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Peptide Sequencing (peptide + sequencing)
Selected AbstractsNonlysine-analog plasminogen modulators promote autoproteolytic generation of plasmin(ogen) fragments with angiostatin-like activityFEBS JOURNAL, Issue 4 2004Shigeki Ohyama We recently discovered several nonlysine-analog conformational modulators for plasminogen. These include SMTP-6, thioplabin B and complestatin that are low molecular mass compounds of microbial origin. Unlike lysine-analog modulators, which increase plasminogen activation but inhibit its binding to fibrin, the nonlysine-analog modulators enhance both activation and fibrin binding of plasminogen. Here we show that some nonlysine-analog modulators promote autoproteolytic generation of plasmin(ogen) derivatives with its catalytic domain undergoing extensive fragmentation (PMDs), which have angiostatin-like anti-endothelial activity. The enhancement of urokinase-catalyzed plasminogen activation by SMTP-6 was followed by rapid inactivation of plasmin due to its degradation mainly in the catalytic domain, yielding PMD with a molecular mass ranging from 68 to 77 kDa. PMD generation was observed when plasmin alone was treated with SMTP-6 and was inhibited by the plasmin inhibitor aprotinin, indicating an autoproteolytic mechanism in PMD generation. Thioplabin B and complestatin, two other nonlysine-analog modulators, were also active in producing similar PMDs, whereas the lysine analog 6-aminohexanoic acid was inactive while it enhanced plasminogen activation. Peptide sequencing and mass spectrometric analyses suggested that plasmin fragmentation was due to cleavage at Lys615-Val616, Lys651-Leu652, Lys661-Val662, Lys698-Glu699, Lys708-Val709 and several other sites mostly in the catalytic domain. PMD was inhibitory to proliferation, migration and tube formation of endothelial cells at concentrations of 0.3,10 µg·mL,1. These results suggest a possible application of nonlysine-analog modulators in the treatment of cancer through the enhancement of endogenous plasmin(ogen) fragment formation. [source] Cloning and characterization of novel snake venom proteins that block smooth muscle contractionFEBS JOURNAL, Issue 11 2002Yasuo Yamazaki In this study, we isolated a 25-kDa novel snake venom protein, designated ablomin, from the venom of the Japanese Mamushi snake (Agkistrodon blomhoffi). The amino-acid sequence of this protein was determined by peptide sequencing and cDNA cloning. The deduced sequence showed high similarity to helothermine from the Mexican beaded lizard (Heloderma horridum horridum), which blocks voltage-gated calcium and potassium channels, and ryanodine receptors. Ablomin blocked contraction of rat tail arterial smooth muscle elicited by high K+ -induced depolarization in the 0.1,1 µm range, but did not block caffeine-stimulated contraction. Furthermore, we isolated three other proteins from snake venoms that are homologous to ablomin and cloned the corresponding cDNAs. Two of these homologous proteins, triflin and latisemin, also inhibited high K+ -induced contraction of the artery. These results indicate that several snake venoms contain novel proteins with neurotoxin-like activity. [source] Specific Ser-Pro phosphorylation by the RNA-recognition motif containing kinase KISFEBS JOURNAL, Issue 14 2000Alexandre Maucuer We present here a first appraisal of the phosphorylation site specificity of KIS (for ,kinase interacting with stathmin'), a novel mammalian kinase that has the unique feature among kinases to possess an RNP type RNA-recognition motif (RRM). In vitro kinase assays using various standard substrates revealed that KIS has a narrow specificity, with myelin basic protein (MBP) and synapsin I being the best in vitro substrates among those tested. Mass spectrometry and peptide sequencing allowed us to identify serine 164 of MBP as the unique site phosphorylated by KIS. Phosphorylation of synthetic peptides indicated the importance of the proline residue at position +1. We also identified a tryptic peptide of synapsin I phosphorylated by KIS and containing a phosphorylatable Ser-Pro motif. Altogether, our results suggest that KIS preferentially phosphorylates proline directed residues but has a specificity different from that of MAP kinases and cdks. [source] Comparative proteomic analysis of primary mouse liver c-Kit,(CD45/TER119), stem/progenitor cellsJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 4 2007Yu-Fei He Abstract Liver stem/progenitor cells play a key role in liver development and maybe also in liver cancer development. In our previous study a population of c-Kit,(CD45/TER119), liver stem/progenitor cells in mouse fetal liver, was successfully sorted with large amount (106,107) by using immuno-magnetic microbeads. In this study, the sorted liver stem/progenitor cells were used for proteomic study. Proteins of the sorted liver stem/progenitor cells and unsorted fetal liver cells were investigated using two-dimensional electrophoresis. A two-dimensional proteome map of liver stem/progenitor cells was obtained for the first time. Proteins that exhibited significantly upregulation in liver stem/progenitor cells were identified by peptide