Gales, Ana Cristina; Simionatto, Simone
This study describes the molecular characteristics and risk factors associated with carbapenem-resistant Klebsiella pneumoniae strains. Risk factors associated with KPC-producing K. pneumoniae strains were investigated in this case-control study from May 2011 to May 2013. Bacterial identification was performed by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS). Antimicrobial susceptibility was determined by broth microdilution. Carbapenemase production was assessed by both modified Hodge test (MHT) and ertapenem hydrolysis using MALDI-TOF MS. The presence of β-lactamase-encoding genes was evaluated by PCR and DNA sequencing. Alterations in genes encoding K. pneumoniae outer membrane proteins were analysed by PCR and DNA sequencing as well as SDS-PAGE. Genetic relatedness among strains was determined by pulsed-field gel electrophoresis. This study included 94 patients. Longer hospitalisation, mechanical ventilation, catheters, and previous surgery were associated with KPC-producing K. pneumoniae. Sixty-eight strains showed resistance to carbapenems. Carbapenemase production was detected by MHT in 67 K. pneumoniae strains and by MALDI-TOF MS in 57. The presence of the blaKPC-2 gene was identified in 57 strains. The blaKPC-2 gene was not found in 11 carbapenem-resistant K. pneumoniae; instead, the blaCTX-M-1-like, blaCTX-M-2-like, blaCTX-M-8 like, blaCTX-M-14-like and blaSHV- like genes associated with OmpK35 and OmpK36 alterations were observed. Thirty-three KPC-producing K. pneumoniae strains were clonally related, and patients infected with these strains had a higher mortality rate (78.78 %). Our results show that KPC-producing K. pneumoniae was associated with several healthcare-related risk factors, including recent surgery.
Kishii, K; Kikuchi, K; Matsuda, N; Yoshida, A; Okuzumi, K; Uetera, Y; Yasuhara, H; Moriya, K
The clinical relevance of Acinetobacter species, other than A. baumannii, as human pathogens has not been sufficiently assessed owing to the insufficiency of simple phenotypic clinical diagnostic laboratory tests. Infections caused by these organisms have different impacts on clinical outcome and require different treatment and management approaches. It is therefore important to correctly identify Acinetobacter species. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has been introduced to identify a wide range of microorganisms in clinical laboratories, but only a few studies have examined its utility for identifying Acinetobacter species, particularly those of the non-Acinetobacter baumannii complex. We therefore evaluated MALDI-TOF MS for identification of Acinetobacter species by comparing it with sequence analysis of rpoB using 123 isolates of Acinetobacter species from blood. Of the isolates examined, we identified 106/123 (86.2%) to species, and 16/123 (13.0%) could only be identified as acinetobacters. The identity of one isolate could not be established. Of the 106 species identified, 89/106 (84.0%) were confirmed by rpoB sequence analysis, and 17/106 (16.0%) were discordant. These data indicate correct identification of 89/123 (72.4%) isolates. Surprisingly, all blood culture isolates were identified as 13 species of Acinetobacter, and the incidence of Acinetobacter pittii was unexpectedly high (42/123; 34.1%) and exceeded that of A. baumannii (22/123; 17.9%). Although the present identification rate using MALDI-TOF MS is not acceptable for species-level identification of Acinetobacter, further expansion of the database should remedy this situation.
