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The standard mass spectrometric technique for the analysis of phosphopeptides on a linear ion trap mass spectrometer is to perform MS/MS followed by MS3 of the putative neutral loss peak observed by MS/MS. This is usually termed data-dependent neutral loss MS3 (DDN-LMS3). The strength of this approach is the sensitivity in full scan MSn of linear ion trap technology and the characteristically strong neutral loss peak, which flags the MS/ MS spectrum as potentially belonging to a phosphopeptide. This approach has been much applied to phospho-proteomic analyses typically involving up-front phosphopeptide enrichment, such as TiO2 chromatography. Indeed, a recent article from Mann and co-workers identified 6600 phosphorylation sites utilizing a hybrid linear ion trap Fourier transform mass spectrometer. However, there are also weaknesses, such as the fact that phosphotyrosine residues rarely produce a neutral loss from the precursor during MS/MS.
Recently, a new dissociation technique, electron transfer dissociation (ETD), has been invented in the lab of Don Hunt. ETD has different but complementary characteristics compared to CID. For example, (1) ETD works best on multiply charged ions of 3+ and above, whereas CID works best on 1+ to 3+ ions; (2) amino acid side chains and important modifications such as phosphorylated amino acid residues are left intact by ETD, producing a rich ladder of c and z ions for sequencing. CID produces mostly b- and y-type ions by collision with gases.
Work reported here compares DDNLMS3 data (and another neutral loss–based technique referred to as multistage activation, also utilized in the work of Mann and colleagues) generated on a series of human kinases with results obtained by data-dependent ETD. The different dissociation techniques were found to be complementary, and a number of novel phosphorylation sites were identified on such well-known kinases as PKB and SAPK2a.