mass fingerprinting and peptide sequencing. Nineteen protein spots corresponding to 12 different proteins were identified as showing significant upregulation in liver stem/progenitor cells and seem to play important roles in such cells in cell metabolism, cell cycle regulation, and stress. An interesting finding is that most of the upregulated proteins were overexpressed in various cancers (11 of 12, including 6 in human hepatocellular carcinoma (HCC)) and involved in cancer development as reported in previous studies. Some of the identified proteins were validated by real-time PCR, Western blotting, and immunostaining. Taken together, the data presented provide a significant new protein-level insight into the biology of liver stem/progenitor cells, a key population of cells that might be also involved in liver cancer development. J. Cell. Biochem. 102: 936,946, 2007. © 2007 Wiley-Liss, Inc. [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] Complete sequences of small acid-soluble proteins from Bacillus globigiiJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 10 2004Jeffrey R. Whiteaker Abstract Three abundant small acid-soluble proteins (SASPs) from spores of Bacillus globigii were sequenced using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry with post-source decay and nanoelectrospray collision-induced dissociation tandem mass spectrometry. The proteins were extracted from spores with 1 M HCl. Scanning electron micrographs of spores before and after acid extraction show that the spores retain their overall structure but have a shriveled texture following the acid treatment. Extracted SASPs were purified by high-performance liquid chromatography and molecular masses of the SASPs were identified at 7068 (SASP-1), 7332 (SASP-2), and 8889 (,-SASP). De novo peptide sequencing was used to determine the protein sequences. The correct ordering of peptide sequences was aided by mapping overlapping enzymatic digests and by comparison with homologous SASPs from Bacillus stearothermophilus. B. globigii is used in many field tests as a surrogate for B. anthracis. Thus complete SASP sequences from B. globigii will facilitate the development of methods for rapid identification of bacteria based on mass spectrometry and the examination of taxonomic relationships between Bacillus species. Copyright © 2004 John Wiley & Sons, Ltd. [source] To b or not to b: The ongoing saga of peptide b ionsMASS SPECTROMETRY REVIEWS, Issue 4 2009Alex G. Harrison Abstract Modern soft ionization techniques readily produce protonated or multiply protonated peptides. Collision-induced dissociation (CID) of these protonated species is often used as a method to obtain sequence information. In many cases fragmentation occurs at amide bonds. When the charge resides on the C-terminal fragment so-called y ions are produced which are known to be protonated amino acids or truncated peptides. When the charge resides on the N-terminal fragment so-called b ions are produced. Often the sequence of y and b ions are essential for peptide sequencing. The b ions have many possible structures, a knowledge of which is useful in this sequencing. The structures of b ions are reviewed in the following with particular emphasis on the variation of structure with the number of amino acid residues in the b ion and the effect of peptide side chain on b ion structure. The recent discovery of full cyclization of larger b ions results in challenges in peptide sequencing. This aspect is discussed in detail. © 2009 Wiley Periodicals, Inc., Mass Spec Rev 28:640,654, 2009 [source] De novo sequencing of peptides by MS/MSPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 4 2010Joerg Seidler Abstract The current status of de novo sequencing of peptides by MS/MS is reviewed with focus on collision cell MS/MS spectra. The relation between peptide structure and observed fragment ion series is discussed and the exhaustive extraction of sequence information from CID spectra of protonated peptide ions is described. The partial redundancy of the extracted sequence information and a high mass accuracy are recognized as key parameters for dependable de novo sequencing by MS. In addition, the benefits of special techniques enhancing the generation of long uninterrupted fragment ion series for de novo peptide sequencing are highlighted. Among these are terminal 18O labeling, MSn of sodiated peptide ions, N-terminal derivatization, the use of special proteases, and time-delayed fragmentation. The emerging electron transfer dissociation technique and the recent progress of MALDI techniques for intact protein sequencing are covered. Finally, the integration of bioinformatic tools into peptide de novo sequencing is demonstrated. [source] Application of electron transfer dissociation (ETD) for the analysis of posttranslational modificationsPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 21 2008Julia Wiesner Abstract Despite major advantages in the field of proteomics, the analysis of PTMs still poses a major challenge; thus far, preventing insights into the role and regulation of protein networks. Additionally, top-down sequencing of proteins is another powerful approach to reveal comprehensive