Ogunlowo, Peter Oladejo; Olley, Mitsan; Springer, Burkhard; Allerberger, Franz; Ruppitsch, Werner
Background Enterobacteriaceae are ubiquitously present in nature and can be found in the intestinal tract of humans and animals as commensal flora. Multidrug-resistant Enterobacteriaceae are increasingly reported and are a threat to public health implicating a need for accurate identification of the isolates to species level. In developing countries, identification of bacteria basically depends on conventional methods: culture and phenotypic methods that hamper the accurate identification of bacteria. In this study, matrix-assisted desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) technique was compared to conventional identification techniques. Materials and Methods In total, 147 Enterobacteriaceae isolates were collected from March to May 2015 from three medical microbiology laboratories of hospitals in Edo state, Nigeria, after being tested according to the individual laboratories standard operating procedures. All isolates were stored at −20°C until tested centrally by MALDI-TOF MS. Results One hundred and forty five (98.6%) isolates had a MALDI Biotyper best score > or =2.0, indicating a secure genus and probable species identification; and 2(1.36%) isolates had a best score <2.0 indicating probable genus identification. Isolates with best scores of > or =2.0 comprised nine genera and 10 species, respectively. A total of 57.2% and 33.1% of isolates identified had agreement between MALDI-TOF MS and conventional techniques for identification at genus and species level, respectively, when analyzing bacteria with MALDI Biotyper best scores > or =2.0. Conclusion The results of our study show that the applied conventional identification techniques for Enterobacteriaceae in the investigated Nigerian hospitals are not very accurate. Use of state-of-the-art identification technologies for microorganisms is necessary to guarantee comparability of bacteriological results. PMID:27703855
Mayer-Scholl, Anne; Murugaiyan, Jayaseelan; Neumann, Jennifer; Bahn, Peter; Reckinger, Sabine; Nöckler, Karsten
Human trichinellosis occurs through consumption of raw or inadequately processed meat or meat products containing larvae of the parasitic nematodes of the genus Trichinella. Currently, nine species and three genotypes are recognized, of which T. spiralis, T. britovi and T. pseudospiralis have the highest public health relevance. To date, the differentiation of the larvae to the species and genotype level is based primarily on molecular methods, which can be relatively time consuming and labor intensive. Due to its rapidness and ease of use a matrix assisted laser desorption / ionization time of flight mass spectrometry (MALDI-TOF MS) reference spectra database using Trichinella strains of all known species and genotypes was created. A formicacid/acetonitrile protein extraction was carried out after pooling 10 larvae of each Trichinella species and genotype. Each sample was spotted 9 times using α-cyano 4-hydoxy cinnamic acid matrix and a MicroFlex LT mass spectrometer was used to acquire 3 spectra (m/z 2000 to 20000 Da) from each spot resulting in 27 spectra/species or genotype. Following the spectra quality assessment, Biotyper software was used to create a main spectra library (MSP) representing nine species and three genotypes of Trichinella. The evaluation of the spectra generated by MALDI-TOF MS revealed a classification which was comparable to the results obtained by molecular methods. Also, each Trichinella species utilized in this study was distinct and distinguishable with a high confidence level. Further, different conservation methods such as freezing and conservation in alcohol and the host species origin of the isolated larvae did not have a significant influence on the generated spectra. Therefore, the described MALDI-TOF MS can successfully be implemented for both genus and species level identification and represents a major step forward in the use of this technique in foodborne parasitology. PMID:26999436
Camoez, M; Sierra, J M; Dominguez, M A; Ferrer-Navarro, M; Vila, J; Roca, I
Early identification of methicillin-resistant Staphylococcus aureus (MRSA) dominant clones involved in infection and initiation of adequate infection control measures are essential to limit MRSA spread and understand MRSA population dynamics. In this study we evaluated the use of matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF/MS) for the automated discrimination of the major MRSA lineages (clonal complexes, CC) identified in our hospital during a 20-year period (1990-2009). A collection of 82 well-characterized MRSA isolates belonging to the four main CCs (CC5, CC8, CC22 and CC398) was split into a reference set (n = 36) and a validation set (n = 46) to generate pattern recognition models using the ClinProTools software for the identification of MALDI-TOF/MS biomarker peaks. The supervised neural network (SNN) model showed the best performance compared with two other models, with sensitivity and specificity values of 100% and 99.11%, respectively. Eleven peaks (m/z range: 3278-6592) with the highest separation power were identified and used to differentiate all four CCs. Validation of the SNN model using ClinProTools resulted in a positive predictive value (PPV) of 99.6%. The specific contribution of each peak to the model was used to generate subtyping reference signatures for automated subtyping using the BioTyper software, which successfully classified MRSA isolates into their corresponding CCs with a PPV of 98.9%. In conclusion, we find this novel automated MALDI-TOF/MS approach to be a promising, powerful and reliable tool for S. aureus typing.
