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Tryptic Digestion (tryptic + digestion)
Selected AbstractsRe-evaluation of intramolecular long-range electron transfer between tyrosine and tryptophan in lysozymesFEBS JOURNAL, Issue 17 2003Evidence for the participation of other residues One-electron oxidation of six different c-type lysozymes from hen egg white, turkey egg white, human milk, horse milk, camel stomach and tortoise was studied by gamma- and pulse-radiolysis. In the first step, one tryptophan side chain is oxidized to indolyl free radical, which is produced quantitatively. As shown already, the indolyl radical subsequently oxidizes a tyrosine side chain to the phenoxy radical in an intramolecular reaction. However this reaction is not total and its stoichiometry depends on the protein. Rate constants also vary between proteins, from 120·s,1 to 1000·s,1 at pH 7.0 and room temperature [extremes are hen and turkey egg white (120·s,1) and human milk (1000·s,1)]. In hen and turkey egg white lysozymes we show that another reactive site is the Asn103,Gly104 peptidic bond, which gets broken radiolytically. Tryptic digestion followed by HPLC separation and identification of the peptides was performed for nonirradiated and irradiated hen lysozyme. Fluorescence spectra of the peptides indicate that Trp108 and/or 111 remain oxidized and that Tyr20 and 53 give bityrosine. Tyr23 appears not to be involved in the process. Thus new features of long-range intramolecular electron transfer in proteins appear: it is only partial and other groups are involved which are silent in pulse radiolysis. [source] Preparation and characterization of PEGylated terlipressinJOURNAL OF APPLIED POLYMER SCIENCE, Issue 6 2010Xiufang Wang Abstract Terlipressin was chemically modified by reaction with succinimidyl propionate- monomethoxy polyethylene glycol (mPEG-SPA). To determine the PEGylated degree, the position and the optimized condition for PEGylated terlipressin, the reactions were monitored in different pH value buffers at different molar ratios by reversed-phase high performance liquid chromatography (RP-HPLC). Tryptic digestion and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was used. The results showed that the amount of mono-PEG-terlipressin was higher at lower pH value and lower content of PEG. Meanwhile, the amount of di-PEG-terlipressin was higher at higher pH value and higher content of PEG under the conditions investigated. The position of PEGylated terlipressin was confirmed by tryptic digestion. © 2010 Wiley Periodicals, Inc. J Appl Polym Sci, 2010 [source] New approach for rapid detection of known hemoglobin variants using LC-MS/MS combined with a peptide database,JOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 3 2007F. Basilico Abstract The identification of hemoglobin (Hb) variants is usually performed by means of different analytical steps and methodologies. Phenotypic methods, such as gel electrophoresis and high performance liquid chromatography, are used to detect the different electrophoretic or chromatographic behaviors of hemoglobin variants in comparison to HbA0 used as a control. These data often need to be combined with mass spectrometry analyses of intact globins and their tryptic peptide mixtures. As an alternative to a ,step-by-step' procedure, we have developed a ,single step' approach for the identification of Hb variants present in biological samples. This is based on the µHPLC-ESI-MS/MS analysis of the peptide mixture generated by a tryptic digestion of diluted Hb samples and an in-house new database containing solely the variant tryptic peptide of known human Hb variants. The experimental results (full MS and MS/MS spectra) are correlated with theoretical mass spectra generated from our in-house-built variant peptide database (Hbp) using the SEQUEST algorithm. Simple preparation of samples and an automated identification of the variant peptide are the main characteristics of this approach, making it an attractive method for the detection of Hb variants at the routine clinical level. We have analyzed 16 different samples, each containing a different known variant of hemoglobin. Copyright © 2006 John Wiley & Sons, Ltd. [source] Identification of proteins directly from tissue: in situ tryptic digestions coupled with imaging mass spectrometryJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 2 2007M. Reid Groseclose Abstract A novel method for on-tissue identification of proteins in spatially discrete regions is described using tryptic digestion followed by matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) with MS/MS analysis. IMS is first used to reveal the protein and peptide spatial distribution in a tissue section and then a serial section is robotically spotted with small volumes of trypsin solution to carry out in situ protease digestion. After hydrolysis, 2,5-Dihydroxybenzoic acid (DHB) matrix solution is applied to the digested spots, with subsequent analysis by IMS to reveal the spatial distribution of the various tryptic fragments. Sequence determination of the tryptic fragments is performed using on-tissue MALDI MS/MS analysis directly from the individual digest spots. This protocol enables protein identification directly from tissue while preserving the spatial integrity of the tissue sample. The procedure is demonstrated with the identification of several proteins in the coronal sections of a rat brain. Copyright © 2007 John Wiley & Sons, Ltd. [source] Site-specific detection of S -nitrosylated PKB ,/Akt1 from rat soleus muscle using CapLC-Q-TOFmicro mass spectrometryJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 9 2005Xiao-Ming Lu Abstract Protein Kinase B,(PKB,, or Akt1) is believed to play a crucial role in programmed cell death, cancer progression and the insulin-signaling cascade. The protein is activated by phosphorylation at multiple sites and subsequently phosphorylates and activates eNOS. Free cysteine residues of the protein may capture reactive, endogenously produced nitric oxide (NO) as S -nitrosothiols. Site-specific detection of S -nitrosylated cysteine residues, usually at low stoichiometry, has been a major challenge in proteomic research largely due to the lack of mass marker for S -nitrosothiols that are very labile under physiologic conditions. In this report we describe a sensitive and specific MS method for detection of S -nitrosothiols in PKB ,/Akt1 in rat soleus muscle. PKB ,/Akt1 was isolated by immunoprecipitation and 2D-gel electrophoresis, subjected to in-gel tryptic digestion, and cysteinyl nitrosothiols were reacted with iodoacetic acids [2-C12/C13 = 50/50] under ascorbate reduction conditions. This resulted in the production of relatively stable carboxymethylcysteine (CMC) immonium ions (m/z 134.019 and m/z 135.019) within a narrow argon collision energy (CE = 30 ± 5 V) in the high MS noise region. In addition, free and disulfide-linked cysteine residues were converted to carboxyamidomethylcysteines (CAM). Tryptic S -nitrosylated parent ion was detected with a mass accuracy of 50 mDa for the two CMC immonium ions at the triggered elution time during capillary liquid chromatography (LC) separation. A peptide containing Cys296 was discriminated from four co-eluting tryptic peptides under lock mass conditions (m/z 785.8426). S -nitrosothiol in the tryptic peptide, ITDFGLBKEGIK (B: CAM, [M + 2H]2+ = 690.86, Found: 690.83), is believed to be present at a very low level, since the threshold for the CMC immonium trigger ions was set at 3 counts/s in the MS survey. The high levels of NO that are produced under stress conditions may result in increased S -nitrosylation of Cys296 which blocks disulfide bond formation between Cys296 and Cys310 and suppresses the biological effects of PKB ,/Akt1. With the procedures developed here, this process can be studied under physiological and pathological conditions. Copyright © 2005 John Wiley & Sons, Ltd. [source] Characterization of covalently inhibited extracellular lipase from Streptomyces rimosus by matrix-assisted laser desorption/ionization time-of-flight and matrix-assisted laser desorption/ionization quadrupole ion trap reflectron time-of-flight mass spectrometry: localization of the active site serine,JOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 12 2004Martin Zehl Abstract A chemical modification approach combined with matrix-assisted laser desorption/ionization (MALDI) mass spectrometry was used to identify the active site serine residue of an extracellular lipase from Streptomyces rimosus R6-554W. The lipase, purified from a high-level overexpressing strain, was covalently modified by incubation with 3,4-dichloroisocoumarin, a general mechanism-based serine protease inhibitor. MALDI time-of-flight (TOF) mass spectrometry was used to probe the nature of the intact inhibitor-modified lipase and to clarify the mechanism of lipase inhibition by 3,4-dichloroisocoumarin. The stoichiometry of the inhibition reaction revealed that specifically one molecule of inhibitor was bound to the lipase. The MALDI matrix 2,6-dihydroxyacetophenone facilitated the formation of highly abundant [M + 