information for biological function. A commonly used fragmentation technique in MS-based peptide sequencing is CID. As CID often fails in PTM-analysis and performs best on doubly-charged, short and middle-sized peptides, confident peptide identification may be hampered. A newly developed fragmentation technique, namely electron transfer dissociation (ETD), supports both, PTM- and top-down analysis, and generally results in more confident identification of long, highly charged or modified peptides. The following review presents the theoretical background of ETD and its technical implementation in mass analyzers. Furthermore, current improvements of ETD and approaches for the PTM-analysis and top-down sequencing are introduced. Alternating both fragmentation techniques, ETD and CID, increases the amount of information derived from peptide fragmentation, thereby enhancing both, peptide sequence coverage and the confidence of peptide and protein identification. [source] Proteomics of human umbilical vein endothelial cells applied to etoposide-induced apoptosisPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 15 2005Arnaud Bruneel Dr. Abstract We have undertaken to continue the proteomic study of human umbilical vein endothelial cells (HUVECs) using the combination of 2-DE, automated trypsin digestion, and PMF analysis after MALDI-TOF MS and peptide sequencing using nano LC-ESI-MS/MS. The overall functional characterization of the 162 identified proteins from primary cultures of HUVECs confirms the metabolic capabilities of endothelium and illustrates various cellular functions more related to cell motility and angiogenesis, protein folding, anti-oxidant defenses, signal transduction, proteasome pathway and resistance to apoptosis. In comparison with controls cells, the differential proteomic analysis of HUVECs treated by the pro-apoptotic topoisomerase inhibitor etoposide further revealed the variation of eight proteins, namely, GRP78, GRP94, valosin-containing protein, proteinase inhibitor 9, cofilin, 37-kDa laminin receptor protein, bovine apolipoprotein, and tropomyosin. These data suggest that etoposide-induced apoptosis of human vascular endothelial cells results from the intricate involvement of multiple apoptosis processes including at least the mitochondrial and the ER stress pathways. The presented 2-D pattern and protein database, as well as the data related to apoptosis of HUVECs, are available at http://www.huvec.com. [source] Carbon starvation survival of Listeria monocytogenes in planktonic state and in biofilm: A proteomic studyPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 10 2003Emmanuelle Helloin Abstract The proteomes of Listeria monocytogenes expressed in suspension and biofilm state, in the presence and absence of a caron source, were analysed by two-dimensional electrophoresis with the help of computer software. The up-regulated proteins in each case were identified by peptide sequencing using electrospray ionisation tandem mass spectrometry and a database search against the Listeria genome was performed. Relevant functions could be attributed to a number of the induced proteins which contibute to the understanding of the mechanisms of starvation survival of L. monocytogenes in planktonic state and in biofilm. [source] An integrated serum proteomic approach capable of monitoring the low molecular weight proteome with sequencing of intermediate to large peptidesRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 17 2009Karen Merrell The low-abundance, low molecular weight serum proteome has high potential for the discovery of new biomarkers using mass spectrometry (MS). Because the serum proteome is large and complex, defining relative quantitative differences for a molecular species between comparison groups requires an approach with robust separation capability, high sensitivity, as well as high mass resolution. Capillary liquid chromatography (cLC)/MS provides both the necessary separation technique and the sensitivity to observe many low-abundance peptides. Subsequent identification of potential serum peptide biomarkers observed in the cLC/MS step can in principle be accomplished by in series cLC/MS/MS without further sample preparation or additional instrumentation. In this report a novel cLC/MS/MS method for peptide sequencing is described that surpasses previously reported size limits for amino acid sequencing accomplished by collisional fragmentation using a tandem time-of-flight MS instrument. As a demonstration of the approach, two low-abundance peptides with masses of ,4000,5000,Da were selected for MS/MS sequencing. The multi-channel analyzer (MCA) was used in a novel way that allowed for summation of 120 fragmentation spectra for each of several customized collision energies, providing more thorough fragmentation coverage of each peptide with improved signal to noise. The peak list from this composite analysis was submitted to Mascot for identification. The two index peptides, 4279,Da and 5061,Da, were successfully identified. The peptides were a 39 amino acid immunoglobulin G heavy chain variable region fragment and a 47 amino acid fibrin alpha isoform C-terminal fragment. The method described here provides