A simple, rapid, straightforward and washing/separation free of in-solution digestion method for microwave-assisted tryptic digestion of proteins (cytochrome c, lysozyme and myoglobin) using bare TiO(2) nanoparticles (NPs) prepared in aqueous solution to serve as multifunctional nanoprobes in electrospray ionization mass spectrometry ...
PubMed
In LC-MS based proteomics, several accelerating trypsination methods have been introduced in order to speed up the protein digestion, which is often considered a bottleneck. Traditionally and most commonly, due to sample heterogeneity, overnight digestion at 37�C is performed in order to digest both easily and ...
Integrated fluidic systems that unite bottom-up and top-down proteomic approaches have the potential to deliver complete protein characterization. To circumvent fraction collection, as is conducted in current blended approaches, a technique to regulate digestion efficiency in a flow-through system is required. The present study examined the concept of regulating ...
tryptic digestion in polyacrylamide matrix. Chromatographia 28:92�94. 7. Fauque, G., A. R. Lino, M
E-print Network
Reduced S-carboxymethylated cobrotoxin was digested with trypsin and chymotrypsin. Nine peptides were isolated from the tryptic digest by a combination of electrophoresis and chromatography on paper. The amino acid sequence in these peptides was determine...
National Technical Information Service (NTIS)
A new strategy for the fast monitoring of peptide biomarkers is described. It is based on the use of accelerated in-solution trypsin digestions under an ultrasonic field provided by high-intensity focused ultrasound (HIFU) and the monitoring of several peptides by selected MS/MS ion monitoring in a linear ion trap mass spectrometer. The performance of the ...
... Digestion of vpB in borate buffers has permitted the isolation of many additional tryptic peptides for amino acid sequence analysis and allowed ...
DTIC Science & Technology
The collagen losses in necrotic woulds are associated with an apparent collagenolysis due to the activation of a collagenolytic enzyme. This enzyme is normally complexed with a protein inhibitor which is more sensitive to tryptic digestion than is the col...
One of the most popular methods to prepare tryptic peptides for bottom-up proteomic analysis is in-gel digestion. To date, there have been few studies comparing various digestion methods. In this study, we compare the efficiency of several popular in-gel digestion methods, along with new technologies that may ...
PubMed Central
Identification of protein glycosylation sites is analytically challenging due to the diverse glycan structures associated with a glycoprotein. Mass spectrometry (MS)-based identification and characterization of glycoproteins has been achieved predominantly with the bottom-up approach, which typically involves the enzymatic cleavage of proteins to peptides prior to LC/MS or LC/MS/MS analysis. ...
This work proposes, for the first time, the use of a high intensity ultrasonic probe to accelerate the tryptic digestion of soybean proteins. Different digestion parameters were optimized: protein extracting solution, reduction, and alkylation conditions (time, concentration, and temperature), trypsin:protein ...
This laboratory has introduced a chemical method for residue-specific protein cleavage and has provided a preliminary assessment of the suitability of microwave-accelerated acid cleavage as a proteomic tool. This report is a continuing assessment of the fate of common protein modifications in microwave-accelerated acid cleavage. We have examined the ...
NASA Astrophysics Data System (ADS)
The strong cross-reactions demonstrated for staphylococcal enterotoxins B (SEB) and C1 (SEC1) by measurement of antigen-binding capacity (J. Immunol. 120:86, 1978) were reflected in well-defined polypeptides obtained by limited tryptic digestion from SEB ...
For bottom-up MS, the digestion step is critical and is typically performed with trypsin. Solvent-assisted digestion in 80% acetonitrile has previously been shown to improve protein sequence coverage at shorter digestion times. This has been attributed to enhanced enzyme digestion efficiency in this solvent. ...
The effects of temperature on ultrasound-assisted tryptic protein digestion were comprehensively investigated using matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry. Three standard proteins, cytochrome c, myoglobin, and bovine serum albumin, were digested at 4�C (ice), room temperature (20-25), 37, ...
Active and inactive products isolated from irradiated lysozyme samples have been subjected to limited proteolysis employing trypsin and to extensive proteolysis employing combined peptic-tryptic hydrolysis. Alterations in conformation or motility of pepti...
... a) Human serum albumin (HSA) reacted with N-acetoxy PhIP The mass list for tryptic-digested HSA-PhIP sample displayed 2,799 values, among ...
Fourteen purified monoclonal IgM cold agglutinins were reduced and alkylated and separated into heavy and light chains. Two dimensional peptide mapping was performed on tryptic digests of the mu chains. Thirteen of the 14 mu chains lacked a peptide, prese...
The development of an AP-MALDI source for a commercial ITMS instrument and its application to the analysis of tryptic digests, posttranslational modifications and noncovalent interactions will be presented. In addition, the use of this instrumentation in the characterization of fragmentation patterns of phosphotyrosine-containing peptides will be ...
Cancer.gov
The optimal reaction of culture media for the cultivation of T-strain Mycoplasma of human origin was investigated. By use of a recently modified tryptic digest medium, the optimal reaction in either agar or fluid medium was found to be pH 6.0. In contrast...
1. On exhaustive digestion of carboxymethylated actin in 6m-urea solutions with carboxypeptidase A, 1 mole of phenylalanine was liberated/43000g. of protein. At a lower urea concentration and in the absence of urea, carboxymethyl-cysteine (CMCys) was also liberated. 2. Three cysteine-containing peptides were identified by the study of peptide `maps' of ...
Staphylococcal entertoxin C1 is converted to a doubly cleaved molecule by trypsin digestion with one of the scissions internal to the disulfide loop and one external to it. The larger, disulfide-containing polypeptide (Mr = 22,000) exhibited excellent bin...
A procedure described for the partial purification of suspensions of the organisms of psittacosis utilizes tryptic digestion, calcium chloride-dextran sulfate precipitation, and centrifugation through a sucrose barrier. More than 40% of the organisms in t...
Efficient protein digestion is a critical step for successful mass spectrometry analysis. Here we describe simultaneous tryptic digestion and gradual unfolding of native proteins by application of a temperature gradient using a single cycle of 5 min or less in a PCR thermocycler. Chemicals typically used for chromatographic techniques ...
and Cleveland digest samples employed the system of Laemmli (11). Samples were mixed with an equal volume has been observed in other systems (21). When platelet microsomes were digested with trypsin the integrity of the 103-kDa protein, while the digested protein had residual ATPase activity. Tryptic diges
A commercially available 9.4 Tesla Fourier transform ion cyclotron resonance (FTICR) mass spectrometer was applied in the analysis of tryptic digests of protein mixtures without any separation. First, the method was demonstrated on a mixture of tryptic digests of equine cytochrome c, equine myoglobin and bovine ...
Localized heating of droplets on an electrowetting-on-dielectric (EWOD) chip has been implemented and shown to accelerate trypsin digestion reaction rates, sample drying, and matrix crystallization for matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). Achieving this involved extending the functionality of previous EWOD droplet-based ...
Negative ion electrospray mass spectra of the peptides produced by tryptic and chymotrypsin digests of bovine insulin, and from the tryptic digest of lysozyme identify at least 80% of the sequences of these proteins. In particular, negative ion mass spectrometry identifies and positions disulfide moieties, and is ...
The determination of bioactive peptides derived from food sources is gaining special attention in last years, due to their ability to promote health and their potential to reduce the risk of chronic diseases. In this work, a new analytical methodology has been developed enabling for the first time the determination of soymetide, a new immunostimulating peptide derived from soybean, in different ...
The silver-staining procedure for detecting proteins in polyacrylamide gels has been modified so that the polypeptides remain suitable for subsequent radioiodination and tryptic peptide analysis. The procedure, which involves a silver-staining/destaining protocol that minimizes crosslinking, is more rapid than many other methods, and can detect as little as 1 ng of protein. ...