Loff, Marché; Mare, Louise; de Kwaadsteniet, Michele; Khan, Wesaal
The aim of this study was to compare standard selective plating, conventional PCR (16S rRNA and species specific primers), MALDI-TOF MS and the 3M™ Molecular Detection System for the routine detection of the pathogens Listeria, Salmonella and Escherichia coli 0157:H7 in wastewater and river water samples. MALDI-TOF MS was able to positively identify 20/21 (95%) of the E. coli isolates obtained at genus and species level, while 16S rRNA sequencing only correctly identified 6/21 (28%) as E. coli strains. None of the presumptive positive Listeria spp. and Salmonella spp. isolates obtained by culturing on selective media were positively identified by MALDI-TOF and 16S rRNA analysis. The species-specific E. coli 0157:H7 PCR described in this present study, was not able to detect any E. coli 0157:H7 strains in the wastewater and river water samples analysed. However, E. coli strains, Listeria spp., L. monocytogenes and Salmonella spp. were detected using species specific PCR. Escherichia coli 0157:H7, Listeria spp. and Salmonella spp. were also sporadically detected throughout the sampling period in the wastewater and river water samples analysed by the 3M™ Molecular Detection System. MALDI-TOF MS, which is a simple, accurate and cost-effective detection method, efficiently identified the culturable organisms, while in the current study both species specific PCR (Listeria spp. and Salmonella spp.) and 3M™ Molecular Detection System could be utilised for the direct routine analysis of pathogens in water sources.
Kok, Jen; Thomas, Lee C; Olma, Thomas; Chen, Sharon C A; Iredell, Jonathan R
Matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) is a novel method for the direct identification of bacteria from blood culture broths. We evaluate for the first time, the performance of the MALDI Sepsityper™ Kit and MS for the identification of bacteria compared to standard phenotypic methods using the manufacturer's specified bacterial identification criteria (spectral scores ≥1.700-1.999 and ≥2.000 indicated identification to genus and species level, respectively). Five hundred and seven positive blood culture broths were prospectively examined, of which 379 (74.8%; 358 monomicrobial, 21 polymicrobial) were identified by MALDI-TOF MS; 195 (100%) and 132 (67.7%) of 195 gram-positive; and 163 (100%) and 149 (91.4%) of 163 gram-negative organisms from monomicrobial blood cultures were correctly identified to genus and species level, respectively. Spectral scores <1.700 (no identification) were obtained in 128/507 (25.2%) positive blood culture broths, including 31.6% and 32.3% of gram-positive and polymicrobial blood cultures, respectively. Significantly more gram-negative organisms were identified compared to gram-positive organisms at species level (p<0.0001). Five blood cultures were misidentified, but at species level only; including four monomicrobial blood cultures with Streptococcus oralis/mitis that were misidentified as Streptococcus pneumoniae. Positive predictive values for the direct identification of both gram-positive and gram-negative bacteria from monomicrobial blood culture broths to genus level were 100%. A diagnostic algorithm for positive blood culture broths that incorporates gram staining and MALDI-TOF MS should identify the majority of pathogens, particularly to genus level.
