Spectra Of Tryptic Peptides Research Articles

Isobaric Labeling Proteomics Allows a High-Throughput Investigation of Protein Corona Orientation.

The formation of the biomolecular corona represents a crucial factor in controlling the biological interactions and trafficking of nanomaterials. In this context, the availability of key epitopes exposed on the surface of the corona, and able to engage the biological machinery, is important to define the biological fate of the material. While the full biomolecular corona composition can be investigated by conventional bottom-up proteomics, the assessment of the spatial orientation of proteins in the corona in a high-throughput fashion is still challenging. In this work, we show that labeling corona proteins with isobaric tags in their native conditions and analyzing the MS/MS spectra of tryptic peptides allow an easy and high-throughput assessment of the inner/outer orientation of the corresponding proteins in the original corona. We put our results in the context of what is currently known of the protein corona of graphene-based nanomaterials. Our conclusions are in line with previous data and were confirmed by in silico calculations.

Approval of the method of electrophoretic separation and identification of some urine proteins in rats with toxic nephropathy

The aim of the article. The aim was to test the method for determining certain protein markers in rat urine by electrophoretic separation followed by mass spectrometric identification for early diagnostic of nephrotoxicity. The objectives of the study included assessing the reproducibility of the protein separation method, comparing rats urine protein profiles normal and after gentamicin sulfate administration, as well as a biochemical and morphological study of kidney biopsy specimens.
 Materials and methods. Gentamicin sulfate (GS) was administered intramuscularly to rats of both sexes at a dose of 60 mg/kg b.w. for consequence 5 days. Daily urine was collected in the background, on the 3rd, 7th and 14th day from the start of the administration of GS in the metabolic cages. We measured the concentrations of total protein and creatinine in urine samples, the level of lipids in the homogenates of the kidneys. Electrophoretic separation of urine proteins was performed in the Mini-PROTEAN Tetra Vertical Electrophoresis. The gels were stained, and the stained zones were excised and enzymatic hydrolysis of the proteins was performed. Spectra of tryptic peptides were recorded on an ultrafleXtreme MALDI-TOF/TOF mass spectrometer (Bruker, Germany). Proteins were identified in the Biotools program by accessing the Mascot database (matrixscience.com).
 Results. Assessment of reproducibility of the results of electrophoretic separation of rat urine proteins showed an acceptable value of the coefficient of variation (on average 8.03%) and the error of analysis (analysis = 14.53%). It that normally in rats of males the alpha-2-uroglobulin precursor dominates in urine and in females immunoglobulin G light chains dominate was show. Against the background of the administration of GS, leukocytes in pathological concentrations appear in the urine in rats of both sexes; in males, the proteomic profile has a strong intraspecific dispersion, including due to sperm contamination; in females, well identified zones of albumin, alpha-1-antitrypsin, aminopeptidase M, beta-2-microglobulin, and transferrin was appear. A biochemical study of kidney homogenates an increase in total cholesterol (males) and triacylglycerides (females) revealed. Pathomorphological changes were similar in male and female rats in the form of fatty degeneration of the proximal tubule nephrothelium and leukocyte infiltration of interstitium and confirmed changes in the spectrum of urine proteins.
 Conclusion. Based on the analysis of the experimental results it was found that the use of the technology of electrophoretic separation of proteins in PAGE under denaturing conditions followed by MALDI mass spectrometric identification (PAGE-MALDI-TOF/TOF) and densitometric determination of the percentage of protein fractions is applicable for the detection of nephrotoxicity. Due to gender differences in the protein spectrum it is preferable to examine the urine of female rats. Pathomorphological changes did not have gender differences.

Proteome Modification in Tomato Plants upon Long-Term Aluminum Treatment.

This study aimed to identify the aluminum (Al)-induced proteomes in tomato (Solanum lycopersicum, "Micro-Tom") after long-term exposure to the stress factor. Plants were treated in Magnavaca's solution (pH 4.5) supplemented with 7.5 μM Al(3+) ion activity over a 4 month period beginning at the emergence of flower buds and ending when the lower mature leaves started to turn yellow. Proteomes were identified using a 8-plex isobaric tags for relative and absolute quantification (iTRAQ) labeling strategy followed by a two-dimensional (high- and low-pH) chromatographic separation and final generation of tandem mass spectrometry (MS/MS) spectra of tryptic peptides on an LTQ-Orbitrap Elite mass spectrometer. Principal component analysis revealed that the Al-treatment had induced systemic alterations in the proteomes from roots and leaves but not seed tissues. The significantly changed root proteins were shown to have putative functions in Al(3+) ion uptake and transportation, root development, and a multitude of other cellular processes. Changes in the leaf proteome indicate that the light reaction centers of photosynthetic machinery are the primary targets of Al-induced stress. Embryo and seed-coat tissues derived from Al-treated plants were enriched with stress proteins. The biological processes involving these Al-induced proteins concur with the physiological and morphological changes, such as the disturbance of mineral homeostasis (higher contents of Al, P, and Fe and reduced contents of S, Zn, and Mn in Al-treated compared to nontreated plants) in roots and smaller sizes of roots and the whole plants. More importantly, the identified significant proteins might represent a molecular mechanism for plants to develop toward establishing the Al tolerance and adaptation mechanism over a long period of stress treatment.