Energy Citations Database
A method has been developed for rapid, sensitive, and accurate tryptic mapping of polypeptides using matrix-assisted laser desorption/ionization time-of-flight mass analysis. The technique utilizes mass spectrometer probe tips which have been activated through the covalent immobilization of trypsin. The enzymatically active probe tips were used for the ...
Enzymes of several classes used in the formulations of cleaning products were characterized by trypsin digestion followed by HPLC with UV detection. A polymeric monolithic column (ProSwift) was used to optimize the separation of both the intact enzymes and their tryptic digests. This column was adequate for the quality control of raw ...
Peptide mapping of underivatized tryptic digests of bovine and chick intestinal calcium-binding proteins has been accomplished by high performance liquid chromatography (HPLC). High precision analysis of nanomolar quantities of peptides were achieved in less than 1 h (recycle time). Peak resolution and definition are superior compared to conventioal ...
A new automated 2D-(SCX/RP)-nano-LC/MSMS method was developed. Separation of the peptides in the first LC dimension was the main focus of this work, and it was optimized using human serum albumin (HSA) and human lung cell lysate tryptic digests. Samples were reduced and alkylated...
EPA Science Inventory
For the first time, we utilized multifunctional nanoparticles composite (NPs composite) for matrix-assisted laser desorption/ionization mass spectrometric (MALDI-MS) analysis of peptides and proteins. Multiwalled carbon nanotubes doped with Cd(2+) ions and modified with cadmium sulfide NPs were synthesized by a chemical reduction method at room temperature. The multifunctional NPs composite ...
The authors have studied the structure and function of the solubilized insulin receptor before and after partial proteolytic digestion to define domains in the ..beta..-subunit that undergo autophosphorylation and contain the tyrosine kinase activity. Wheat germ agglutinin purified insulin receptor from Fao cells was digested briefly at 22/sup 0/C with low ...
An inseparable mixture of two cysteine proteinases, isolated from the digestive tract of the American lobster, was investigated by ionspray mass spectrometry (ISP-MS), using a combination of infusion of intact proteins with on-line liquid chromatography--mass spectrometry (LC--MS) and LC--MS--MS analyses of tryptic digests. These data ...
Black lipid membranes were formed with oxidized cholesterol in the presence of either the acetylcholine receptor, purified from the electric organ of the electric ray Torpedo californica or its tryptic digest. In both cases, conductance of cations increased and was dependent on the concentration of the receptor protein. Conductance of Ca/sup + +/ was ...
Hemoglobin-based oxygen carriers (HBOCs) of bovine hemoglobin (Hb) or human Hb origin were developed for replacement or augmentation of blood during transfusion and have the potential to increase oxygen-carrying capacity of circulating blood and thus improve tissue oxygen delivery. Due to their potential for increasing oxygen-carrying capacity of circulating blood, they are excellent candidates ...
Plasma biomarkers studies are based on the differential expression of proteins between different treatment groups or between diseased and control populations. Most mass spectrometry-based methods of protein quantitation, however, are based on the detection and quantitation of peptides, not intact proteins. For peptide-based protein quantitation to be accurate, the digestion ...
The purpose of this study was to identify the fragment of the hen egg-white lysozyme (HEL) molecule presented by macrophages to helper T cells. This was investigated by using T-cell hybridomas and macrophages prefixed in paraformaldehyde. We previously had shown that such prefixed macrophages could present a tryptic digest of HEL. The ...
An automated online multidimensional liquid chromatography system coupled to ESI-based tandem mass spectrometry was used to assess the effectiveness of TiO2 in the enrichment of phosphopeptides from tryptic digests of protein mixtures. By monitoring the enrichment of phosphopeptides, an optimized set of loading, wash, and elution conditions were realized ...
An automated online multidimensional liquid chromatography system coupled to ESI-based tandem mass spectrometry was used to assess the effectiveness of TiO2 in the enrichment of phosphopeptides from tryptic digests of protein mixtures. By monitoring the enrichment of phosphopeptides, an optimized set of loading, wash, and elution ...
Magnetic iron(II, III) oxide (magnetite, Fe(3)O(4)) nanoparticles were used to selectively enrich phosphopeptides from tryptic digests of bovine beta-casein and from tryptic digest mixtures containing bovine beta-casein, cytochrome c, bovine serum albumin, and horse heart myoglobin. The magnetic property of the ...
Detergents have been widely used for the solubilization of membrane proteins and the improvement of their digestion. In this paper, we have evaluated the application of sodium deoxycholate (SDC) to the solubilization and digestion of rat hippocampal plasma membrane (PM) proteins. For in-solution digestion, rat hippocampal PM fraction ...
A simple mass spectrometric approach for the discovery and validation of biomarkers in human plasma was developed by targeting non-glycopeptides adjacent to glycosylation sites in an N-linked glycoprotein, one of the most important biomarkers for early detection, prognoses, and disease therapies. The discovery and validation of novel biomarkers requires complex sample pre-treatment steps, such as ...
To identify the DNA binding site(s) in Escherichia coli DNA polymerase I (pol I) (Klenow fragment), the authors have used an active-site-directed reagent, phenylglyoxal (PG), which specifically reacts with arginine residues. Preincubation of DNA pol I with PG resulted in the loss of polymerase, 3'-5'-exonuclease, and DNA binding functions. Furthermore, the presence of DNA but not ...
A and up to 96% labeling of BSA (Table 1). Myoglobin was labeled with reagent 9 at 6-8%. The background% modification of myoglobin. Tryptic digest and subsequent ESI-MS analysis of the fragments of all proteins was additionally labeled (Figure 3). Reagent 10 modified myoglobin at W15 at a very low level, consistent
The amino acid sequence of Pseudomonas fluorescens azurin has been determined. The protein consists of a single peptide chain of 128 residues. There is one intra-chain disulphide bridge. The sequence was determined by isolation of the soluble tryptic peptides, and by exhaustive examination of the products of chymotryptic and peptic digestion. The sequence ...
epidermal keratinocytes (keratin filaments) and of baby hamster kidney(BHK-21) cells were studied to de of the keratin IF subunits. Intact IF were subjected to limited tryptic digestion, and structurally identical a (smaller, most basic), respectively. Bovine epidermal keratin IF consist of at least 10 different subunits
Institute of Allergy and Infectious Diseases, (NIH/DHHS through interagency agreement Y1-AI-4894 for concurrent LC-MS and fractionation LC Column Flow-Split 12T FTICR-MS 1. On-line LC-MS Silicon chip/well 3. On-plate tryptic digestion HPLC System Silicon chip bearing a 20 x 20 array of 5�m nanospray
We herein demonstrate superparamagnetic Fe3O4 nanoparticles coated with nitrilotriacetic acid derivative (NTA) that can bind with different immobilized metal ions are capable of probing diverse target species. Immobilized Ni(II) on the surfaces of the NTA-magnetic nanoparticles have the capability of selectively trapping histidine (His)-tagged proteins such as a mutated streptopain tagged with 6x ...
Tryptic digests of human serum albumin (HSA) and human lung epithelial cell lysates were used as test samples in a novel proteomics study. Peptides were separated and analyzed using 2D-nano-LC/MSMS with strong cation exchange (SCX) and reverse phase (RP) chromatography and contin...
to the corresponding sulfinic acid (+32 Da). The formation of sulfinic acid and sulfenic acid in cysteine- containing. The resulting metalloprotein was active in the hydrogenation of 2-(acetamido)acrylic acid and N-acyl alanineH of the solution to acid. Tryptic digestion of modified papain in gel The selected
Primate antiserums to human leukemia cells can detect antigens specific for lymphocytic leukemia cells or antigens present on certain myeloid leukemia cells. The antigen specific for lymphocytic leukemia cells is destroyed by treatment with neuraminidase or trypsin. Tryptic digests of lymphocytic leukemia cells contain the antigen, which has a high ...