2H]2+ ions with good resolution compared to other matrices in a linear TOF instrument. This allowed the detection of two different inhibitor-modified lipase species. Exact localization of the modified amino acid residue was accomplished by tryptic digestion followed by low-energy collision-induced dissociation peptide sequencing of the detected 2-(carboxychloromethyl)benzoylated peptide by means of a MALDI quadrupole ion trap reflectron TOF instrument. The high sequence coverage obtained by this approach allowed the confirmation of the site specificity of the inhibition reaction and the unambiguous identification of the serine at position 10 as the nucleophilic amino acid residue in the active site of the enzyme. This result is in agreement with the previously obtained data from multiple sequence alignment of S. rimosus lipase with different esterases, which indicated that this enzyme exhibits a characteristic Gly-Asp-Ser-(Leu) motif located close to the N-terminus and is harboring the catalytically active serine residue. Therefore, this study experimentally proves the classification of the S. rimosus lipase as GDS(L) lipolytic enzyme. Copyright © 2004 John Wiley & Sons, Ltd. [source] Peptide profile of human acquired enamel pellicle using MALDI tandem MSJOURNAL OF SEPARATION SCIENCE, JSS, Issue 3 2008Rui Vitorino Abstract The present study proposes a strategy for human in vivo acquired enamel pellicle (AEP) peptidome characterisation based on sequential extraction with guanidine and TFA followed by MALDI-TOF/TOF identification. Three different nanoscale analytical approaches were used: samples were subjected to tryptic digestion followed by nano-HPLC and mass spectrometry (MS and MS/MS) analysis. Undigested samples were analysed by LC-MS (both linear and reflector modes) and LC-MS/MS analysis, and samples were subjected to nano-HPLC followed by on-plate digestion and mass spectrometry (MS and MS/MS) analysis. The majority of the identifications corresponded to peptide/protein fragments of salivary protein, belonging to the classes: acidic PRPs, basic PRPs, statherin, cystatins S and SN and histatin 1 (all also identified in intact form). Overall, more than 90 peptides/proteins were identified. Results clearly show that peptides with acidic groups are enriched in the TFA fraction while peptides with no acidic or phosphate groups are prevalent on the guanidine extract. Also, phosphorylated peptides were observed mainly on the TFA fraction. Fragments present in the AEP show a predominance of cleavage points located at Arg, Tyr and Lys residues. Obtained data suggest that proteolytic activity could influence AEP formation and composition. [source] Production and characterization of an allergen panel for component-resolved diagnosis of celery allergyMOLECULAR NUTRITION & FOOD RESEARCH (FORMERLY NAHRUNG/FOOD), Issue S2 2008Merima Bublin Abstract In celery a relevant food allergen source, three allergens have been identified so far: Api g 1 and Api g 4, and one glycosylated protein, Api g 5. For component-resolved food allergy diagnosis high amounts of well-defined allergens are needed. Depending on the individual celery allergen, protocols for heterologous production and purification from natural source, respectively, were established to obtain homogenous protein batches. Afterwards the purified recombinant allergens, Api g 1, Api g 4 and natural Api g 5 were characterized regarding their structural integrity and immunological activity. Therefore, several methods were applied. Proteins were identified by partial N-terminal sequencing, protein mass was verified by MS and sequence integrity by MALDI-TOF and N-terminal sequencing after tryptic digestion. Presence of isoforms in natural allergen preparations was identified by 2-DE. Secondary and tertiary structures were evaluated by circular dichroism (CD) spectroscopy and NMR analysis. Finally, IgE binding capacity was verified using selected sera from celery allergic patients in IgE immunoblots and IgE ELISA. These well-defined celery allergens will be used to prove the concept of component-resolved diagnosis and will contribute to improve food allergy diagnosis in the future. [source] A quantitative strategy to detect changes in accessibility of protein regions to chemical modification on heterodimerizationPROTEIN SCIENCE, Issue 7 2009Mathias Dreger Abstract We describe a method for studying quantitative changes in accessibility of surface lysine residues of the PB1 subunit of the influenza RNA polymerase as a result of association with the PA subunit to form a PB1-PA