Amino acid sequences of peptides from a tryptic digest of a urea-soluble protein fraction (U.S.3) from oxidized wool.
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ABSTRACT:
SUBMITTER: Corfield MC
PROVIDER: S-EPMC1270331 | biostudies-other | 1967 Mar
REPOSITORIES: biostudies-other
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Project description:1. A chymotryptic digest of the protein fraction U.S.3. from oxidized wool was separated into 51 peptide fractions by chromatography on a column of cation-exchange resin. 2. The less acidic fractions were separated into their component peptides by a combination of cation-exchange-resin chromatography, paper chromatography and paper electrophoresis. 3. The amino acid sequences of 34 of these peptides were elucidated, and those of 14 others partially determined. 4. Overlaps between the tryptic and chymotryptic peptides from fraction U.S.3 have enabled ten extended amino acid sequences to be deduced, the longest containing 20 amino acid residues. 5. The relevance of the results to the structures of the helical and non-helical regions of wool is discussed.
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Project description:1. Cysteic acid peptides from various digests of calf rennin were purified by diagonal paper electrophoresis. 2. The amino acid sequence of these peptides accounts for 38 amino acids around three unique disulphide bridges in rennin. 3. One bridge connects two acidic regions of the chain, one forms a loop of five residues and the other a loop of six residues. 4. These bridges are homologous with those of hog pepsin. 5. Tryptic peptides from the C-terminus of rennin account for 22 residues, 17 of which are homologous with the C-terminus of pepsin. 6. Altogether, sequences accounting for 94 of the 270 residues in rennin are described and the degree of homology with pepsin approximates to 70%.
| S-EPMC1271253 | biostudies-other
Project description:Improvements in the mass accuracy and resolution of mass spectrometers have greatly aided mass spectrometry-based proteomics in profiling complex biological mixtures. With the use of innovative bioinformatics approaches, high mass accuracy and resolution information can be used for filtering chemical noise in mass spectral data. Using our recent algorithmic developments, we have generated the mass distributions of all theoretical tryptic peptides composed of 20 natural amino acids and with masses limited to 3.5 kDa. Peptide masses are distributed discretely, with well-defined peak clusters separated by empty or sparsely populated trough regions. Accurate models for peak centers and widths can be used to filter peptide signals from chemical noise. We modeled mass defects, the difference between monoisotopic and nominal masses, and peak centers and widths in the peptide mass distributions. We found that peak widths encompassing 95% of all peptide sequences are substantially smaller than previously thought. The result has implications for filtering out larger stretches of the mass axis. Mass defects of peptides exhibit an oscillatory behavior which is damped at high mass values. The periodicity of the oscillations is about 14 Da which is the most common difference between the masses of the 20 natural amino acids. To determine the effects of amino acid modifications on our findings, we examined the mass distributions of peptides composed of the 20 natural amino acids, oxidized Met, and phosphorylated Ser, Thr, and Tyr. We found that extension of the amino acid set by modifications increases the 95% peak width. Mass defects decrease, reflecting the fact that the average mass defect of natural amino acids is larger than that of oxidized Met. We propose that a new model for mass defects and peak widths of peptides may improve peptide identifications by filtering chemical noise in mass spectral data.
| S-EPMC3312599 | biostudies-literature
Project description:A major limitation of mass spectrometry-based proteomics is inefficient and differential ionization during electrospray ionization (ESI). This leads to problems such as increased limits of detection and incomplete sequence coverage of proteins. Incomplete sequence coverage is especially problematic for analyses that require the detection and identification of specific peptides from a protein, such as the analysis of post-translational modifications. We describe here the development and use of aldehyde-based chemistry for the alkylation of peptide primary amines to increase peptide hydrophobicity, providing increased ionization efficiency and concomitant signal enhancement. When employed to modify the peptide products of protein tryptic digests, increased sequence coverage is obtained from combined modified and unmodified digests. To evaluate the utility of alkylation of peptides for selected reaction monitoring (SRM) assays, we alkylated a peptide from the protein Oct4, known to play a role in regulating stem cell differentiation. Increased chromatographic retention and ionization efficiency is observed for the alkylated Oct4 peptide compared to its unmodified form.
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Project description:The in vitro deuteroacetylation of histones obtained from biological sources has been used previously in bottom-up mass spectrometry analyses to quantitate the percent of endogenous acetylation of specific lysine sites and/or peptides. In this report, derivatization of unmodified lysine residues on histones is used in combination with high performance mass spectrometry, including combined HPLC MS/MS, to distinguish and quantitate endogenously acetylated isoforms occurring within the same tryptic peptide sequence and to extend this derivatization strategy to other post-translational modifications, specifically methylation, dimethylation and trimethylation. The in vitro deuteroacetylation of monomethylated lysine residues is observed, though dimethylated or trimethylated residues are not derivatised. Comparison of the relative intensities ascribed to the deuteroacetylated and monomethylated species with the deuteroacetylated but unmethylated analog, provides an opportunity to estimate the percent of methylation at that site. In addition to the observed fragmentation patterns, the very high mass accuracy available on the Orbitrap mass spectrometer can be used to confirm the structural isoforms, and in particular to distinguish between trimethylated and acetylated species.