EH and sEH sequences are similar to a bacterial haloalkane dehalogenase and other related proteins was key of haloalkane dehalogenase (54, 63). Electrospray mass spectrometric analysis of the tryptic digestion of murine the /-hydrolase fold family. For example, haloalkane dehalogenases have tryptophans (53), whereas esterases have
Desorption electrospray ionization mass spectrometry (DESI-MS) was investigated as a method to detect and identify peptides from tryptic digests of cytochrome c and myoglobin separated on ProteoChrom HPTLC Silica gel 60 F(254s) plates and ProteoChrom HPTLC Cellulose sheets. Full-scan mass spectra and data-dependent tandem mass spectra were acquired in ...
Desorption electrospray ionization mass spectrometry was investigated as a method to detect and identify peptides from tryptic digests of cytochrome c and myoglobin separated on ProteoChrom HPTLC Silica gel 60 F254s plates and ProteoChrom HPTLC Cellulose sheets. Both full scan mass spectra and data-dependent tandem mass spectra were acquired in separate ...
Aquaporin (AQP) family plays a pivotal role in fluid secretion and absorption, especially in the digestive system and secretory glands. Within this family, AQP8 was reported to be widely expressed in the epithelia of the digestive tract, liver, and pancreas. In two parallel experimental platforms with different analytical and comparative approaches, in-gel ...
Tryptic digestion of proteins in trypsin loaded porous silica has been shown to be highly efficient. Enzymatic silica-reactors were prepared by immobilizing trypsin into macroporous ordered siliceous foam (MOSF) and into mesoporous SBA-15 silica which has a smaller pore size. The tryptic products from the silica reactors were analyzed ...
Differential recovery of peptides due to nonspecific adsorption can seriously compromise reproducibility and quality of proteomic data for peptide analyses by liquid chromatography-mass spectrometry (LC-MS). This study demonstrates large variations in reproducibility and quantitation of LC-MS data for peptides derived from tryptic digests of BSA upon ...
Phycoerythrobilin peptides were prepared from Porphyridium cruentum B-phycoerythrin by tryptic digestion and isolated under mild conditions without alteration in the spectroscopic properties of the bilin. High-resolution /sup 1/H NMR spectral analysis of the smallest trypsin fragment derived from the ..cap alpha.. subunit of B-phycoerythrin, the ..cap ...
An analogue of the replicase translational operator of bacteriophage R17, that contains a 5-bromouridine at position -5 (RNA 1), complexes with a dimer of the coat protein and photocrosslinks to the coat protein in high yield upon excitation at 308 nm with a xenon chloride excimer laser. Tryptic digestion of the crosslinked nucleoprotein complex followed ...
Hydrodynamic disintegration of activated sludge resulted in organic matter and polymers transfer from the solid phase into the liquid phase. Disintegration by hydrodynamic cavitation had a positive effect on the degree and rate of excess sludge anaerobic digestion. Also, addition of a part of anaerobic digested sludge containing adapted microorganisms ...
In this study, we investigated a novel ionic liquid matrix (ILM), namely, the 1,1,3,3-tetramethylguanidinium salt of 2,4,6-trihydroxyacetophenone (THAP). This matrix[1,1,3,3-tetramethylguanidinium 2,4,6-trihydroxyacetophenone (GTHAP)] turned out to be well suited for the matrix-assisted laser desorption/ionization mass spectrometric analysis of glycopeptides and glycans, and overcame the ...
The structural organization of the steroid-binding protein of the IM-9 cell glucocorticoid receptor was investigated by using one- and two-dimensional gel electrophoresis of proteolytic receptor fragments. One-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of receptor fragments isolated after trypsin digestion of immunopurified (/sup ...
In this paper, a general procedure is described for the generation of peptide maps of proteins with monolithic silica-based columns. The peptide fragments were obtained by tryptic digestion of various cytochrome c species with purification of the tryptic fragments achieved by reversed-phase high-performance liquid chromatographic ...
In contrast to lower phosphorylation states (e.g., the tryptic monophosphopeptide FQpSEEQQQTEDELQDK from bovine -casein), the specific detection of multi-phosphorylated peptides (e.g. the tetraphosphopeptide RELEELNVPGEIVEpSLpSpSpSEESITR from tryptic digestion of bovine -casein) has often been problematic for liquid chromatography-mass ...
18-Crown-6 ether (18C6) is evaluated as a shift reagent for multidimensional ion mobility spectrometry-mass spectrometry (IMS-IMS-MS) analyses of tryptic protein digests. In this approach, 18C6 is spiked into the solution-phase mixture and noncovalent peptide-crown ion complexes are formed by electrospraying the mixture into the gas phase. After an initial ...
Desorption electrospray ionization mass spectrometry (DESI-MS) was demonstrated as a method to detect and identify peptides from two-dimensional separations of cytochrome c and myoglobin tryptic digests on ProteoChrom HPTLC Cellulose sheets. Data-dependent tandem mass spectra were acquired during lane scans across the TLC plates. Peptides and the ...
A gel absorption-based sample preparation method for shotgun analysis of membrane proteome has been developed. In this new method, membrane proteins solubilized in a starting buffer containing a high concentration of sodium dodecyl sulfate (SDS) were directly entrapped and immobilized into gel matrix when the membrane protein solution was absorbed by the vacuum-dried polyacrylamide gel. After the ...
A synthetic approach to model the analytical complexity of biological proteolytic digests has been developed. Combinatorial peptide libraries ranging in length between 9 and 12 amino acids that represent typical tryptic digests were designed, synthesized, and analyzed. Individual libraries and mixtures thereof were studied by replicate ...
A synthetic approach to model the analytical complexity of biological proteolytic digests has been developed. Combinatorial peptide libraries ranging in length between nine and twelve amino acids that represent typical tryptic digests were designed, synthesized and analyzed. Individual libraries and mixtures thereof were studied by ...
We perform two-dimensional capillary electrophoresis on fluorescently labeled proteins and peptides. Capillary sieving electrophoresis was performed in the first dimension and micellar electrokinetic capillary chromatography was performed in the second. A cellular homogenate was labeled with the fluorogenic reagent FQ and separated using the system. This homogenate generated a pair of ridges; the ...
1. A tryptic digest of the protein fraction U.S.3 from oxidized wool has been separated into 32 peptide fractions by cation-exchange resin chromatography. 2. Most of these fractions have been resolved into their component peptides by a combination of the techniques of cation-exchange resin chromatography, paper chromatography and paper electrophoresis. 3. ...
This work brings together some contributions for the use of nonaqueous media for proteomic analysis, for both capillary electrophoresis (CE) separation and the preparation of tryptic digests. First, a ternary nonaqueous buffer consisting of 60/30/10 v/v methanol/acetonitrile/acetic acid with 12.5 mmol/L ammonium acetate was optimized for CE separation of ...
Tyr(P)-containing proteins were purified from extracts of insulin-treated rat hepatoma cells (H4-II-E-C3) by antiphosphotyrosine immunoaffinity chromatography. Two major insulin-stimulated, Tyr(P) proteins were recovered: an M/sub r/ 95,000 protein (identified as the insulin receptor ..beta.. subunit by its immunoprecipitation by a patient-derived anti-insulin receptor serum and several ...
... BAM Media M154: Trypticase (Tryptic) Soy Broth. January 2001. Bacteriological Analytical Manual. M154 Trypticase (Tryptic) Soy Broth. ...