heterodimer. Our method combines two established methods: (i) the chemical modification of surface lysine residues of native proteins by N -hydroxysuccinimidobiotin (NHS-biotin) and (ii) the stable isotope labeling of amino acids in cell culture (SILAC) followed by tryptic digestion and mass spectrometry. By linking the chemical modification with the SILAC methodology for the first time, we obtain quantitative data on chemical modification allowing subtle changes in accessibility to be described. Five regions in the PB1 monomer showed altered reactivity to NHS-biotin when compared with the [PB1-PA] heterodimer. Mutational analysis of residues in two such regions,at K265 and K481 of PB1, which were about three- and twofold, respectively, less accessible to biotinylation in the PB1-PA heterodimer compared with the PB1 monomer, demonstrated that both K265 and K481 were crucial for polymerase function. This novel assay of quantitative profiling of biotinylation patterns (Q-POP assay) highlights likely conformational changes at important functional sites, as observed here for PB1, and may provide information on protein,protein interaction interfaces. The Q-POP assay should be a generally applicable approach and may detect novel functional sites suitable for targeting by drugs. [source] Proteomic analysis of Aspergillus nidulans cultured under hypoxic conditionsPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 1 2009Motoyuki Shimizu Abstract The fungus Aspergillus nidulans reduces nitrate to ammonium and simultaneously oxidizes ethanol to acetate to generate ATP under hypoxic conditions in a mechanism called ammonia fermentation (Takasaki, K. et al.. J. Biol. Chem. 2004, 279, 12414,12420). To elucidate the mechanism, the fungus was cultured under normoxic and hypoxic (ammonia fermenting) conditions, intracellular proteins were resolved by 2-DE, and 332 protein spots were identified using MALDI MS after tryptic digestion. Alcohol and aldehyde dehydrogenases that play key roles in oxidizing ethanol were produced at the basal level under hypoxic conditions but were obviously provoked by ethanol under normoxic conditions. Enzymes involved in gluconeogenesis, as well as the tricarboxylic and glyoxylate cycles, were downregulated. These results indicate that the mechanism of fungal energy conservation is altered under hypoxic conditions. The results also showed that proteins in the pentose phosphate pathway as well as the metabolism of both nucleotide and thiamine were upregulated under hypoxic conditions. Levels of xanthine and hypoxanthine, deamination products of guanine and adenine were increased in DNA from hypoxic cells, indicating an association between hypoxia and intracellular DNA base damage. This study is the first proteomic comparison of the hypoxic responses of A. nidulans. [source] Flow cytometry-assisted purification and proteomic analysis of the corticotropes dense-core secretory granulesPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 18 2008Daniel J. Gauthier Abstract The field of organellar proteomics has emerged as an attempt to minimize the complexity of the proteomics data obtained from whole cell and tissue extracts while maximizing the resolution on the protein composition of a single subcellular compartment. Standard methods involve lengthy density-based gradient and/or immunoaffinity purification steps followed by extraction, 1-DE or 2-DE, gel staining, in-gel tryptic digestion, and protein identification by MS. In this paper, we present an alternate approach to purify subcellular organelles containing a fluorescent reporter molecule. The gel-free procedure involves fluorescence-assisted sorting of the secretory granules followed by gentle extraction in a buffer compatible with tryptic digestion and MS. Once the subcellular organelle labeled, this procedure can be done in a single day, requires no major modification to any instrumentation and can be readily adapted to the study of other organelles. When applied to corticotrope secretory granules, it led to a much enriched granular fraction from which numerous proteins could be identified through MS. [source] Lectin precipitation using phytohemagglutinin-L4 coupled to avidin,agarose for serological biomarker discovery in colorectal cancerPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 16 2008Yong-Sam Kim Abstract N -acetylglucosaminyltransferase V (GnT-V) has been reported to be upregulated in malignant cancer cells, and its targets have been sought after with regard to biomarker identification. The low capacity and high false positive