McMullen, Allison R.; Wallace, Meghan A.; Pincus, David H.; Wilkey, Kathy
Invasive fungal infections have a high rate of morbidity and mortality, and accurate identification is necessary to guide appropriate antifungal therapy. With the increasing incidence of invasive disease attributed to filamentous fungi, rapid and accurate species-level identification of these pathogens is necessary. Traditional methods for identification of filamentous fungi can be slow and may lack resolution. Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) has emerged as a rapid and accurate method for identification of bacteria and yeasts, but a paucity of data exists on the performance characteristics of this method for identification of filamentous fungi. The objective of our study was to evaluate the accuracy of the Vitek MS for mold identification. A total of 319 mold isolates representing 43 genera recovered from clinical specimens were evaluated. Of these isolates, 213 (66.8%) were correctly identified using the Vitek MS Knowledge Base, version 3.0 database. When a modified SARAMIS (Spectral Archive and Microbial Identification System) database was used to augment the version 3.0 Knowledge Base, 245 (76.8%) isolates were correctly identified. Unidentified isolates were subcultured for repeat testing; 71/319 (22.3%) remained unidentified. Of the unidentified isolates, 69 were not in the database. Only 3 (0.9%) isolates were misidentified by MALDI-TOF MS (including Aspergillus amoenus [n = 2] and Aspergillus calidoustus [n = 1]) although 10 (3.1%) of the original phenotypic identifications were not correct. In addition, this methodology was able to accurately identify 133/144 (93.6%) Aspergillus sp. isolates to the species level. MALDI-TOF MS has the potential to expedite mold identification, and misidentifications are rare. PMID:27225405
Background Burkholderia (B.) pseudomallei and B. mallei are genetically closely related species. B. pseudomallei causes melioidosis in humans and animals, whereas B. mallei is the causative agent of glanders in equines and rarely also in humans. Both agents have been classified by the CDC as priority category B biological agents. Rapid identification is crucial, because both agents are intrinsically resistant to many antibiotics. Matrix-assisted laser desorption/ionisation mass spectrometry (MALDI-TOF MS) has the potential of rapid and reliable identification of pathogens, but is limited by the availability of a database containing validated reference spectra. The aim of this study was to evaluate the use of MALDI-TOF MS for the rapid and reliable identification and differentiation of B. pseudomallei and B. mallei and to build up a reliable reference database for both organisms. Results A collection of ten B. pseudomallei and seventeen B. mallei strains was used to generate a library of reference spectra. Samples of both species could be identified by MALDI-TOF MS, if a dedicated subset of the reference spectra library was used. In comparison with samples representing B. mallei, higher genetic diversity among B. pseudomallei was reflected in the higher average Eucledian distances between the mass spectra and a broader range of identification score values obtained with commercial software for the identification of microorganisms. The type strain of B. pseudomallei (ATCC 23343) was isolated decades ago and is outstanding in the spectrum-based dendrograms probably due to massive methylations as indicated by two intensive series of mass increments of 14 Da specifically and reproducibly found in the spectra of this strain. Conclusions Handling of pathogens under BSL 3 conditions is dangerous and cumbersome but can be minimized by inactivation of bacteria with ethanol, subsequent protein extraction under BSL 1 conditions and MALDI-TOF MS analysis being faster than
Nur Afifah, Diana; Rustanti, Ninik; Anjani, Gemala; Syah, Dahrul; Yanti; Suhartono, Maggy T.
This paper presents the proteomics study which includes separation, identification and characterization of proteins. The experiment on Indonesian fermented food such as extracellular fibrinolytic protease from Bacillus licheniformis RO3 and Bacillus pumilus 2.g isolated from red oncom and tempeh gembus was conducted. The experimental works comprise the following steps: (1) a combination of one- and two-dimensional electrophoresis analysis, (2) mass spectrometry analysis using MALDI-TOF-MS and (3) investigation using protein database. The result suggested that there were new two protein fractions of B. licheniformis RO3 and three protein fractions of B. pumilus 2.g. These result has not been previously reported.
Background The routine diagnosis of Streptococcus spp. and other mastitis associated gram-positive, catalase-negative cocci is still based upon biochemical tests and serological methods, which frequently provide ambiguous identification results. We therefore aimed to establish an accurate identification system for differential diagnosis of mastitis associated Streptococcus spp. and related species using biophysical techniques such as Fourier-transform infrared (FTIR) spectroscopy and MALDI – TOF/MS. Results Based on a panel of 210 isolates from cases of bovine mastitis, an unsupervised FTIR spectral reference library was established and an artificial neural network (ANN) - assisted identification system was developed. All bacterial isolates were previously identified by species-specific PCR and/or 16S rRNA gene sequence analysis. An overall identification rate of 100% at species level for 173 strains unknown to the ANN and the library was achieved by combining ANN and the spectral database, thus demonstrating the suitability of our FTIR identification system for routine diagnosis. In addition, we investigated the potential of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for the identification of mastitis associated Streptococcus spp. and related bacteria. Using the Microflex LT System, MALDI Biotyper software™ (V3.3) we achieved an accuracy rate of 95.2%. A blind study, including 21 clinical samples from dairy cows, revealed a 100% correct species identification rate for FTIR and 90.5% for MALDI-TOF MS, indicating that these techniques are valuable tools for diagnosis. Conclusions This study clearly demonstrates that FTIR spectroscopy as well as MALDI-TOF MS can significantly improve and facilitate the identification and differentiation of mastitis associated Streptococcus spp. and related species. Although the FTIR identification system turned out being slightly superior to MALDI-TOF MS in terms of identification
Liu, Yi; Marszalek, Tomasz; Müllen, Klaus; Pisula, Wojciech; Feng, Xinliang
A series of trisbenzothieno[1,2:7,8:13,14]hexa-peri-hexabenzocoronenes were synthesized and characterized by a combination of NMR, 2D NMR, MALDI-TOF MS, UV/Vis absorption spectroscopy, and 2D-WAXS measurement. By structural modulation like decoration of electro-donating alkoxyl chain, and conversion from an electron-rich thiophene ring into an electron-poor thiophene-S,S-dioxide moiety, the highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) levels of the hexabenzocoronenes derivatives can be effectively tuned which is further verified by the DFT calculations and cyclic voltammetry.