Center for Food Safety and Applied Nutrition (CFSAN)
... BAM Media M152: Trypticase (Tryptic) Soy Agar. January 2001. Bacteriological Analytical Manual. M152 Trypticase (Tryptic) Soy Agar. ...
In this study we present a rapid method for tryptic digestion of proteins using micro-columns with enzyme immobilized on perfusion chromatography media. The performance of the method is exemplified with acyl-CoA-binding protein and reduced carbamidomethylated bovine serum albumin. The method proved to be significantly faster and yielded a better sequence ...
Trypsin rapidly inactivated the catalytic activities of spinach ribulose bisphosphate carboxylase/oxygenase but the stoichiometry of binding of the reaction-intermediate analogue carboxyarabinitol bisphosphate was only slightly reduced after proteolysis. Electrophoretic analysis indicated that several forms of the large subunit were generated during proteolysis but that the small subunit was ...
The spatial distribution of proteins in tissue sections can be used to identify potential markers for pathological processes. Tissue sections are often subjected to enzymatic digestion before matrix-assisted laser desorption/ionization (MALDI) imaging. This study is targeted at improving the on-tissue identification of tryptic peptides by accurate mass ...
The sampling, ionization and detection of tryptic peptides separated in one-dimension on reversed-phase high-performance thin layer chromatography (HPTLC) plates was performed using liquid microjunction surface sampling probe electrospray ionization mass spectrometry. Tryptic digests of five proteins [cytochrome c, myoglobin, ...
The sampling, ionization and detection of tryptic peptides separated in one-dimension on reversed phase HPTLC plates was performed using liquid microjunction surface sampling probe electrospray ionization mass spectrometry. Tryptic digests of five proteins (cytochrome c., myoglobin, beta-casein, lysozyme, and bovine serum albumin) were ...
Partial proteolytic digestion of the mammary prolactin (PRL) receptor was used to generate receptor fragments and analyze their immunoreactivity and PRL binding properties. Tryptic digestion of the PRL receptor produced two immunoreactive fragments (Mr approximately 30,000 and approximately 15,000) that reacted with a monoclonal ...
A protocol for improved extraction of peptides from in-gel protein digests, using a combination of the acid labile surfactant, sodium deoxycholate (SDC) and C18 Empore� membranes, is presented. This approach results in better mass spectrum quality, higher numbers of identified peptide peaks and improved identification scores compared to standard tryptic ...
The use of histidine as a protein digestion buffer followed by isoelectric trapping separations using 'membrane separated wells for isoelectric focusing and trapping' (MSWIFT) and mass spectrometry (MS) analysis is described. Tryptic digestion of bovine serum albumin (BSA) performed in histidine buffered solutions yields similar amino ...
Glycoprotein structure determination and quantification by MS requires efficient isolation of glycopeptides from a proteolytic digest of complex protein mixtures. Here we describe that the use of acids as ion-pairing reagents in normal-phase chromatography (IP-NPLC) considerably increases the hydrophobicity differences between non-glycopeptides and glycopeptides, thereby ...
Peptide mass fingerprinting (PMF) of protein digests is a widely-accepted method for protein identification in MS-based proteomic studies. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI) is the technique of choice in PMF experiments. The success of protein identification in a PMF experiment is directly related to the amount of amino acid sequence ...
For label-free expression profiling of tissue proteomes, efficient protein extraction, thorough and quantitative sample cleanup and digestion procedures, as well as sufficient and reproducible chromatographic separation, are highly desirable but remain challenging. However, optimal methodology has remained elusive, especially for proteomes that are rich in membrane proteins, ...
A 2-D separation platform was constructed using micro strong cation-exchange liquid chromatography (?-SCXLC) and reversed-phase pressurized capillary electrochromatography (RP-pCEC) for the analysis of complex samples. Samples were fractionated by the first-dimension ?-SCXLC with a linear solvent gradient and then injected into the second-dimension RP-pCEC for further separation. The ...
The template activities of the 60-70S RNA complex and of the 30-40S subunit RNA species of Rous sarcoma virus were tested in a cell-free protein-synthesizing system from mouse ascites Krebs II cells. Stimulation of protein synthesis over the endogenous background was about 2-fold with 30-40S viral RNA and about 1.3-fold with 60-70S viral RNA as template. Analysis by sodium dodecyl sulfate-gel ...
We report on the preparation and performance of a high efficiency 70 cm ´ 20 µm i.d. silica-based monolithic capillary column. With a mobile phase delivery pressure of 5000 psi, this monolithic column provides flow rates as low as ~40 nL/min at an LC linear velocity of ~0.24 cm/s. The resultant columns provided a separation peak capacity of ~420 under conditions of on-line coupling micro solid ...
The pyruvate dehydrogenase complex was purified to homogeneity from baker's yeast (Saccharomyces cerevisiae). Yeast cells were disrupted in a Manton-Gaulin laboratory homogenizer. The pyruvate dehydrogenase complex was purified by fractionation with polyethylene glycol, isoelectric precipitation, ultracentrifugation and chromatography on hydroxylapatite. Final purification of the yeast ...
The mechanism of nonsense codon mistranslation at one particular amber mutation, trpE278, was characterized by determining the amino acid sequence in the region of readthrough of the TrpE278 polypeptide. The amino acid sequence of this region was found to be identical to the wild type sequence except for residue 71, which was Lys. The domain structure of the native TrpE polypeptide was probed by ...
Oxidation of thiol proteins, which results in conversion of cysteine residues to cysteine sulfenic, sulfinic or sulfonic acids, is an important posttranslational control of protein function in cells. To facilitate the analysis of this process with MALDI-MS, we have developed a method for selective enrichment and identification of peptides containing cysteine sulfonic acid (sulfopeptides) in ...
Light (L) chains of the lambda type are rare in mouse immunoglobulins. One lambda chain, the L chain of myeloma protein 315 (L315), differs in amino acid sequence at many positions in the COOH-terminal domain from all other lambda chains whose sequences have been determined (called lambda1 chains). To determine whether chains of the L315 type (called lambda2) occur in normal mouse immunoglobulins, ...
Peptic-tryptic digests of alcohol-soluble proteins from flours of 10 accessions of Trificum monococcum with contrasting storage protein compositions and bread-making characteristics were found unable to agglutinate K562(S) cells even at a peptide concentration as high as 14 g/L, agglutination being strongly correlated with toxicity in celiac disease. When ...
RNA-protein cross-links were introduced into brome mosaic virus in situ by using the heterobifunctional agent p-azidophenylglyoxal. An improved RNA isolation method, without phenol extraction, was used to isolate RNA cross-linked with protein. RNA of the covalently linked complex was acid-digested and the oligonucleotides still attached to protein were 5'-end-labelled with ...
Juvenile hormone esterase (JHE, EC 3.1.1.1) from whole Drosophila melanogaster prepupae has previously been purified by selective precipitations, isoelectric focussing and two column chromatography steps. JHE bands from dried silver-stained SDS-PAGE gels of that material were digested with trypsin. The masses of the tryptic digest ...
Because of its good biocompatibility, high surface-to-volume ratio, and distinct surface electrical properties, porous anodic alumina (PAA) membrane has been used to selectively enrich phosphopeptides from a mixture of synthetic peptides and tryptic digest product of beta-casein by a direct MALDI-TOF MS analysis. As we reported previously, PAA membrane has ...
Data-dependent selective external ion ejection with improved resolution is demonstrated with a 3.5 tesla FTICR instrument employing DREAMS (Dynamic Range Enhancement Applied to Mass Spectrometry) technology. To correct for the fringing rf-field aberrations each rod of the selection quadrupole has been segmented into three sections, so that ion excitation and ejection was performed by applying ...