rates of 2-DE gel-based lectin blots using phytohemagglutinin-L4 (L-PHA) prompted us to develop a novel protocol for identifying GnT-V targets, in which serum proteins were subjected to immunodepletion, alkylation, and lectin precipitation using L-PHA coupled to avidin,agarose bead complexes, and tryptic digestion. Proteins captured by L-PHA conjugates were analyzed by a nano-LC-FT-ICR/LTQ MS. Here, we report 26 candidate biomarkers for colorectal cancer (CRC) that show 100% specificity and sensitivities of greater than 50%. Not only can these candidate proteins be used as analytes for validation, but the novel protocol described herein can be applied to biomarker discovery in nonCRCs. [source] The secretome of Pleurotus sapidusPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 18 2005Holger Zorn Dr. Abstract Due to their unique capability to attack lignified biopolymers, extracellular enzymes of white-rot fungi enjoy an increasing interest in various fields of white biotechnology. The edible fungus Pleurotus sapidus was selected as a model organism for the analysis of the secretome by means of 2-DE. For enzyme production, the fungus was grown in submerged cultures either on peanut shells or on glass wool as a carrier material. Identification of the secreted enzymes was performed by tryptic digestion, ESI-MS/MS ab initio sequencing, and homology searches against public databases. The spectrum of secreted enzymes comprised various types of hydrolases and lignolytic enzymes of the manganese peroxidase/versatile peroxidase family. While peptidases were secreted mainly by the cultures grown on peanut shells, versatile peroxidase type enzymes dominated in the cultures grown on glass wool. [source] Determination of the site-specific and isoform-specific glycosylation in human plasma-derived antithrombin by IEF and capillary HPLC-ESI-MS/MSPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 15 2005Alexander Plematl Abstract The glycan structures of the major and more than ten minor populated isoforms of antithrombin (AT) were determined after separation of the isoforms by IEF using IPG strips. The bands excised from the gel were reduced, derivatized by iodoacetamide and submitted to tryptic digestion. The digest was analyzed by RP-HPLC-ESI-MS equipped with a quadrupole ion-trap mass analyzer. MS/MS experiments allowed establishing the monosaccharide compositions in the glycopeptides. For the major isoform of ,-AT four identical biantennary glycans with two terminal sialic acids (SA) each, a total of eight SA, were found in full agreement with the literature. In the IEF-band containing this major isoform (pI 5.18) a further, much less abundant, isoform was detected showing a fucosylation on the glycan attached to Asn155 but being of otherwise identical structure as described above. The isoforms with pI 5.10 were found to include one triantennary glycan, all antennas carrying terminal SA. The occurrence of triantennary structure is site specific, involving the peptides with Asn135 and Asn155, alternately. At pI 5.24 we found those four isoforms that carry the glycans like the main-isoform of ,-AT but missing one terminal SA. There was no site specificity found for the mono-sialo structure. The isoform at pI 5.31 is the major isoform of ,-AT containing three identical biantennary structures being fully sialylated. No isoforms (above 0.5% abundance) with two glycans only or three glycans other than ,-AT were detected. Fucosylation was found in the main isoform with an abundance of about 5%, and as expected with all the other isoforms with a comparable abundance. [source] Comparison of alternative analytical techniques for the characterisation of the human serum proteome in HUPO Plasma Proteome ProjectPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 13 2005Xiaohai Li Abstract Based on the same HUPO reference specimen (C1-serum) with the six proteins of highest abundance depleted by immunoaffinity chromatography, we have compared five proteomics approaches, which were (1) intact protein fractionation by anion-exchange chromatography followed by 2-DE-MALDI-TOF-MS/MS for protein identification (2-DE strategy); (2) intact protein fractionation by 2-D HPLC followed by tryptic digestion of each fraction and microcapillary RP-HPLC/microESI-MS/MS identification (protein 2-D HPLC fractionation strategy); (3) protein digestion followed by automated online microcapillary 2-D HPLC (strong cation-exchange chromatography (SCX)-RPC) with IT microESI-MS/MS; (online shotgun strategy); (4) same as (3) with the SCX step performed