Clinton, Lani Kai; Bankowski, Matthew J; Shimasaki, Teppei; Sae-Ow, Wichit; Whelen, A Christian; O'Connor, Norman; Kim, Wesley; Young, Royden
A 54-year-old female with a prosthetic mitral valve presented with a 3-day history of dizziness, subjective fever, and chills. Blood cultures were positive for a pleomorphic Gram-positive rod. Initial phenotypic testing could only support the identification of a Corynebacterium species. Nucleic acid sequencing (16S rRNA) and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) were conclusive for Corynebacterium diphtheriae. Definitive phenotypic testing classified the strain as nontoxigenic C. diphtheriae biotype Gravis.
Naumczuk, Beata; Hyz, Karolina; Kawęcki, Robert; Bocian, Wojciech; Bednarek, Elżbieta; Sitkowski, Jerzy; Wielgus, Ewelina; Kozerski, Lech
Using DOSY NMR and MALDI-TOF MS techniques, we present evidence that quaternary trimethylammonium salts of topotecan, [TPT-NMe3 ](+) X(-) (X = CF3SO3, HCOO), bind covalently the natural DNA oligomer upon near UV irradiation in water under physiological conditions. It is shown that formate salt is very reactive at pH 7 and requires short irradiation time. This weak irradiation at 365 nm paves the way for a new application of TPT derivatives in clinical use, which can dramatically increase the therapeutic effects of a medicine.
Shi, Chenyi; Deng, Chunhui; Zhang, Xiangmin; Yang, Pengyuan
In this work, we synthesized highly water-dispersible multiwalled carbon nanotubes@polydopamine (MWCNTs@PDA) core-shell composites by a facile in situ oxidative polymerization. The composites were successfully applied as a novel matrix for the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis of various water-soluble small molecule compounds. It was found that MWCNTs@PDA composites have a higher sensitivity and peak intensities for small molecules detection.
Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry interlaboratory comparison of mixtures of polystyrene with different end groups: statistical analysis of mass fractions and mass moments.
