Targeted SCX based peptide fractionation for optimal sequencing by collision induced, and electron transfer dissociation
Ontology highlight
ABSTRACT:
INSTRUMENT(S): LTQ FT, instrument model, LTQ XL
ORGANISM(S): Drosophila Melanogaster (fruit Fly)
TISSUE(S): Embryo, Early Embryonic Cell
SUBMITTER: 
Henk W.P. van den Toorn
PROVIDER: PRD000083 | Pride | 2013-06-18
REPOSITORIES: pride
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Project description:The identification of peptides presented by human leukocyte antigen (HLA) class I is tremendously important for the understanding of antigen presentation mechanisms under healthy or diseased conditions. Currently, mass spectrometry-based methods represent the best methodology for the identification of HLA class I-associated peptides. However, the HLA class I peptide repertoire remains largely unexplored because the variable nature of endogenous peptides represents difficulties in conventional peptide fragmentation technology. Here, we substantially enhanced (about threefold) the identification success rate of peptides presented by HLA class I using combined electron-transfer/higher-energy collision dissociation (EThcD), reporting over 12,000 high-confident (false discovery rate <1%) peptides from a single human B-cell line. The direct importance of such an unprecedented large dataset is highlighted by the discovery of unique features in antigen presentation. The observation that a substantial part of proteins is sampled across different HLA alleles, and the common occurrence of HLA class I nested sets, suggest that the constraints of HLA class I to comprehensively present the health states of cells are not as tight as previously thought. Our dataset contains a substantial set of peptides bearing a variety of posttranslational modifications presented with marked allele-specific differences. We propose that EThcD should become the method of choice in analyzing HLA class I-presented peptides.
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Project description:Electron transfer dissociation (ETD) and collision-induced dissociation (CID) were used to investigate underivatized, metal-cationized oligosaccharides formed via electrospray ionization (ESI). Reducing and non-reducing sugars were studied including the tetrasaccharides maltotetraose, 3α,4β,3α-galactotetraose, stachyose, nystose, and a heptasaccharide, maltoheptaose. Univalent alkali, divalent alkaline earth, divalent and trivalent transition metal ions, and a boron group trivalent metal ion were adducted to the non-permethylated oligosaccharides. ESI generated [M + Met]+, [M + 2Met]2+, [M + Met]2+, [M + Met - H]+, and [M + Met - 2H]+ most intensely along with low intensity nitrate adducts, depending on the metal and sugar ionized. The ability of these metal ions to produce oligosaccharide adduct ions by ESI had the general trend: Ca(II) > Mg(II) > Ni(II) > Co(II) > Zn(II) > Cu(II) > Na(I) > K(I) > Al(III) ≈ Fe(III) ≈ Cr(III). Although trivalent metals were utilized, no triply charged ions were formed. Metal cations allowed for high ESI signal intensity without permethylation. ETD and CID on [M + Met]2+ produced various glycosidic and cross-ring cleavages, with ETD producing more cross-ring and internal ions, which are useful for structural analysis. Product ion intensities varied based on glycosidic-bond linkage and identity of monosaccharide sub-unit, and metal adducts. ETD and CID showed high fragmentation efficiency, often with complete precursor dissociation, depending on the identity of the adducted metal ion. Loss of water was occasionally observed, but elimination of small neutral molecules was not prevalent. For both ETD and CID, [M + Co]2+ produced the most uniform structurally informative dissociation with all oligosaccharides studied. The ETD and CID spectra were complementary. Graphical Abstract ᅟ.
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Project description:Electron transfer dissociation (ETD) is a recently introduced mass spectrometric technique which has proven to be an excellent tool for the elucidation of labile post-translational modifications such as phosphorylation and O-GlcNAcylation of serine and threonine residues. However, unlike collision induced dissociation (CID), which has been studied for decades, the intricacies of ETD-based fragmentation have not yet been firmly established or systematically addressed. In this analysis, we have systematically compared the CID and ETD fragmentation patterns for the large majority of the peptides that do not contain such labile modifications. Using a standard 48 protein mix, we were able to measure false-positive rates for the experiments and also assess a large number of peptides for a detailed comparison of CID and ETD fragmentation pattern. Analysis of approximately 19,000 peptides derived from both standard proteins and complex protein samples revealed that (i) CID identified 50% more peptides than ETD; (ii) ETD resulted in approximately 20% increase in amino acid sequence coverage over CID; and (iii) combining CID and ETD fragmentation increased the sequence coverage for an average tryptic peptide to 92%. Interestingly, our analysis revealed that nearly 60% of all ETD-identified peptides carried two positive charges, which is in sharp contrast to what has been generally accepted. We also present a novel strategy for automatic validation of peptide assignments based on identification of a peptide by consecutive CID and ETD fragmentation in an alternating mode.
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Project description:We present a workflow using an ETD-optimised version of Mascot Percolator and a modified version of SLoMo (turbo-SLoMo) for analysis of phosphoproteomic data. We have benchmarked this against several database searching algorithms and phosphorylation site localisation tools and show that it offers highly sensitive and confident phosphopeptide identification and site assignment with PSM-level statistics, enabling rigorous comparison of data acquisition methods. We analysed the Plasmodium falciparum schizont phosphoproteome using for the first time, a data-dependent neutral loss-triggered-ETD (DDNL) strategy and a conventional decision-tree method. At a posterior error probability threshold of 0.01, similar numbers of PSMs were identified using both methods with a 73% overlap in phosphopeptide identifications. The false discovery rate associated with spectral pairs where DDNL CID/ETD identified the same phosphopeptide was <1%. 72% of phosphorylation site assignments using turbo-SLoMo without any score filtering, were identical and 99.8% of these cases are associated with a false localisation rate of <5%. We show that DDNL acquisition is a useful approach for phosphoproteomics and results in an increased confidence in phosphopeptide identification without compromising sensitivity or duty cycle. Furthermore, the combination of Mascot Percolator and turbo-SLoMo represents a robust workflow for phosphoproteomic data analysis using CID and ETD fragmentation.Biological significanceProtein phosphorylation is a ubiquitous post-translational modification that regulates protein function. Mass spectrometry-based approaches have revolutionised its analysis on a large-scale but phosphorylation sites are often identified by single phosphopeptides and therefore require more rigorous data analysis to unsure that sites are identified with high confidence for follow-up experiments to investigate their biological significance. The coverage and confidence of phosphoproteomic experiments can be enhanced by the use of multiple complementary fragmentation methods. Here we have benchmarked a data analysis pipeline for analysis of phosphoproteomic data generated using CID and ETD fragmentation and used it to demonstrate the utility of a data-dependent neutral loss triggered ETD fragmentation strategy for high confidence phosphopeptide identification and phosphorylation site localisation.
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Project description:We recently introduced a novel scheme combining electron-transfer and higher-energy collision dissociation (termed EThcD), for improved peptide ion fragmentation and identification. We reasoned that phosphosite localization, one of the major hurdles in high-throughput phosphoproteomics, could also highly benefit from the generation of such EThcD spectra. Here, we systematically assessed the impact on phosphosite localization utilizing EThcD in comparison to methods employing either ETD or HCD, respectively, using a defined synthetic phosphopeptide mixture and also using a larger data set of Ti(4+)-IMAC enriched phosphopeptides from a tryptic human cell line digest. In combination with a modified version of phosphoRS, we observed that in the majority of cases EThcD generated richer and more confidently identified spectra, resulting in superior phosphosite localization scores. Our data demonstrates the distinctive potential of EThcD for PTM localization, also beyond protein phosphorylation.
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