Neutral Loss Scan

Most protein phosphorylation events occur at serine and threonine residues. Upon CID, serine and threonine phosphopeptides exhibit a characteristic loss of H3PO4 not observed for unmodified peptides [e.g., 24, 25].

This specific feature can be used to detect Ser- and Thr-phosphopeptides. Figure 6.11 shows the spectra obtained when a sample of protein kinase A was in-gel digested with elastase and subjected to nanoESI-MS analysis using scanning for neutral loss of 98 Da. In this way, specific detection of singly charged phosphopeptides is achieved [26]. Digestion with elastase instead of the usual trypsin was selected, because elastase generates peptides with a smaller molecular weight, and because the loss of H3PO4 was found to proceed most effectively for small phosphopeptides with molecular weights between 400 and 1000 Da. The phosphopeptide candidates spotted in this way in the digest mixture are then selected for product ion scanning for sequencing and for pinpointing the phosphorylation site.

Fig. 6.11 NanoESI-MS analysis of an elastase digest of protein kinase A. (a) Survey MS; (b) neutral loss scan (98 Da) selectively showing the singly charged phosphopeptides present in the mixture (triple quadrupole analysis).

128 | 6 Covalent Protein Modification Analysis by Electrospray Tandem Mass Spectrometry PO3 Marker Fragment

In negative ion mode CID, Ser-,Thr-, and Tyr-phosphopeptides generate a PO- fragment at m/z 79, which can be used for recognition of the corresponding phosphopeptides in LC-MS by skimmer CID [27, 28] or by nanoESI-MS and precursor ion scanning [29]. Following their recognition in negative ion mode, the corresponding molecular ion signals are selected in positive ion mode for their characterization by MS/MS, since negative ion product ion spectra of have a poor sequence information content [19].

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