the ability both to survey thousands of serum molecules and to couple that with markedly enhanced cLC/MS/MS peptide sequencing capabilities, providing a promising technique for serum biomarker discovery. Copyright © 2009 John Wiley & Sons, Ltd. [source] Sequence- and site-specific photodissociation at 266,nm of protonated synthetic polypeptides containing a tryptophanyl residueRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 22 2004Joo Yeon Oh Photodissociation at 266,nm of protonated synthetic polypeptides containing a tryptophanyl residue was investigated using a homebuilt tandem time-of-flight mass spectrometer equipped with a matrix-assisted laser desorption/ionization source. Efficient photodissociation of the protonated peptides was demonstrated. Most of the intense peaks in the laser-induced tandem mass spectra were sequence ions. Furthermore, sequence ions due to cleavages at all the peptide bonds were observed; this is a feature of the technique that is particularly useful for peptide sequencing. Fragmentations at both ends of the tryptophanyl residue were especially prevalent, which can be useful for location of the tryptophanyl chromophore in a peptide. Copyright © 2004 John Wiley & Sons, Ltd. [source] Identification of substituted sites on MUC5AC mucin motif peptides after enzymatic O-glycosylation combining ,-elimination and fixed-charge derivatizationRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 1 2002X. Czeszak A strategy for determination of O-glycosylation site(s) in glycopeptides has been developed using model compounds obtained by enzymatic glycosylation (by human GaNTase-T2 isoform) on peptides derived from the human MUC5AC mucin tandem repeat motif. The ,-elimination-addition reaction (using dimethylamine and concomitantly ethanethiol) on the formerly glycosylated sites through a Michael-type condensation produced efficient deglycosylation with appropriate chemical modification. After N-terminal derivatization by a phosphonium group, peptide sequencing was then carried out by nanospray tandem mass spectrometry experiments. The highly predictable fragmentation pathways of these fixed-charge phosphonium derivatives enable straightforward recognition of glycosylation site(s) based on the mass increment of +44,Da for originally glycosylated threonine compared to the mass of fragments containing nonglycosylated residues. Copyright ©,2001 John Wiley & Sons, Ltd. [source] Atmospheric pressure matrix-assisted laser desorption/ionization ion trap mass spectrometry of sulfonic acid derivatized tryptic peptidesRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 23 2001T. Keough Atmospheric pressure matrix-assisted laser desorption/ionization (AP-MALDI) and ion trap mass spectrometry have been used to study the fragmentation behavior of native peptides and peptide derivatives prepared for de novo sequencing applications. Sulfonic acid derivatized peptides were observed to fragment more extensively and up to 28 times more efficiently than the corresponding native peptides. Tandem mass spectra of native peptides containing aspartic or glutamic acids are dominated by cleavage on the C-terminal side of the acidic residues. This significantly limits the amount of sequence information that can be derived from those compounds. The MS/MS spectra of native tryptic peptides containing oxidized Met residues show extensive loss of CH3SOH and little sequence-specific fragmentation. On the other hand, the tandem mass spectra of derivatized peptides containing Asp, Glu and oxidized Met show much more uniform fragmentation along the peptide backbone. The AP-MALDI tandem mass spectra of some derivatized peptides were shown to be qualitatively very similar to the corresponding vacuum MALDI postsource decay mass spectra, which were obtained on a reflector time-of-flight instrument. However, the ion trap mass spectrometer offers several advantages for peptide sequencing relative to current reflector time-of-flight instruments including improved product ion mass measurement accuracy, improved precursor ion selection and MSn. These latter capabilities were demonstrated with solution digests of model proteins and with in-gel digests of 2D-gel separated proteins. Copyright © 2001 John Wiley & Sons, Ltd. [source] Location of crosslinks in chemically stabilized horseradish peroxidase: Implications for design of crosslinksBIOTECHNOLOGY & BIOENGINEERING, Issue 4 2001Anne Marie O'Brien Abstract The bifunctional compound, ethylene-glycol bis(N -hydroxysuccinimidylsuccinate) (EGNHS), stabilizes horseradish peroxidase C (HRP) by reaction with the enzyme's lysine residues. In this study we compare native and modified HRP by proteolytic fragmentation, peptide sequencing, and mass spectroscopy, and identify the sites of modification. Most significantly, EGNHS is shown to form a crosslink between Lys232 and Lys241 of HRP and modifies Lys174 without formation of a crosslink. These findings are in agreement with the lysine side-chain reactivities predicted from the surface accessibility of the amino groups, and the maximal span of 16 Å of the EGNHS crosslinker. © 2001 John Wiley & Sons, Inc. Biotechnol Bioeng 76: 277,284, 2001. [source] | |||||||||||||