Guttman, Charles M; Wetzel, Stephanie J; Flynn, Kathleen M; Fanconi, Bruno M; VanderHart, David L; Wallace, William E
A matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) interlaboratory comparison was conducted on mixtures of synthetic polymers having the same repeat unit and closely matching molecular mass distributions but with different end groups. The interlaboratory comparison was designed to see how well the results from a group of experienced laboratories would agree on the mass fraction, and molecular mass distribution, of each polymer in a series of binary mixtures. Polystyrenes of a molecular mass near 9000 u were used. Both polystyrenes were initiated with the same butyl initiator; however, one was terminated with -H (termed PSH) and the other was terminated with -CH2CH2OH (termed PSOH). End group composition of the individual polymers was checked by MALDI-TOF MS and by nuclear magnetic resonance (NMR). Five mixtures were created gravimetrically with mass ratios between 95:5 and 10:90 PSOH/PSH. Mixture compositions where measured by NMR and by Fourier transform infrared spectrometry (FT-IR). NMR and FT-IR were used to benchmark the performance of these methods in comparison to MALDI-TOF MS. Samples of these mixtures were sent to any institution requesting it. A total of 14 institutions participated. Analysis of variance was used to examine the influences of the independent parameters (participating laboratory, MALDI matrix, instrument manufacturer, TOF mass separation mode) on the measured mass fractions and molecular mass distributions for each polymer in each mixture. Two parameters, participating laboratory and instrument manufacturer, were determined to have a statistically significant influence. MALDI matrix and TOF mass separation mode (linear or reflectron) were found not to have a significant influence. Improper mass calibration, inadequate instrument optimization with respect to high signal-to-noise ratio across the entire mass range, and poor data analysis methods (e.g., baseline subtraction and peak integration
Cho, Donghyun; Hong, Sangsu; Shim, Sangdeok
We present the employment of few layer graphene (FLG) as a matrix for the analysis of low molecular weight polymeric compounds using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The practicality of FLG as a matrix for MALDI experiments is demonstrated by analyzing low molecular weight polymers, polar polyethylene glycol (PEG) of 1000 Da and nonpolar polymethylmethacrylate (PMMA) of 650 Da. The high quality MS spectra without low-mass interference signals without any further sampling procedure were acquired.
Ding, Chunlai; Fan, Caiwei; Jiang, Ganquan; Pan, Xiangqiang; Zhang, Zhengbiao; Zhu, Jian; Zhu, Xiulin
Vinyl acetate is polymerized in the living way under the irradiation of blue light-emitting diodes (LEDs) or sunlight without photocatalyst at ambient temperature. 2-(Ethoxycarbonothioyl)sulfanyl propanoate is exclusively added and acts as initiator and chain transfer agent simultaneously in the current system. Poly(vinyl acetate) with well-regulated molecular weight and narrow molecular weight distribution (Đ < 1.30) is synthesized. Near quantitative end group fidelity of polymer is demonstrated by nuclear magnetic resonance (NMR) and matrix-assisteed laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS).
Characterization of Achromobacter Species in Cystic Fibrosis Patients: Comparison of bla(OXA-114) PCR Amplification, Multilocus Sequence Typing, and Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry.
Rodrigues, Elenice R A; Ferreira, Alex G; Leão, Robson S; Leite, Cassiana C F; Carvalho-Assef, Ana Paula; Albano, Rodolpho M; Marques, Elizabeth A
Molecular methodologies were used to identify 28 Achromobacter spp. from patients with cystic fibrosis (CF). Multilocus sequence typing (MLST) identified 17 Achromobacter xylosoxidans isolates (all bla(OXA-114) positive), nine Achromobacter ruhlandii isolates (all bla(OXA-114) positive), one Achromobacter dolens isolate, and one Achromobacter insuavis isolate. All less common species were misidentified as A. xylosoxidans by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Chronic colonization by clonally related A. ruhlandii isolates was demonstrated.
Ghosh, Kaushik; Chandra, Krishnendu; Roy, Sadhan K; Mondal, Subhas; Maiti, Debabrata; Das, Debsankar; Ojha, Arnab K; Islam, Syed S
A water-soluble polysaccharide was isolated from the aqueous extract of the stem of Lagenaria siceraria. The polysaccharide was found to be constituted of methyl d-galacturonate, 2-O-methyl-D-xylose, and d-xylose in a ratio of 1:1:1. On the basis of total acid hydrolysis, methylation analysis, periodate oxidation, NMR studies ((1)H, (13)C, 2D-COSY, TOCSY, NOESY, HSQC, and HMBC), and MALDI-TOF MS analysis, the structure of the repeating unit of the polysaccharide is determined as.
Almagro-Molto, M; Eder, W; Schubert, S
We report on two new cases Bordetella trematum in chronic ulcers. Literature shows that this species is infrequently isolated from clinical samples. Only a few cases have been reported so far. B. trematum may be underdiagnosed because of its problematic identification. Today, MALDI-TOF MS allows for an accurate discrimination of these bacteria. Overall, B. trematum was typically associated with severe skin and soft tissue infections, particularly in diabetic patients. The intrinsic resistance to some antibiotics commonly used to treat these infections makes the isolation of B. trematum significant.