Identification of Phosphopeptides

Peptide Mapping If the sequence of the protein is known, molecular mass determination of the peptide fragments in the unfractionated protein digest can rapidly identify the presence of phosphopeptides. Protein digest is analyzed by MALDI-MS (or ESI-MS), and phosphopeptides are recognized from the mass shift of 80 Da (or a multiple of 80 Da). 

Phosphatase Treatment The specificity of phosphatases for the removal of the phosphate group has also been exploited to identify phosphopeptides and proteins selectively [38]. The molecular mass of phosphopeptides decreases by 80 Da for each phospho unit after the phosphatase treatment. The reaction is monitored by MALDI-MS [39]. The peptide maps can be compared before and after phosphatase treatment to identify phosphopeptides in the digest [40]. Sensitivity is a major issue in such experiments. One way to improve sensitivity is to perform dephosphorylation on the MALDI target [40]. Another way is to use immobilized phosphatase packed in a smaU-diameter colnmn in-line with an LC/ESI-MS or CE/ESI-MS system [39,41], 

Precursor-Ion Scan This procedure makes use of tandem mass spectrometry, and is implemented mainly on a triple quadrupole instrument. Phosphopep-tide ions are generated by nano-ESI [44,45]. Phosphoserine-, phosphoihreonine-, and phosphotyrosine-containing peptides readily form a structurally diagnostic PO3 ion of m/z 79 under CID conditions precursor-ion scan of m/z 79 in the 

A similar derivatization strategy also exists for the selective detection of phosphoserine- and phosphothreonine-containing peptides in the positive-ion 

Neutral-Loss Scan This procedure is similar to precursor-ion scanning except that it monitors a characteristic neutral loss using a triple-quadrupole tandem instrument. All three types of O-phosphorylated peptides exhibit the loss of 98 

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Gram FI, Schmitz R, Zuber JF, Baumann G, Identification of phosphopeptide ligands for the Src-homology 2 (SH2) domain of Grb2 by phage display, Eur. J. Biochem., 246(3) 633-637, 1997. 

M. Adamczyk, J.C. Gebler, J. Wu, Identification of phosphopeptides by chemical modification with an isotopic tag and ion trap MS, Rapid Commun. Mass Spectrom., 16(2002)999. 

A general protocol for mass spectrometric analysis of phosphoproteins is illustrated in Figure 15 various steps of this protocol are cleavage of purified phosphoproteins, isolation and preferential enrichment of phosphopeptides, selective detection of phosphopeptides in the digest, identification of the phosphorylation sites using tandem mass spectrometry, and identification of phosphopeptides/proteins through a database search. 

Several versions of the precursor-ion scan can be found in the literature for selective identification of phosphopeptides. In one version, the precursor-ion scan of m/z 79 is performed in the negative-ion mode.100 In another version, termed phosphotyrosine-specific immonium-ion (PSI) scanning, only phosphotyrosine-containing peptides are detected by monitoring the CID-produced immonium ion of m/z 216.043 in the positive-ion mode.101,102... 

In precursor scan mode, the functions of Qj and are reversed. is set to a particular m/z, while Qj scans. This allows identification of all precursor ions that yield a particular product. A relevant example of such an application is the identification of phosphopeptides (Neubauer and Mann, 1999). Since phosphopeptides will lose PO3 fragments in the collision-induced dissociation step, Q3 is set at m/z 79 and Qj scanned. This allows identification of peptides that contain phosphate. Figure 3.10 includes the product and precursor ion spectra of such phosphopeptides. 

The selectivity of Ga(III)- IMAC for phosphopeptides, combined with the specificity of mass determination by MALDI-MS often enables the identification of phosphopeptide candidates, by comparing the predicted proteolytic peptide masses to experimentally determined mass values. However, the high mass... 

T. T. Yip and W. Hutchens, Mapping and sequence-specific identification of phosphopeptides in unfractionated protein digest mixtures by matrix-assisted laser desorp-tion/ionization time-of-flight mass spectrometry, FEBS Lett. 308, 149-153 (1992). 

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