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Modified Peptides (modified + peptide)
Selected AbstractsMass spectrometric characterization of the covalent modification of the nitrogenase Fe-protein in Azoarcus sp.FEBS JOURNAL, Issue 13 2009Nitrogenase Fe-protein modification was analyzed in the endophytic ,-proteobacterium Azoarcus sp. BH72. Application of modern MS techniques localized the modification in the peptide sequence and revealed it to be an ADP-ribosylation on Arg102 of one subunit of nitrogenase Fe-protein. A double digest with trypsin and endoproteinase Asp-N was necessary to obtain an analyzable peptide because the modification blocked the trypsin cleavage site at this residue. Furthermore, a peptide extraction protocol without trifluoroacetic acid was crucial to acquire the modified peptide, indicating an acid lability of the ADP-ribosylation. This finding was supported by the presence of a truncated version of the original peptide with Arg102 exchanged by ornithine. Site-directed mutagenesis verified that the ADP-ribosylation occurred on Arg102. With our approach, we were able to localize a labile modification within a large peptide of 31 amino acid residues. The present study provides a method suitable for the identification of so far unknown protein modifications on nitrogenases or other proteins. It represents a new tool for the MS analysis of protein mono-ADP-ribosylations. [source] Structural studies and model membrane interactions of two peptides derived from bovine lactoferricinJOURNAL OF PEPTIDE SCIENCE, Issue 7 2005Leonard T. Nguyen Abstract The powerful antimicrobial properties of bovine lactoferricin (LfcinB) make it attractive for the development of new antimicrobial agents. An 11-residue linear peptide portion of LfcinB has been reported to have similar antimicrobial activity to lactoferricin itself, but with lower hemolytic activity. The membrane-binding and membrane-perturbing properties of this peptide were studied together with an amidated synthetic version with an added disulfide bond, which was designed to confer increased stability and possibly activity. The antimicrobial and cytotoxic properties of the peptides were measured against Staphylococcus aureus and Escherichia coli and by hemolysis assays. The peptides were also tested in an anti-cancer assay against neuroblastoma cell lines. Vesicle disruption caused by these LfcinB derivatives was studied using the fluorescent reporter molecule calcein. The extent of burial of the two Trp residues in membrane mimetic environments were quantitated by fluorescence. Finally, the solution NMR structures of the peptides bound to SDS micelles were determined to provide insight into their membrane bound state. The cyclic peptide was found to have greater antimicrobial potency than its linear counterpart. Consistent with this property, the two Trp residues of the modified peptide were suggested to be embedded deeper into the membrane. Although both peptides adopt an amphipathic structure without any regular ,-helical or ß-sheet conformation, the 3D-structures revealed a clearer partitioning of the cationic and hydrophobic faces for the cyclic peptide. Copyright © 2004 European Peptide Society and John Wiley & Sons, Ltd. [source] A general precursor ion-like scanning mode on quadrupole-TOF instruments compatible with chromatographic separationPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 1 2006Ricarda Niggeweg Abstract MS protein identification and quantitation are key proteomic techniques in biological research. Besides identification of proteins, MS is used increasingly to characterize secondary protein modifications. This often requires trimming the analytical strategy to a specific type of modification. Direct analysis of protein modifications in proteomic samples is often hampered by the limited dynamic range of current analytical tools. Here we present a fast, sensitive, multiplexed precursor ion scanning mode , implemented on a quadrupole-TOF instrument , that allows the specific detection of any modified peptide or molecule that reveals itself by a specific fragment ion or pattern of fragment ions within a complex proteomic sample. The high mass accuracy of the TOF mass spectrometer is available for the marker ion specificity and the precursor ion mass determination. The method is compatible with chromatographic separation. Fragment ions and intact molecular ions are acquired quasi-simultaneously by continuously switching the collision energy between elevated and low levels. Using this technique many secondary modifications can be analyzed in parallel; however, the number of peptides carrying a specific modification that can be analyzed successfully is limited by the chromatographic resolution or, more generally, by the depth of the resolved time domain. [source] Characterization of N -palmitoylated human growth hormone by in situ liquid,liquid extraction and MALDI tandem mass spectrometryJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 6 2007Emmanuelle Sachon Abstract Acylation is a common post-translational modification found in secreted proteins and membrane-associated proteins, including signal transducing and regulatory proteins. Acylation is also explored in the pharmaceutical and biotechnology industry to increase the stability and lifetime of protein-based products. The presence of acyl moieties in proteins and peptides affects the physico-chemical properties of these species, thereby modulating protein stability, function, localization and molecular interactions. Characterization of protein acylation is a challenging analytical task, which includes the precise definition of the acylation sites in proteins and determination of the identity and molecular heterogeneity of the acyl moiety at each individual site. In this study, we generated a chemically modified human growth hormone (hGH) by incorporation of a palmitoyl moiety on the N, group of a lysine residue. Monoacylation of the hGH protein was confirmed by determination of the intact molecular weight by mass spectrometry. Detailed analysis of protein acylation was achieved by analysis of peptides derived from hGH by protease treatment. However, peptide mass mapping by MALDI MS using trypsin and AspN proteases and standard sample preparation methods did not reveal any palmitoylated peptides. In contrast, in situ liquid,liquid extraction (LLE) performed directly on the MALDI MS metal target enabled detection of acylated peptide candidates by MALDI MS and demonstrated that hGH was N -palmitoylated at multiple lysine residues. MALDI MS and MS/MS analysis of the modified peptides mapped the N -palmitoylation sites to Lys158, Lys172 and Lys140 or Lys145. This study demonstrates the utility of LLE/MALDI MS/MS for mapping and characterization