Old proteins and the Achilles heel of mass spectrometry. The role of proteomics in the etiology of human cataract

Proteomics may have enabled the root cause of a major human-blinding condition, age-related cataract, to be established. Cataract appears to result from the spontaneous decomposition of long-lived macromolecules in the human lens, and recent proteomic analysis has enabled both the particular crystallins, and the specific sites of amino acid modification within each polypeptide, to be identified. Analysis of proteins from cataract lenses has demonstrated that there are key sites on some structural proteins that show a consistently greater degree of deterioration than age-matched normal lenses. Proteomic analysis, using MS, revealed that the most abundant posttranslational modification of aged lens proteins is racemization. This is somewhat ironic, since structural isomers can be viewed as the "Achilles heel" of MS and there are typically few, if any, differences in the MS/MS spectra of tryptic peptides containing one d-amino acid. It is proposed that once a certain level of spontaneous PTM at key sites occurs, that protein-protein interactions are disrupted, and binding of complexes to cell membranes takes place that impairs cell-to-cell communication. These findings may apply more widely to age-related human diseases, in particular where the deterioration of long-lived proteins is a crucial component in the etiology.

UniNovo: a universal tool for de novo peptide sequencing

Motivation: Mass spectrometry (MS) instruments and experimental protocols are rapidly advancing, but de novo peptide sequencing algorithms to analyze tandem mass (MS/MS) spectra are lagging behind. Although existing de novo sequencing tools perform well on certain types of spectra [e.g. Collision Induced Dissociation (CID) spectra of tryptic peptides], their performance often deteriorates on other types of spectra, such as Electron Transfer Dissociation (ETD), Higher-energy Collisional Dissociation (HCD) spectra or spectra of non-tryptic digests. Thus, rather than developing a new algorithm for each type of spectra, we develop a universal de novo sequencing algorithm called UniNovo that works well for all types of spectra or even for spectral pairs (e.g. CID/ETD spectral pairs). UniNovo uses an improved scoring function that captures the dependences between different ion types, where such dependencies are learned automatically using a modified offset frequency function.Results: The performance of UniNovo is compared with PepNovo+, PEAKS and pNovo using various types of spectra. The results show that the performance of UniNovo is superior to other tools for ETD spectra and superior or comparable with others for CID and HCD spectra. UniNovo also estimates the probability that each reported reconstruction is correct, using simple statistics that are readily obtained from a small training dataset. We demonstrate that the estimation is accurate for all tested types of spectra (including CID, HCD, ETD, CID/ETD and HCD/ETD spectra of trypsin, LysC or AspN digested peptides).Availability: UniNovo is implemented in JAVA and tested on Windows, Ubuntu and OS X machines. UniNovo is available at http://proteomics.ucsd.edu/Software/UniNovo.html along with the manual.Contact: kwj@ucsd.edu or ppevzner@ucsd.eduSupplementary information: Supplementary data are available at Bioinformatics online.

Alternative methods for verifying the results of the mass spectrometric identification of peptides in shotgun proteomics

Database search is the most popular approach used for the identification of peptides in contemporary shotgun proteomics; it utilizes only mass spectrometric data. In this work, we introduce three criteria for the verification of peptide identification; these are based on the analysis of data orthogonal to tandem mass spectra. The first one utilizes chromatographic retention times of peptides. The development of such approaches has been hindered by the relatively low accuracy of retention time prediction algorithms. In this work, we suggest the use of two independent models of the liquid chromatography of peptides, which increase the reliability of the results. The second criterion utilizes the mean number of missed tryptic cleavages per peptide. The third one results from the analysis of the difference between theoretical and experimentally measured peptide masses. The proposed criteria were applied to the tandem mass spectra of tryptic peptides from rat kidney tissue, which were processed by the Mascot search engine. All the criteria showed that Mascot significantly overestimated the reliability of an identification. This conclusion was supported by the PeptideProphet algorithm.

SwedCAD, a Database of Annotated High-Mass Accuracy MS/MS Spectra of Tryptic Peptides

A database of high-mass accuracy tryptic peptides has been created. The database contains 15 897 unique, annotated MS/MS spectra. It is possible to search for peptides according to their mass, number of missed cleavages, and sequence motifs. All of the data contained in the database is downloadable, and each spectrum can be visualized. An example is presented of how the database can be used for studying peptide fragmentation. Fragmentation of different types of missed cleaved peptides has been studied, and the results can be used to improve identification of these types of peptides.