A high-throughput approach for biomolecule analysis is demonstrated for a mixture of peptides from tryptic digest of four proteins as well as a tryptic digests of human plasma. In this method a chip-based electrospray autosampler coupled to a hybrid ion mobility (IMS) mass spectrometer (MS) is utilized to achieve ...
The principal objective of this study was the evaluation of two-dimensional gel electrophoresis (2-DE) in combination with MALDI-TOF MS, after tryptic digest with regard to suitability for qualitative characterization and identification of therapeutic recombinant monoclonal antibodies trastuzumab and rituximab. Moreover, the impact of post-translational ...
Although there are now multiple methods for analysis of membrane proteomes there is relatively little systematic characterization of proteomic workflows for membrane proteins. The Asia Oceania Human Proteome Organisation (AOHUPO) has therefore embarked on a Membrane Proteomics Initiative using a large range of workflows. Here we describe characterization of the Membrane Proteomics Initiative mouse ...
Two procedures were proposed for the specific recovery of fluorescent derivatives of glycoprotein-derived oligosaccharides and tryptic glycopeptides using certain plant lectins. The first was based on the salting out of oligosaccharide-lectin conjugates with ammonium sulfate. Oligosaccharides specifically bound to lectins were recovered free from lectins using ethanol ...
The toxicity of two varieties of bread wheat, one poor in alpha and beta gliadins and the other poor in alpha, beta, gamma, and omega gliadins, has been tested. The peptic-tryptic digest of these wheats was assessed using coeliac mucosa in an in vitro organ culture system. A significantly lower toxicity was found in respect of bread wheat containing all ...
Four unique carboxymethylcysteine-containing peptides were isolated from tryptic and chymotryptic digests of trout muscle actin carboxymethylated with iodo[2-14C]acetic acid in 6m-guanidinium chloride. The amino acid sequences of these peptides were determined and showed a high degree of homology with the corresponding sequences from ...
Phytohemagglutinin, the lectin of the common bean Phaseolus vulgaris, has a high mannose and a modified (fucosylated) oligosaccharide on each polypeptide. Fractionation by high performance liquid chromatography of tryptic digests of (/sup 3/H)fucose or (/sup 3/H)glucosamine labeled phytohemagglutinin, followed by amino acid sequencing of the isolated ...
Five glycosaminoglycans were isolated from tryptic digestion of both cancerous and normal tissues of the human pancreas and were assayed by determining the carbohydrate content of materials. Separation of these five polymers was achieved by Dowex 1-X2 column chromatography and fractionation with Benedict's solution. They were identified as hyaluronic acid, ...
An ink jet printer valve and nozzle were used to deliver matrix and sample from an electrophoresis capillary onto a MALDI plate. The system was evaluated by separation of a set of standard peptides. That separation generated up to 40,000 theoretical plates in less than three minutes. Detection limits were 500 amol using an ABI TOF-TOF instrument and 2 fmol for an ABI Q-TOF instrument. Over 70% ...
The assignment of protein glycosylation sites and their microheterogeneities are of biological importance, yet such characterization is still considered to be analytically very challenging. Several approaches have been recently developed to improve the characterization of glycosylation sites of proteins, including lectin and HILIC enrichment-based methods coupled to mass spectrometry. However, ...
To separate proteins with a wide distribution of pIs under the conditions compatible to online tryptic digestion (with preferable pH=8.0), weak anion and cation exchange chromatography (WAX/WCX) mixed-bed microcolumn has been developed. With a mixture of five proteins with pIs ranging from 4.2 to 11.4, the effect of WAX/WCX ratio on the separation ...
An endogenous brain neuropeptide with 104 amino acid residues that modulates ..gamma..-aminobutyric acid receptor function was termed DBI because it displaces diazepam from its specific brain binding sites. Tryptic digestion of DBI generates an octadecaneuropeptide (ODN) that is more potent than the parent compound in the displacement of specifically bound ...
The influence of the mobile-phase composition and the pressure on the chromatographic separation of the peptides from the enzymatic digest of myoglobin was studied under linear conditions. The retention behavior of these tryptic peptides was measured under isocratic conditions with different mobile-phase compositions, ranging from 9 to 28% (v/v) ...
Conditions for carrying out chemically targeted identification of peptides containing phosphorylated or glycosylated serine residues have been investigated. Ba(OH)2 was used at ambient temperature to catalyze the ?-elimination reaction at 25�C. Nucleophilic addition of 2-aminoethanethiol was performed in both parallel and tandem experiments. The method was demonstrated by ...
The circulating concentration of a biomarker for congestive heart failure, Brain (B-type) Natriuretic Peptide (BNP-32), is measured using ELISA based assays in order to rapidly diagnose and monitor disease progression. The lack of molecular specificity afforded by these assays has recently come into question as emerging studies indicate there are potentially multiple heterogeneous forms of BNP in ...
Alpha-zirconium phosphate nanoplatelets (alpha-ZrPN) were studied as a binding agent for phosphopeptides. Nanoplatelets of alpha-zirconium phosphate were incubated overnight with zirconium oxychloride, followed by centrifugation, and washed twice with water followed by an aqueous solution of 80% acetonitrile to form the binding agent. Alpha-ZrPN were able specifically to capture ...
Monolithic columns having long hydrocarbon chains were prepared by in-situ polymerization in capillary fused silica tubing. The capillary columns were coupled with a newly developed carbon fiber electrospray ionization (ESI) emitter for proteomic analysis using sheathless capillary HPLC-ESI mass spectrometry (MS). The sample loading capacity and chromatographic performance of the styrene-based ...
Replicate injections of a myoglobin tryptic digest, ultrafiltrates of human serum, and ultrafiltrates of human plasma made on a splitless nanoscale liquid chromatography system coupled to a Fourier-transform ion cyclotron resonance mass spectrometer were utilized to assess analytical reproducibility. The mean (across 19 tryptic ...
Protein disulfide isomerase (PDI) has been identified in a protein extract from the venom duct of the marine snail C. amadis. In-gel tryptic digestion of a thick protein band at approximately 55 kDa yields a mixture of peptides. Analysis of tryptic fragments by MALDI-MS/MS and LC-ESI-MS/MS methods permits sequence assignment. Three ...
Tryptic peptides derived from the regulatory subunits of brain and heart cAMP-dependent protein kinase II were mapped by reverse phase HPLC. At 280 nm, 15 unique peptides were found only in the heart RII digest, while 5 other peptides were obtained only from brain RII. At 210 nm, 13 brain-RII specific and 15 heart-RII specific tryptic ...
A patent application for an accelerated digestion process for releasing trichinellae from meat (usually pork) as excysted and encysted worms in transparent fluid whereby they may be easily identified and/or counted visually or automatically is described. ...
Mass spectrometry-based multiple reaction monitoring (MRM) quantitation of proteins can dramatically impact the discovery and quantitation of biomarkers via rapid, targeted, multiplexed protein expression profiling of clinical samples. A mixture of 45 peptide standards, easily adaptable to common plasma proteomics work flows, was created to permit absolute quantitation of 45 endogenous proteins in ...
The peptide, HLLVMKGAPER, which contains Lysine 501 of the {alpha} polypeptide can be released from intact sodium and potassium ion activated adenosinetriphosphatase by tryptic digestion. An immunoadsorbent directed against the carboxy-terminal, -GAPER, has been constructed. Sealed, right-side-out vesicles, prepared from canine renal kidneys, were labeled ...
This paper presents a rapid method based on MALDI-TOF-MS peptide mass finger printing (PMF) for identification of somatropin. The protein was digested with either trypsin or endoproteinase Lys-C prior to MALDI-MS analysis. The identification was then performed by comparing the measured mass spectra with those predicted in-silico. The cysteine residues at positions 182, 190 and ...
