| Autor: |
Froehlich JW; Department of Urology and Urological Diseases Research Center, Boston Children's Hospital and Harvard Medical School, Boston, Massachusetts, USA., Dodds ED, Wilhelm M, Serang O, Steen JA, Lee RS |
| Jazyk: |
angličtina |
| Zdroj: |
Molecular & cellular proteomics : MCP [Mol Cell Proteomics] 2013 Apr; Vol. 12 (4), pp. 1017-25. Date of Electronic Publication: 2013 Feb 25. |
| DOI: |
10.1074/mcp.M112.025494 |
| Abstrakt: |
Determining which glycan moieties occupy specific N-glycosylation sites is a highly challenging analytical task. Arguably, the most common approach involves LC-MS and LC-MS/MS analysis of glycopeptides generated by proteases with high cleavage site specificity; however, the depth achieved by this approach is modest. Nonglycosylated peptides are a major challenge to glycoproteomics, as they are preferentially selected for data-dependent MS/MS due to higher ionization efficiencies and higher stoichiometric levels in moderately complex samples. With the goal of improving glycopeptide coverage, a mass defect classifier was developed that discriminates between peptides and glycopeptides in complex mixtures based on accurate mass measurements of precursor peaks. By using the classifier, glycopeptides that were not fragmented in an initial data-dependent acquisition run may be targeted in a subsequent analysis without any prior knowledge of the glycan or protein species present in the mixture. Additionally, from probable glycopeptides that were poorly fragmented, tandem mass spectra may be reacquired using optimal glycopeptide settings. We demonstrate high sensitivity (0.892) and specificity (0.947) based on an in silico dataset spanning >100,000 tryptic entries. Comparable results were obtained using chymotryptic species. Further validation using published data and a fractionated tryptic digest of human urinary proteins was performed, yielding a sensitivity of 0.90 and a specificity of 0.93. Lists of glycopeptides may be generated from an initial proteomics experiment, and we show they may be efficiently targeted using the classifier. Considering the growing availability of high accuracy mass analyzers, this approach represents a simple and broadly applicable means of increasing the depth of MS/MS-based glycoproteomic analyses. |
| Databáze: |
MEDLINE |
| Externí odkaz: |
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