of acylation sites in proteins and peptides and the importance of optimizing sample preparation methods for mass spectrometry-based determination of substoichiometric, multi-site protein modifications. Copyright © 2007 John Wiley & Sons, Ltd. [source] Investigation of tyrosine nitration in proteins by mass spectrometryJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 6 2001Ann-Sofi Petersson Abstract In vivo nitration of tyrosine residues is a post-translational modification mediated by peroxynitrite that may be involved in a number of diseases. The aim of this study was to evaluate possibilities for site-specific detection of tyrosine nitration by mass spectrometry. Angiotensin II and bovine serum albumin (BSA) nitrated with tetranitromethane (TNM) were used as model compounds. Three strategies were investigated: (i) analysis of single peptides and protein digests by matrix-assisted laser desorption/ionization (MALDI) peptide mass mapping, (ii) peptide mass mapping by electrospray ionization (ESI) mass spectrometry and (iii) screening for nitration by selective detection of the immonium ion of nitrotyrosine by precursor ion scanning with subsequent sequencing of the modified peptides. The MALDI time-of-flight mass spectrum of nitrated angiotensin II showed an unexpected prompt fragmentation involving the nitro group, in contrast to ESI-MS, where no fragmentation of nitrated angiotensin II was observed. The ESI mass spectra showed that mono- and dinitrated angiotensin II were obtained after treatment with TNM. ESI-MS/MS revealed that the mononitrated angiotensin II was nitrated on the side-chain of tyrosine. The dinitrated angiotensin II contained two nitro groups on the tyrosine residue. Nitration of BSA was confirmed by Western blotting with an antibody against nitrotyrosine and the sites for nitration were investigated by peptide mass mapping after in-gel digestion. Direct mass mapping by ESI revealed that two peptides were nitrated. Precursor ion scanning for the immonium ion for nitrotyrosine revealed two additional partially nitrated peptides. Based on the studies with the two model compounds, we suggest that the investigation of in vivo nitration of tyrosine and identification of nitrated peptides might be performed by precursor ion scanning for the specific immonium ion at m/z 181.06 combined with ESI-MS/MS for identification of the specific nitration sites. Copyright © 2001 John Wiley & Sons, Ltd. [source] Computational analysis of unassigned high-quality MS/MS spectra in proteomic data setsPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 14 2010Kang Ning Abstract In a typical shotgun proteomics experiment, a significant number of high-quality MS/MS spectra remain "unassigned." The main focus of this work is to improve our understanding of various sources of unassigned high-quality spectra. To achieve this, we designed an iterative computational approach for more efficient interrogation of MS/MS data. The method involves multiple stages of database searching with different search parameters, spectral library searching, blind searching for modified peptides, and genomic database searching. The method is applied to a large publicly available shotgun proteomic data set. [source] Glutathione- S -transferase pi as a model protein for the characterisation of chemically reactive metabolitesPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 2 2008Rosalind E. Jenkins Dr. Abstract Chemically reactive metabolites (CRMs) are thought to be responsible for a number of adverse drug reactions through modification of critical proteins. Methods that defined the chemistry of protein modification at an early stage would provide invaluable tools for drug safety assessment. Here, human GST pi (GSTP) was exploited as a model target protein to determine the chemical, biochemical and functional consequences of exposure to the hepatotoxic CRM of paracetamol (APAP), N -acetyl- p -benzoquinoneimine (NAPQI). Site-specific, dose-dependent modification of Cys47 in native and His-tagged GSTP was revealed by MS, and correlated with inhibition of glutathione (GSH) conjugating activity. In addition, the adaptation of iTRAQ labelling technology to define precisely the quantitative relationship between covalent modification and protein function is described. Multiple reaction monitoring (MRM)-MS of GSTP allowed high sensitivity detection of modified peptides at physiological levels of exposure. Finally, a bioengineered mutant cytochrome P450 with a broad spectrum of substrate specificities was used in an in vitro reaction system to bioactivate APAP: in this model, GSTP trapped the CRM and exhibited both reduced enzyme activity and site-specific modification of the protein. These studies provide the foundation for the development of novel test systems to predict the toxicological potential of CRMs produced by new therapeutic agents. [source] In vacuo isotope coded alkylation technique (IVICAT); an N-terminal stable isotopic label for quantitative liquid chromatography/mass spectrometry proteomics,RAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 16 2006Brigitte L. Simons We present a new isotopic labeling strategy to modify the N-terminal amino group of peptides in a quantifiable reaction without the use of expensive reagents or solvents. The In Vacuo Isotope Coded Alkylation Technique (IVICAT) is a methylation reaction, carried out at low pressure (<100,mTorr), that results in a stable quaternary trimethylammonium group, thus adding a permanent positive charge at the N-terminus of peptides without modifying the , -amino groups of lysine. The methylation reaction increases the signal intensity of modified peptides in matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and liquid chromatography (LC)/MS and the isotopic peak pair differs by 9 mass units which can be easily resolved by either instrument. N-terminally trimethylated peptides exhibit collision-induced dissociation (CID) mass spectra that differ from their unmodified analogues by an enhanced b -ion series in MS2 spectra due to the fixed positive charge. Using LC/MS/MS with an LTQ mass analyzer for quantification, the experimentally determined ratios of H9 - to D9 -trimethyl-labeled peptides of , -casein provided accurate estimates of the actual ratios with low % error. IVICAT labeling also accurately quantified proteins in rat kidney inner medullary collecting duct cell types, as judged by comparison with relative quantification by subsequent immunoblotting experiments. IVICAT labeling, when used in conjunction with the new proteomics software QUIL, can accurately report relative protein abundances and increase the sequence coverage of proteins of tissue proteomes. Published in 2006 by John Wiley & Sons, Ltd. [source] | ||||||||||