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单击此处编辑母版标题样式,单击此处编辑母版文本样式,第二级,第三级,第四级,第五级,单击此处编辑母版标题样式,单击此处编辑母版文本样式,第二级,第三级,第四级,第五级,Large Scale Localization of ProteinPhosphorylation by Use of Electron Capture Dissociation Mass Spectrometry,Steve M. M. Sweet, Christopher M. Bailey, Debbie L. Cunningham,John K. Heath, and Helen J. Cooper,From the Cancer Research UK Growth Factor Group, School of Biosciences, College of Life and Environmental Sciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, United Kingdom,Content,Introduction of background,Experiment procedures,Analysis of Results,Conclusions,1.,Introduction of background,Damage to structure,In contrast to tradition mathods, labile modifications are usually retained intact upon ECD peptide backbone cleavage.,Low precision,Lack of study,ECD provides more extensive sequence coverage in polypeptides，and at higher electron energies even isoleucine and leucine are distinguishable,No large scale comparison of CID and ECD for phosphoprotein analysis has been carried out.,2. Experiment procedures,Cell Culture,Invitrogen,Cell lysis,Centrifugation,Determined by,Coomassie,Acidification,and desalted,Strong Cation Exchange,(SCX) Chromatography,Phosphopeptide,Enrichment,LC-MS/MS,Data-dependent CID and,ECD (DD-CID-ECD),Data Analysis,3. Analysis of Results,Summary of phosphopeptides identified from both DD-CID-ECD and NL-ECD experiments. The results indicate that phosphopeptides constituted 4045% of the starting mixture. It should be noted that not every phosphopeptide identified by CID triggered an ECD event.,FIG. 1. Overlap between DTAs leading to identifications by ECD and CID. All 4763 identifications (IDs) are from paired CID/ECD events .FIG. 2. Overlap between distinct phosphopeptides identified by ECD and CID. All 1220 identifications are from paired CID/ECDevents.,FIG. 3. Binned distribution of SLoMo scores. The x axis has log scale. All identifications are from paired CID/ECD events. Identifications with only one possible site of localization are not included.,FIG. 4. Overlap between distinct, well localized (SLoMo 19) phosphopeptides identified by ECD and CID. All 725 identifications are from paired CID/ECD events. Identifications with only one possible localization are not included.,FIG. 5. Example of isomeric phosphopeptide co-elution. a, ECD mass spectrum showing first and third serine phosphorylation (SLoMo score, 21.5). b, CID mass spectrum showing evidence for threonine phosphorylation (SLoMo score, 24.3). The site of phosphorylation indicated by SLoMo is shown uppermost inset in b.,FIG. 6. Multiply phosphorylated peptides identified and localized from ECD and CID mass spectra and sorted by charge state. All identifications are from paired events. Identifications with only one possible localization are not included.,4. Conclusion,I,n this study we analyzed 6080 paired CID/ECD mass spectra, 1892 of which led to phosphopeptide identifications by CID, ECD, or both. This data set allowed a comparison of the relative merits of LTQ CID and FT-ICR ECD mass spectra for phosphopeptide identification and site localization.,In conclusion, our results indicate that combined ECD and CID analysis results in high confidence phosphopeptide identifications and phosphorylation site localization. Hybrid mass spectrometers, such as the LTQ-FT used in this work, are primarily used to measure precursor ions with high mass accuracy while carrying out rapid CID (at lower resolution). We showed that there are potential advantages to using both parts of the hybrid instrument during peptide fragmentation to acquire orthogonal MS/MS data.,Thanks for your attention!,Please correct me critically,
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