The complex of digestive proteinases in caterpillars of the greater wax moth Galleria mellonella was studied. Using chromogenic substrates and inhibitor analysis, it was found that serine proteinases play a key role in this complex. Three anionic and two cationic forms of trypsin and one anionic and one cationic form of chymotrypsin were identified by zymography in the midgut ...
Quantitative proteomics requires novel analytical methodology to fill the gap related to absolute protein abundance in different physiological conditions. In this paper, we demonstrate a proof-of-concept study for absolute protein quantification. 1,4,7,10-Tetraazacyclododecane-1,4,7-trisacetic acid-10-maleimidoethylacetamide (MMA-DOTA) loaded with Eu was used to label lysozyme, insulin, and ...
Simon P. Schurr, Dianne P. O'Leary, Andr\\'e Tits ... Optimization Software and Modeling Systems ... Optimization for Simulation: LAD Accelerator ...
Mechanisms for accelerated biodegradation of cellulosic substrates have been examined, especially analyses and optimization of pre-treatment of the fiber prior to microbial digestion. Methodology has been developed for enumeration of cellulolytic microorg...
We demonstrate that the microwave-assisted protein enzymatic digestion (MAPED) method can be successfully applied to the mass spectrometric characterization of proteins captured on the affinity surfaces of protein chips. The microwave-assisted on-chip tryptic digestion method was developed using a domestic microwave, completing the ...
The effect of raw and anaerobically digested sludge on heat inactivation of poliovirus was investigated. Raw sludge was found to be very protective of poliovirus plaque-forming ability at all temperatures studied, but digested sludge had variable effects that were highly dependent upon the experimental conditions. In low concentrations and at relatively ...
Rat brain membrane benzodiazepine receptors (BZR) were photoaffinity labelled specifically (in presence or absence of 6 ..mu..M clonazepam) with 10 nM /sup 3/H-flunitrazepam (FNZ). Digestion of the FNZ-labelled, membrane-bound BZR with 200 ..mu..g trypsin/mg membrane protein yielded H/sub 2/O-soluble BZR-fragments of molecular mass (M/sub r/) 34, 31, 28, 24, 21, 18, 16, 12, 10 ...
Na, K-ATPase is believed to be the receptor for digitalis glycosides. The authors have previously documented that C17 side group of the cardenolide molecule is crucial to ..cap alpha.. subunit receptor binding. They have attempted to identify the structure of this binding site by labelling the enzyme with a /sup 3/H-labelled photoactive probe localized in the C17 side group of the genin molecule. ...
A recently published model of the human erythrocyte hexose transporter deduced from the protein's primary structure proposes that the transporter is organized into two membrane domains comprising 77% of the protein's mass and three hydrophilic domains, a short segment that includes the polypeptide's N-terminus and two larger segments, one lying between the membrane ...
T-antigen (the simian virus 40 A cistron protein) was purified by immunoprecipitation and electrophoresis on polyacrylamide gels from monkey kidney CV-1 cells infected with simian virus S (SV-S), dl1263, or dl1265 and digested with trypsin. The tryptic peptides, labeled with [35S]methionine, [35S]cysteine, or [3H]proline, were fractionated either by ...
Analysis of a tryptic digest of a recombinant D83A mutant of the murine dioxin receptor (D83A-DR) by CapHPLC-MALDI-TOF/TOF-MS/MS revealed a peptide corresponding to cleavage at a lysine that had either been mutated to an arginine or modified by dimethylation or formylation (Ser-Phe-Phe-Ala-Val-Ala-Leu-Me2K/FormK/Arg). High mass accuracy data were obtained ...
Rapid screening for phosphopeptides within complex proteolytic digests involving electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR-MS) in the negative ion mode with infrared multiphoton dissociation (IRMPD) accompanied by improved phosphopeptide sensitivity and selectivity is demonstrated with the tryptic ...
Here we present a demonstration of the proof of principle that absolute concentration of a protein within a mixture of other proteins can be measured with SI traceability. The method used was based on tryptic digestion of a protein followed by quantification using double exact matching isotope dilution mass spectrometry (IDMS) of the peptides released. To ...
The regulatory site peptide sequence of phosphorylated inactive pyruvate, orthophosphate dikinase from maize leaf tissue was determined by automated Edman degradation analysis of /sup 32/P-labeled peptides purified by reversed-phase high performance liquid chromatography. The overlapping phosphopeptides were products of a digestion of the (..beta..-/sup 32/P)ADP-inactivated ...
The authors have purified the two major isozymes of the L-isoaspartyl/D-aspartyl protein methyltransferase from both human and bovine erythrocytes. These four enzymes all have polypeptide molecular weights of approximately 26,500 and appear to be monomers in solution. Each of these enzymes cross-reacts with antibodies directed against protein carboxyl methyltransferase I from bovine brain. Their ...
Mammalian liver mitochondrial carbamoyl phosphate synthetase, a polypeptide of 160 kDa, is activated allosterically by N-acetyl-L-glutamate. The analogue of this activator N-(chloroacetyl)-L-({sup 14}C)glutamate has been found to serve as a photoaffinity label for this enzyme. The specificity was demonstrated by the drastic reduction in the radioactivity bound to the protein when (a) an excess of ...
Formalin-fixed paraffin-embedded (FFPE) tissues have been used to discover disease-associated protein changes using mass spectrometry. Protein post-translational modifications such as glycosylation are known to associate with disease development. In this study, we investigated whether FFPE tissues preserve such modifications and therefore can be used as specimen of choice to identify the ...
The mass defect of a substance can be used in mass spectral analysis to identify peaks as likely belonging to a compound class, such as peptides, if the mass defect is within the known range for that compound class. For peptides, a range of possible mass defects was calculated previously, using a set of theoretical peptides, where all possible amino acid combinations were considered (Mann , M. ...
The NAD(P)H-nitrate reductase complex (overall-NR) of Chlamydomonas reinhardii exhibits two partial activities: NAD(P)H-cytochrome c reductase (diaphorase) and reduced benzyl viologen-NR (terminal-NR). Mild tryptic digestion of the enzyme complex resulted in the loss of both overall and terminal-NR activities, whereas diaphorase activity remained ...
The protein covalently bound to the 5' termini of adenovirus type 2 DNA has been purified from virus labeled with (/sup 35/S)methionine, using exclusion chromatography of disrupted virions to isolate the DNA-protein complex, which is then digested with DNase. The terminal protein isolated from mature virus is most effectively labeled if the cells are exposed to (/sup ...
A novel chemically modified insulin, -NB29-DHC-insulin (where DHC-insulin stands for dehydrocholyl-insulin), was prepared by covalent linkage of DHC (dehydrocholic acid) to the -amino group of LysB29 without any protecting agent and analysed by PAGE and reversed-phase HPLC. DHC-insulin was identified by MALDI-TOF-MS (matrix-assisted laser-desorption ionization-time-of-flight MS) coupled with ...
In-gel digestion is an attractive route in mass spectrometry-based proteomic analysis, which, however, often suffers from a certain amount of sample loss mainly due to insufficient protein digestion and peptide extraction. To address this, herein we establish a partially degradable gel-assisted protein digestion and peptide recovery ...
In this report, trypsin was immobilized on silica-coated fiberglass core in microchip to form a core-changeable bioreactor for highly efficient proteolysis. To prepare the fiber core, a layer of organic-inorganic hybrid silica coating was prepared on the surface of a piece of glass fiber by a sol-gel method with tetraethoxysilane (TEOS) and 3-aminopropyltriethoxysilane (APTES) as precursors. ...