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Project description:The fragmentation characteristics of peptides derivatized at the side-chain epsilon-amino group of lysyl residues via reductive amination with benzaldehyde have been examined using collision-induced dissociation (CID) tandem mass spectrometry. The resulting MS/MS spectra exhibit peaks representing product ions formed from two independent fragmentation pathways. One pathway results in backbone fragmentation and commonly observed sequence ion peaks. The other pathway corresponds to the unsymmetrical, heterolytic cleavage of the C(zeta)-N(epsilon) bond that links the benzyl derivative to the side-chain lysyl residue. This results in the elimination of the derivative as a benzylic or tropylium carbocation and a (n - 1)(+)-charged peptide product (where n is the precursor ion charge state). The frequency of occurrence of the elimination pathway increases with increasing charge of the precursor ion. For the benzyl-modified tryptic peptides analyzed in this study, peaks representing products from both of these pathways are observed in the MS/MS spectra of doubly-charged precursor ions, but the carbocation elimination pathway occurs almost exclusively for triply-charged precursor ions. The experimental evidence presented herein, combined with molecular orbital calculations, suggests that the elimination pathway is a charge-directed reaction contingent upon protonation of the secondary epsilon-amino group of the benzyl-derivatized lysyl side chain. If the secondary epsilon-amine is protonated, the elimination of the carbocation is observed. If the precursor is not protonated at the secondary epsilon-amine, backbone fragmentation persists. The application of appropriately substituted benzyl analogs may allow for selective control over the relative abundance of product ions generated from the two pathways.
| S-EPMC2927711 | biostudies-literature
Project description:This work describes the mass distribution of all theoretically possibly tryptic peptides made of 20 amino acids, up to the mass of 3 kDa, with resolution of 0.001 Da. We characterize regions between the peaks of the distribution, including gaps (forbidden zones) and low-populated areas (quiet zones). We show how the gaps shrink over the mass range and when they completely disappear. We demonstrate that peptide compositions in quiet zones are less diverse than those in the peaks of the distribution and that by eliminating certain types of unrealistic compositions the gaps in the distribution may be increased. The mass distribution is generated using a parallel implementation of a recursive procedure that enumerates all amino acid compositions. It allows us to enumerate all compositions of tryptic peptides below 3 kDa in 48 min using a computer cluster with 12 Intel Xeon X5650 CPUs (72 cores). The results of this work can be used to facilitate protein identification and mass defect labeling in mass spectrometry-based proteomics experiments.
| S-EPMC3184890 | biostudies-literature
Project description:1. The half-cystine content of ovotransferrin, measured as cysteic acid, was 31mol/80000g of protein. 2. The amino acid sequences of cysteic acid-containing peptides from performic acid-oxidized ovotransferrin were studied. 3. 34 unique cysteic acid residues were identified. 4. It is concluded that hen ovotransferrin does not consist of two identical halves or subunits.
| S-EPMC1185389 | biostudies-other
Project description:Partially tryptic peptides are often identified in shotgun proteomics using trypsin as the proteolytic enzyme; however, their sources have been controversial. Herein, we investigate the impact of in-source fragmentation on shotgun proteomics profiling of three biological samples: a standard protein mixture, a mouse brain tissue homogenate, and mouse plasma. Because the in-source fragments of peptide ions have the same LC elution time as their parental peptides, partially tryptic peptide ions from in-source fragmentation can be distinguished from other partially tryptic peptides based on their elution time differences from those computationally predicted data. The percentage of partially tryptic peptide identifications resulting from in-source fragmentation in a standard protein digest was observed to be ?60%. In more complex mouse brain or plasma samples, in-source fragmentation contributed to a lesser degree of 1-3% of all identified peptides due to the limited dynamic range of LC-MS/MS measurements. The other major source of partially tryptic peptides in complex biological samples is presumably proteolytic cleavage by endogenous proteases in the samples. Our work also provides a method to identify such proteolytic-derived partially tryptic peptides due to endogenous proteases in the samples by removing in-source fragmentation artifacts from the identified peptides.
| S-EPMC3584699 | biostudies-literature
Project description:On-tissue digestion has become the preferred method to identify proteins in mass spectrometry (MS) imaging. In this study, we report advances in data acquisition and protein identification for MS imaging after on-tissue digestion. Tryptic peptides in a coronal mouse brain section were measured at 50 ?m pixel size and revealed detailed histological structures, e.g., the ependyma (consisting of one to two cell layers), which was confirmed by H&E staining. This demonstrates that MS imaging of tryptic peptides at or close to cellular resolution is within reach. We also describe a detailed identification workflow which resulted in the identification of 99 proteins (with 435 corresponding peptides), based on comparison with LC-MS/MS data and in silico digest. These results were obtained with stringent parameters, including high mass accuracy in imaging mode (RSME <?3 ppm) and at least two unique peptides per protein showing consistent spatial distribution. We identified almost 50% of proteins with at least four corresponding peptides. As there is no agreed approach for identification of proteins after on-tissue digestion yet, we discuss our workflow in detail and make the corresponding mass spectral data available as "open data" via ProteomeXchange (identifier PXD003172). With this, we would like to contribute to a more effective discussion and the development of new approaches for tryptic peptide identification in MS imaging. From an experimental point of view, we demonstrate the improvement due to the combination of high spatial resolution and high mass resolution/mass accuracy on a measurement at 25 ?m pixel size in mouse cerebellum tissue. A whole body section of a mouse pub imaged at 50 ?m pixel size (40 GB, 230,000 spectra) demonstrates the stability of our protocol. For this data set, we developed a workflow that is based on conversion to the common data format imzML and sequential application of freely available software tools. In combination, the presented results for spatial resolution, protein identification, and data processing constitute significant improvements for the field of on-tissue digestion. Graphical abstract MS imaging of coronal mouse brain cerebellum with a pixel size of 25 ?m: A Optical image, B myelin staining, C H&E staining, and D MS image overlay (RGB) of tryptic peptides m/z?=?726.4045?±?0.005, HGFLPR + H+ (red), m/z?=?536.3173?±?0.005, AKPAK + Na+ (green), and m/z?=?994.5436?±?0.005, WRQLIEK + Na+ (blue).
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