offline (offline shotgun strategy) and (5) same as (4) with the SCX fractions reanalysed by optimised nanoRP-HPLC-nanoESI-MS/MS (offline shotgun-nanospray strategy). All five approaches yielded complementary sets of protein identifications. The total number of unique proteins identified by each of these five approaches was (1) 78, (2) 179, (3) 131, (4) 224 and (5) 330 respectively. In all, 560 unique proteins were identified. One hundred and sixty-five proteins were identified through two or more peptides, which could be considered a high-confidence identification. Only 37 proteins were identified by all five approaches. The 2-DE approach yielded more information on the pI -altered isoforms of some serum proteins and the relative abundance of identified proteins. The protein prefractionation strategy slightly improved the capacity to detect proteins of lower abundance. Optimising the separation at the peptide level and improving the detection sensitivity of ESI-MS/MS were more effective than fractionation of intact proteins in increasing the total number of proteins identified. Overall, electrophoresis and chromatography, coupled respectively with MALDI-TOF/TOF-MS and ESI-MS/MS, identified complementary sets of serum proteins. [source] The human plasma proteome: Analysis of Chinese serum using shotgun strategyPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 13 2005Ping He Abstract We have investigated the serum proteome of Han-nationality Chinese by using shotgun strategy. A complete proteomics analysis was performed on two reference specimens from a total of 20,healthy donors, in which each sample was made from ten-pooled male or female serum, respectively. The methodology used encompassed (1),removal of six high-abundant proteins; (2),tryptic digestion of low- and high-abundant proteins of serum; (3),separation of peptide mixture by RP-HPLC followed by ESI-MS/MS identification. A total of 944,nonredundant proteins were identified under a stringent filter condition (Xcorr,,,1.9, ,2.2, and ,3.75, ,Cn,,,0.1, and Rsp,,,4.0) in both pooled male and female samples, in which 594 and 622,entire proteins were found, respectively. Compared with the total 3020 protein identifications confirmed by more than one laboratory or more than one specimen in HUPO Plasma Proteome Project (PPP) participating laboratories recently, 206,proteins were identified with at least two distinct peptides per protein and 185,proteins were considered as high-confidence identification. Moreover, some lower abundance serum proteins (ng/mL range) were detected, such as complement,C5 and CA125, routinely used as an ovarian cancer marker in plasma and serum. The resulting nonredundant list of serum proteins would add significant information to the knowledge base of human plasma proteome and facilitate disease markers discovery. [source] Proteome analysis of hepatocellular carcinoma cell strains, MHCC97-H and MHCC97-L, with different metastasis potentialsPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 4 2004Shi-Jian Ding Abstract To better understand the mechanism underlying hepatocellular carcinoma (HCC) metastasis and to search for potential markers for HCC prognosis, differential proteome analysis on two HCC cell strains with high and low metastatic potentials, MHCC97-H and MHCC97-L, was conducted using two-dimensional (2-D) gel electrophoresis followed by matrix-assisted laser desorption/time of flight mass spectrometry and liquid chromatography ion trap mass spectrometry. Image analysis of silver-stained 2-D gels revealed that 56 protein spots showed significant differential expression in MHCC97-H and MHCC97-L cells (Student's t -test, P < 0.05) and 4 protein spots were only detected in MHCC97-H cells. Fourteen protein spots were further identified using in-gel tryptic digestion, peptide mass fingerprinting and tandem mass spectrometry. The expressions of pyruvate kinase M2, ubiquitin carboxy-terminal hydrolase L1, laminin receptor 67 kDa, S100 calcium-binding protein A4, thioredoxin and cytokeratin 19 were elevated in MHCC97-H cells. However, manganese superoxide dismutase, calreticulin precursor, cathepsin D, lactate dehydrogenase B, non-metastatic cell protein 1, cofilin 1 and calumenin precursor were down-regulated in MHCC97-H cells. Intriguingly, most of these identified proteins have been reported to be associated with tumor metastasis. The functional implications of alterations in the levels of these proteins are discussed. [source] Desulfurization of cysteine-containing peptides resulting from sample preparation for protein