Statistical Analysis of Peptide Electron Transfer Dissociation Fragmentation Mass Spectrometry

Chalkley, Robert J.; Medzihradszky, Katalin F.; Lynn, Aenoch J.; Baker, Peter R.; Burlingame, Alma L.

doi:10.1021/ac9018582

Cited by 55 publications

(74 citation statements)

References 20 publications

(33 reference statements)

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“…In our analysis, the proximity of the sites of proton attachment and subsequent electron capture/transfer seems to also play a role in the success of locating clustered sites of O-glycosylation and not just an indiscriminate charge density. This also has been reported in broader studies for doubly charged precursor peptide cations (50). However, the bias is said to decrease with higher charge states.…”

Section: Application Of Ecd/etd To Clustered Sites Of O-glycosylationsupporting

confidence: 67%

Clustered O-Glycans of IgA1

Takahashi

Wall

Suzuki

et al. 2010

Molecular & Cellular Proteomics

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Glycosylation is one of the most common post-translational modifications of proteins. It is estimated that over half of mammalian proteins are glycosylated. Patients with several autoimmune disorders, chronic inflammatory diseases, and some infectious diseases exhibit abnormal glycosylation of serum immunoglobulins and other glycoproteins (1-5). The biological functions of these modifications in health and disease have become a significant area of interest in biomedical research (6). A subset of these glycoproteins has clustered sites of O-glycosylation with serine-and threonine-rich stretches within the amino acid sequence. Mucins, such as membrane-associated MUC1, are perhaps the best known family of proteins that are heavily O-glycosylated. Their altered expression and aberrant glycosylation have made them potential targets as biomarkers for early detection of cancer (7). Immunoglobulin A1 (IgA1) 1 contains both O-and N-glycans (Fig. 1). Aberrant O-glycosylation of IgA1 is involved in the pathogenesis of IgA nephropathy (IgAN) and the closely related Henoch-Schö nlein purpura nephritis (1, 8). Interestingly, the aberrantly glycosylated molecules, IgA1 in IgAN and MUC1 in cancer, are recognized by the immune system as neoepitopes as evidenced by formation of specific antibodies (9 -11). Mucin-like bacterial surface proteins exhibit similar properties: the molecules have clustered bacterial O-glycans that mediate cellular adhesion, and blocking antibodies target these glycan-containing epitopes (12).An O-glycosylated protein from a single source contains a population of variably O-glycosylated isoforms that show a distinct distribution of microheterogeneity of the O-glycan chains in terms of number, sites of attachment, and composition. Characterizing these clustered sites and understanding how the distributions change under different biological conditions or disease states are an analytical challenge. Enzymatic or chemical release of O-glycans is not selective. The heterogeneity, composition, and quantitative aspects of different O-glycan chains can be assessed and quantified by gas chromatographic and/or mass spectrometric techniques.

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Section: Application Of Ecd/etd To Clustered Sites Of O-glycosylationsupporting

confidence: 67%

Clustered O-Glycans of IgA1

Takahashi

Wall

Suzuki

et al. 2010

Molecular & Cellular Proteomics

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“…These Z-type carbohydrate fragment ions have also been found in electron capture dissociation (ECD) and appear to be due to radical-site-induced elimination processes within the glycan [24,25]. Another alternative reason was that trypsin digestion leads to more doubly charged peptides, which are not well dissociated in ETD [26]. Although ETD was not as efficient as CID in the fucosylation identification, the combination of the two methods increased the coverage and confidence of fucosylation identification.…”

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confidence: 99%

Development of a combined chemical and enzymatic approach for the mass spectrometric identification and quantification of aberrant N-glycosylation

Chen

Wang

Tan

et al. 2012

Journal of Proteomics

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“…We recently performed a study to characterize how frequently the different fragment ion types are detected in ETD spectra when analyzing complex digest mixtures produced by proteolytic enzymes or chemical cleavage reagents of different sequence specificity (16). These results were analyzed with respect to precursor charge state and location of basic residues, which were both shown to be significant factors in controlling the fragment ion types observed.…”

mentioning

confidence: 99%

“…Based on this statistical analysis of ETD data from a diverse range of peptides (16), in the present study, a new scoring system was developed and implemented in the search engine Batch-Tag within Protein Prospector that adjusts the weighting for different fragment ion types based on the precursor charge state and the presence of basic amino acid residues at either peptide terminus. The results using this new scoring system were compared with the previous generation of Batch-Tag, which used ion score weightings based on the average frequency of observation of different fragment types in ETD spectra of tryptic peptides and used the same scoring irrespective of precursor charge and sequence.…”

mentioning

confidence: 99%

Improving Software Performance for Peptide Electron Transfer Dissociation Data Analysis by Implementation of Charge State- and Sequence-Dependent Scoring

Baker

Medzihradszky

Chalkley

2010

Molecular & Cellular Proteomics

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The use of electron transfer dissociation (ETD) fragmentation for analysis of peptides eluting in liquid chromatography tandem mass spectrometry experiments is increasingly common and can allow identification of many peptides and proteins in complex mixtures. Peptide identification is performed through the use of search engines that attempt to match spectra to peptides from proteins in a database. However, software for the analysis of ETD fragmentation data is currently less developed than equivalent algorithms for the analysis of the more ubiquitous collision-induced dissociation fragmentation spectra. In this study, a new scoring system was developed for analysis of peptide ETD fragmentation data that varies the ion type weighting depending on the precursor ion charge state and peptide sequence. The recently developed fragmentation approach of electron transfer dissociation (ETD) 1 has become a genuine alternative to the more ubiquitous collision-induced dissociation (CID) for high throughput and high sensitivity proteomic analysis (1-3).ETD (4) and the related fragmentation process electron capture dissociation (ECD) (5) have been demonstrated to have particular advantages for the analysis of large peptides and small proteins (6 -8) as well as the analysis of peptides bearing labile post-translational modifications (9 -11). The results achieved through ETD and ECD analysis have been shown to be highly complementary to those obtained through CID fragmentation analysis, both through increasing confidence in particular identifications of peptides and also by allowing identification of extra components in complex mixtures (10, 12, 13). As CID and ETD can be sequentially or alternatively performed on precursor ions in the same mass spectrometric run, it is expected that the combined use of these two fragmentation analysis techniques will become increasingly common to enable more comprehensive sample analysis.Software for analysis of CID spectra is significantly more advanced than that for ECD/ETD data. This is partly because the behavior of peptides under CID fragmentation is better characterized and understood so software has been developed that is better able to predict the fragment ions expected. The fragment ion types observed in ETD and ECD are largely known (5,14,15), but information about the frequency and peak intensities of the different ion types observed is less well documented.We recently performed a study to characterize how frequently the different fragment ion types are detected in ETD spectra when analyzing complex digest mixtures produced by proteolytic enzymes or chemical cleavage reagents of different sequence specificity (16). These results were analyzed with respect to precursor charge state and location of basic residues, which were both shown to be significant factors in controlling the fragment ion types observed. The results showed that ETD spectra of doubly charged precursor ions produced very different fragment ions depending on the location of a basic residue in the sequence.Based on th...

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Statistical Analysis of Peptide Electron Transfer Dissociation Fragmentation Mass Spectrometry

Cited by 55 publications

References 20 publications

Clustered O-Glycans of IgA1

Clustered O-Glycans of IgA1

Development of a combined chemical and enzymatic approach for the mass spectrometric identification and quantification of aberrant N-glycosylation

Improving Software Performance for Peptide Electron Transfer Dissociation Data Analysis by Implementation of Charge State- and Sequence-Dependent Scoring

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