Selective detection of phosphopeptides from complex biological samples is a challenging and highly relevant task in many proteomics applications. In this study, a novel phosphopeptide enrichment approach based on the strong interaction of Fe(3)O(4)@Al(2)O(3) magnetic core-shell microspheres with phosphopeptides has been developed. With a well-defined core-shell structure, the Fe(3)O(4)@Al(2)O(3) ...
to accelerate exact caching systems, by employing a multi-level cache ( Figure 3.4). A digest cache3-1 Chapter 3 Efficient Packet Classification with Digest Caches Francis Chang, Wu-chang Feng, Wu-chi Feng Systems Software Laboratory, OGI School of Science and Engineering at OHSU Kang Li Department
Proteomic analysis typically has been performed using proteins digested with trypsin because of the excellent fragmentation patterns they produce in collision induced dissociation (CID). For analyses in which high protein coverage is desirable, such as global monitoring of post-translational modifications, additional sequences can be seen using parallel ...
A poly (acrylamide-co-methylenebisacrylamide) (poly (AAm-co-MBA)) monolith was prepared by thermal polymerization in the 100 or 250 ?m i.d. capillary. The monolithic support was activated by ethylenediamine followed by glutaraldehyde. Trypsin was then introduced to form an immobilized enzyme reactor (IMER). The prepared IMER showed a reliable mechanical stability and permeability (permeability ...
... BAM Media M186: Trypicase (Tryptic) Soy Broth with ferrous sulfate. January 2001. ... M186 Trypicase (Tryptic) Soy Broth with ferrous sulfate. Trypticase peptone, 17 ...
There is increasing evidence for the importance of the secretory immune system in the gut. In studies of local antibody production it is important to have satisfactory methods for measuring immunoglobulin concentrations and to be aware of the errors which may occur. Studies on immunoglobulin measurement in intestinal secretion by the radial immunodiffusion method are reported, showing the effects ...
The genus Nepenthes comprises carnivorous plants that digest insects in pitcher fluid to supplement their nitrogen uptake. In a recent study, two acid proteinases (nepenthesins I and II) were purified from the pitcher fluid. However, no other enzymes involved in prey digestion have been identified, although several enzyme activities have been reported. To ...
Advanced glycation end-product (AGE)/peptides, arising from in vivo digestion of glycated proteins, are biologically important compounds, due to their reactivity against circulating and tissue proteins. For information on their possible structure, in vitro glycation of HSA and its further enzymatic digestion were performed. The resulting ...
A previous study from our laboratory suggested that 3-bromo-1,4-dihydroxy-2-butanone 1,4-bisphosphate is an affinity label for spinach ribulosebisphosphate carboxylase. To identify the essential residues that react with the reagent we have isolated and characterized the labeled peptides that are present in tryptic digests of inactivated enzyme but lacking ...
A polyacrylate-based monolithic column bearing cationic functionalities and designed for capillary electrochromatography (CEC) has been prepared via photopolymerization of a mixture of hexyl acrylate, butanediol diacrylate, 2-(acryloyloxy) ethyltrimethyl ammonium chloride (monomers), azobisisobutyronitrile (photoinitiator), acetonitrile, phosphate buffer, and ethanol (porogens). The polymerization ...
To determine the epitopic structure for an anti-Siaalpha2-6GalNAcalpha-Ser/Thr (anti-sialyl Tn) monoclonal antibody, MLS 132, ovine submaxillary mucin (OSM) was digested with the combination of trypsin and thermolysin and the digest fractionated by immunoaffinity column chromatography and HPLC. From tryptic digest, ...
Protein citrullination results from enzymatic deimination of peptidylarginine and plays an important role in health and disease. Despite increasing scientific interest, the identity and function of citrullinated proteins in vivo remain widely unknown. Thorough proteomic studies could contribute to a better understanding of the role of this posttranslational modification but will require tools for ...
Tryptic peptides which account for all five cysteinyl residues in ribulosebisphosphate carboxylase/oxygenase from Rhodospirillum rubrum have been purified and sequenced. Collectively, these peptides contain 94 of the approximately 500 amino acid residues per molecule of subunit. Due to one incomplete cleavage at a site for trypsin and two incomplete chymotryptic-like ...
The authors have studied the sensitivity of sulfhydryl groups of a highly purified p21 protein of the v-ras/sup H/ oncogene to a thiol-specific reagent, N-ethylmaleimide (NEM). Approximately 70% of GTP binding and autokinase activities of p21 were inactivated by NEM, and excessive amounts of GTP or GDP protected p21 activities. Thiol titration revealed the presence of one fast reactive cycteine ...
This investigation describes the separation of tryptic peptides by capillary reversed-phase high-performance liquid chromatography (RP-HPLC) with eluents in the intermediate pH range, followed by in-line electrospray ionisation tandem mass spectrometry (ESI-MS/MS) analysis. For these purposes, gradient elution procedures with an aqueous eluent containing 20 mM ammonium ...
Recombinant porcine (rpST) and bovine somatotropins (rbST) synthesized in Escherichia coli contain the amino acid, epsilon-N-acetyllysine. This amino acid was initially discovered in place of the normal lysine144 in a modified reversed-phase HPLC (RP-HPLC) species of rpST. Mass spectrometry and amino acid sequencing of a tryptic peptide isolated from this RP-HPLC purified ...
Using the technique of UV-mediated cross-linking of nucleotides to their acceptor sites, we have labeled calf terminal deoxynucleotidyltransferase (TdT) with (/sup 32/P)dTTP. The specificity of dTTP cross-linking at the substrate binding site in TdT is demonstrated by the competitive inhibition of the cross-linking reaction by other deoxynucleoside triphosphates, and ATP and its analogues, ...
Being able to rapidly and sensitively detect specific enzymatic products is important when screening biological samples for enzymatic activity. We present a simple method for assaying protease activity in the presence of protease inhibitors (PIs) by measuring tryptic peptide accumulation on copolymer pMALDI target chips using a dual fluorescence/MALDI-TOF-MS read-out. The ...
Fe3O4@ZrO2 microspheres with well-defined core-shell structure were prepared and applied for the highly selective enrichment of phosphopeptides from tryptic digest product of proteins. To successfully coat iron oxide microspheres with uniform zirconia shell, magnetic Fe3O4 microspheres were first synthesized via a solvothermal reaction, followed by being ...
An approach based on staggered multistep elution solid-phase extraction (SPE) capillary electrophoresis/tandem mass spectrometry (CE/MS/MS) was developed in the analysis of digested protein mixtures. On-line coupling of SPE with CE/MS was achieved using a two-leveled two-cross polydimethylsiloxane (PDMS)-based interface. Multistep elution SPE was used prior to CE to provide an ...
Two molecular forms of the muscarinic cholinergic receptor have been consistently observed in a variety of species, albeit in variable amounts. Proteins which are specifically labeled by (/sup 3/H)propylbenzilylcholine mustard ((/sup 3/H)PrBCM) were observed at 94kDa and 80kDa upon SDS-PAGE of membrane proteins prepared from brains and hearts of trout, frog, turtle, chicken, rat, and pig. They ...
Due to hydrophobicity, structural analysis of integral membrane proteins poses a formidable challenge for current mass spectrometry-based proteomics approaches. Herein, we demonstrate results from optimized sample preparation and enzymatic proteolysis procedures for the complete primary structure determination of a targeted integral membrane protein, lens aquaporin 0 (AQP0). Plasma membrane from ...
A combination of electrospray ionization mass spectrometry (ESIMS), fast atom bombardment mass spectrometry (FABMS), and sodium dodecylsulfate-polyacrylamide get electrophoresis (SDS-PAGE) was used to determine the homegeneity of chromatographic fractions of [beta]-crystallins, separated by reversed-phase HPLC. The molecular weight of the major component of [beta]-crystallins, [beta]B2, was found ...