characterization by mass spectrometryRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 3 2010Zhouxi Wang In this study, we have examined two cysteine modifications resulting from sample preparation for protein characterization by mass spectrometry (MS): (1) a previously observed conversion of cysteine into dehydroalanine, now found in the case of disulfide mapping and (2) a novel modification corresponding to conversion of cysteine into alanine. Using model peptides, the conversion of cysteine into dehydroalanine via , -elimination of a disulfide bond was seen to result from the conditions of typical tryptic digestion (37°C, pH 7.0,9.0) without disulfide reduction and alkylation. Furthermore, the surprising conversion of cysteine into alanine was shown to occur by heating cysteine-containing peptides in the presence of a phosphine (tris(2-carboxyethyl)phosphine hydrochloride (TCEP)). The formation of alanine from cysteine, investigated by performing experiments in H2O or D2O, suggested a radical-based desulfurization mechanism unrelated to , -elimination. Importantly, an understanding of the mechanism and conditions favorable for cysteine desulfurization provides insight for the establishment of improved sample preparation procedures of protein analysis. Copyright © 2010 John Wiley & Sons, Ltd. [source] Quantum dots , electrospray ionization mass spectrometry: 3-mercaptopropanic acid capped CdS quantum dots as accelerating and enrichment probes for microwave tryptic digestion of proteinsRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 22 2009Kamlesh Shrivas First page of article [source] Accelerated tryptic digestion of proteins in plasma for absolute quantitation using a protein internal standard by liquid chromatography/tandem mass spectrometryRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 5 2009Fumin Li First page of article [source] A Robust Protein Host for Anchoring Chelating Ligands and OrganocatalystsCHEMBIOCHEM, Issue 4 2008Manfred T. Reetz Prof. Dr. Abstract In order to put the previously proposed concept of directed evolution of hybrid catalysts (proteins that harbor synthetic transition-metal catalysts or organocatalysts) into practice, several prerequisites must be met. The availability of a robust host protein that can be expressed in sufficiently large amounts, and that can be purified in a simple manner is crucial. The thermostable enzyme tHisF from Thermotoga maritima, which constitutes the synthase subunit of a bi-enzyme complex that is instrumental in the biosynthesis of histidine, fulfills these requirements. In the present study, fermentation has been miniaturized and parallelized, as has purification of the protein by simple heat treatment. Several mutants with strategically placed cysteines for subsequent bioconjugation have been produced. One of the tHisF mutants, Cys9Ala/Asp11Cys, was subjected to bioconjugation by the introduction of a variety of ligands for potential metal ligation, of a ligand/metal moiety, and of several organocatalytic entities that comprise a flavin or thiazolium salts. Characterization by mass spectrometry and tryptic digestion was achieved. As a result of this study, a platform for performing future directed evolution of these hybrid catalysts is now available. [source] Identification of proteins directly from tissue: in situ tryptic digestions coupled with imaging mass spectrometryJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 2 2007M. Reid Groseclose Abstract A novel method for on-tissue identification of proteins in spatially discrete regions is described using tryptic digestion followed by matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) with MS/MS analysis. IMS is first used to reveal the protein and peptide spatial distribution in a tissue section and then a serial section is robotically spotted with small volumes of trypsin solution to carry out in situ protease digestion. After hydrolysis, 2,5-Dihydroxybenzoic acid (DHB) matrix solution is applied to the digested spots, with subsequent analysis by IMS to reveal the spatial distribution of the various tryptic fragments. Sequence determination of the tryptic fragments is performed using on-tissue MALDI MS/MS analysis directly from the individual digest spots. This protocol enables protein identification directly from tissue while preserving the spatial integrity of the tissue sample. The procedure is demonstrated with the identification of several proteins in the coronal sections of a rat brain. Copyright © 2007 John Wiley & Sons, Ltd. [source] | ||||||||||||||||||||||||||||