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Orbitrap_SciLib
Reputable Mentor II
Reputable Mentor II
Jedrychowski MP, Huttlin EL, Haas W, Sowa ME, Rad R, Gygi SP.
Mol Cell Proteomics. 2011 Dec;10(12):M111.009910.
Protein phosphorylation modulates a myriad of biological functions, and its regulation is vital for proper cellular activity. Mass spectrometry (MS) is the enabling tool for phosphopeptide analysis, where recent instrumentation advances in both speed and sensitivity in linear ion trap and orbitrap technologies may yield more comprehensive phosphoproteomic analyses in less time. Protein phosphorylation analysis by MS relies on structural information derived through controlled peptide fragmentation. Compared to traditional, ion-trap-based CID (collision-induced dissociation), a more recent type of fragmentation termed HCD (higher energy collisional dissociation) provides beam type CID MS/MS with detection of fragment ions at high resolution in the orbitrap mass analyzer. Here we compared HCD to traditional CID for large-scale phosphorylation analyses of murine brain under three separate experimental conditions. These included a same-precursor analysis where CID and HCD scans were performed back-to-back, separate analyses of a phosphotyrosine peptide immunoprecipitation (IP) experiment, and separate whole phosphoproteome analyses. HCD generally provided higher search engine scores with more peptides identified, thus out-performing CID for back-to-back experiments for most metrics tested. However, for phosphotyrosine IPs and in a full phosphoproteome study of mouse brain, the greater acquisition speed of CID-only analyses provided larger data sets. We reconciled our results with those in direct contradiction from Nagaraj N, DSouza RCJ et. al. (J. Proteome Res. 9:6786, 2010). We conclude, for large-scale phosphoproteomics, CID fragmentation with rapid detection in the ion trap still produced substantially richer data sets, but the back-to-back experiments demonstrated the promise of HCD and orbitrap detection for the future.

http://www.mcponline.org/content/early/2011/09/13/mcp.M111.009910.full.pdf
Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA.
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