This work explores the use of 20-microm-i.d. polymeric polystyrene-divinylbenzene monolithic nanocapillary columns for the LC-ESI-MS analysis of tryptic digest peptide mixtures. In contrast to the packing of microparticles, capillary columns were prepared, without the need of high pressure, in fused-silica capillaries, by thermally induced in situ ...
Structural properties of the retinal extracellular-matrix glycolipoprotein interphotoreceptor retinoid-binding protein (IRBP) from human, monkey and bovine retinas have been compared. SDS/polyacrylamide-gel-electrophoretic analysis of limited tryptic and Staphylococcus aureus-V8-proteinase digests show virtually identical patterns for the monkey and human ...
Proteomic-based description of varieties of barley (Hordeum vulgare L.) is a very important task especially in the food and brewing industry. This study is focused on major storage proteins in barley--hordeins--as a group of proteins soluble in alcohol/water mixtures that shows up significant changes in proteomic profiles for different varieties of barley. Unusual amino acid composition of ...
The application of Fourier transform ion cyclotron resonance (FTICR) mass spectrometry to the analysis of polypeptide mixtures resulting from proteolytic digestion is described. A new 11.5-T FTICR mass spectrometer has been applied for the analysis of tryptic digestion mixtures of the protein bovine serum albumin (BSA). The improved ...
Proteolysis is a central component of most proteomics methods. Unfortunately much of the information relating to the structural diversity of proteins is lost during digestion. This paper describes a method in which the native proteome of yeast was subjected to preliminary fractionation by size exclusion chromatography (SEC) prior to trypsin digestion of ...
The major glycoprotein of the human erythrocyte membrane has been isolated by treatment with lithium di-iodosalicylate and found to be a single polypeptide chain with a molecular weight of about 50,000. This molecule, which is 60% carbohydrate and 40% protein, carries multiple blood-group antigens, the receptors for influenza viruses, and various plant agglutinins. Four unique ...
A study is presented on chemical modification of the three subunit Paracoccus denitrificans bc(1) complex. N-(Ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline (EEDQ) treatment caused a loss of the proton pumping activity of liposome-reconstituted bc(1) complex. A similar effect, which is referred to as the decoupling effect, resulted upon reaction of N,N'-dicyclohexylcarbodiimide (DCCD) with the ...
BackgroundStable isotope probing (SIP) technique was developed to link function, structure and activity of microbial cultures metabolizing carbon and nitrogen containing substrates to synthesize their biomass. Currently, available methods are restricted solely to the estimation of fully saturated heavy stable isotope incorporation and convenient methods with sufficient accuracy are still missing. ...
A proteomic approach to the rapid identification of bacteria is presented, which relies on the solubilization of a limited number of proteins from intact cells combined with on-probe tryptic digestion. Within 20 min, complete cleavage products of a limited set of bacterial proteins with molecular masses of about 4-125 kDa were obtained by on-probe ...
Serotonin-bonded silica was developed for specific extraction of sialic-acid-containing glycans and glycopeptides. Serotonin, coupled by reductive amination with aldehyde silica particles via its ethylamino group, has strong affinity to sialic acid in glycan chain termini. Sialylated glycans trapped on serotonin silica particles are released by washing with ammonium acetate solution, providing ...
This review highlights the methodological and instrumental developments in microchip electrophoresis (MCE)-mass spectrometry (MS) from 1997. In MCE-MS, the development of ionization interface is one of the most important issues to realize highly sensitive detection and high separation efficiency. Among several interfaces, electrospray ionization (ESI) has been mainly employed to MCE-MS since a ...
In the serum of a Japanese patient with familial amyloidotic polyneuropathy (FAP), we demonstrated the presence of a prealbumin variant using a single amino acid substitution of a methionine residue for a valine at position 30. We have developed a highly sensitive and specific method for quantitative analysis of the prealbumin variant in the sera of FAP patients by using radioimmunoassay for a ...
The B27K-DTrI insulin (human insulin with B28-30 removed and B27 Thr replaced by Lys) was reported to have superior monomeric property with 80% insulin activity in vivo. It has potential use as a new fast-acting analog of insulin. We cloned the monomeric insulin B27 DTrI precursor (MIP) into the pTWIN1 vector, and prepared by intein mediated expression in Escherichia coli. After ...
Most of the cellular processes are regulated by reversible phosphorylation of proteins, which in turn plays a critical role in the regulation of gene expression, cell division, signal transduction, metabolism, differentiation, and apoptosis. Mass spectrometry of phosphopeptides obtained from tryptic protein digests has become a powerful tool for ...
The TripleTOF 5600 System, a hybrid quadrupole time-of-flight mass spectrometer, was evaluated to explore the key figures of merit in generating peptide and protein identifications that included spectral acquisition rates, data quality, proteome coverage, and biological depth. Employing a Saccharomyces cerevisiae tryptic digest, careful consideration of ...
This paper reports the identification and quantification of superoxide dismutase in the liver of Mus musculus and Mus spretus mice using a metallomics analytical approach. The approach consisted of using orthogonal chromatographic systems coupled to ICP-MS and UV detectors. Size-exclusion fractionation of the cytosolic extracts was followed by anion-exchange chromatographic separation of Cu- and ...
A 39-residue peptide from the tryptic digestion of bovine blood clotting factor X has been isolated by specific adsorption on barium citrate. The amino- and carboxyl-terminal sequences of the peptide were determined and compared to the vitamin K-dependent Ca2+-binding region from bovine prothrombin. The factor X peptide was found to contain ...
The authors report the construction of a plasmid-based expression vector that carries the murine metallothionein gene promoter, the human preprorenin gene, the Tn5 phosphotransferase gene, and a complete bovine papilloma virus genome. Murine cells transformed with this vector constitutively secrete high levels of human prorenin as determined by immunoprecipitation of culture media with anti-human ...
Dissociation of protonated peptides via infrared multiphoton dissociation (IRMPD) provides more extensive sequence information than is obtained with collisionally activated dissociation (CAD) in a quadrupole ion trap due to the lack of the CAD low m/z cutoff and the ability to form secondary and higher order fragments with the non-resonant photoactivation technique. In addition, IRMPD is shown to ...
In this paper, we discuss the challenge of large-scale quantification of a proteome, referring to our programme that aims to define the absolute quantity, in copies per cell, of at least 4000 proteins in the yeast Saccharomyces cerevisiae. We have based our strategy on the well-established method of stable isotope dilution, generating isotopically labelled peptides using QconCAT technology, in ...
We describe a 4-column, high-pressure capillary liquid chromatography (LC) system for robust, high-throughput LC-MS(/MS) analyses. This system performs multiple LC separations in parallel, but staggers each of them such that the data-rich region of each separation is sampled sequentially. By allowing nearly continuous data acquisition, this design maximizes the use of the mass spectrometer. Each ...
Infrared multiphoton dissociation (IRMPD) in a quadrupole ion trap coupled to high-performance liquid chromatography allows the selective dissociation of phosphorylated peptides in mixtures following chromatographic separation. This method is shown to be effective for differentiation of phosphorylated peptides from unphosphorylated ones; only the abundances of the phosphorylated species are ...
The carbon monoxide dehydrogenase from the photosynthetic bacterium Rhodospirillum rubrum was purified over 600-fold by DEAE-cellulose chromatography, heat treatment, hydroxylapatite chromatography, and preparative scale gel electrophoresis. In vitro, this enzyme catalyzed a two-election oxidation of CO to form CO/sub 2/ as the product. The reaction was dependent on the addition of an electron ...
An array of monolithic poly(styrene/divinylbenzene) capillaries with individual column thermostats was constructed to demonstrate its utility for the separation of nucleic acids, proteins, and tryptic digests in combination with UV absorbance detection. Because of polymerization-related variation in surface area of monolithic columns, the concentration of ...
