Phosphorylation Site Identification:
Identification of phosphorylation sites is becoming an increasingly popular request. However the success rate has been somewhat limited due to the nature of protein phosphorylation. The important question is to what extent any one site is phosphorylated. When there is stochiometric incorporation usually there is a clear band or spot shift from unphosphorylated protein which is most evident for smaller molecular weight proteins. These projects typically have a higher success rate. Often times one will want to identify in vivo phosphorylation sites, which more often than not have a very low level of incorporation at any one site. Even if a high level of radioactivity (P32) or western signal is evident it is usually representative of a chemically insignificant portion of the total protein which may be below the detection limit of the mass spectrometer. Since the incorporation may not be stochiometric my approach is to first enrich for phosphopetides after tryptic digestion using a TiO2 affinity capture procedure. The enriched mixture is then analyzed by either MALDI-TOF MS or LC ESI MS/MS mass spectrometry. In the case of MALDI-TOF MS analyses phosphopeptides show a characteristic neutral loss of phosphate (-98) for S/T phosphorylation. Alternatively resorting to other proteases other than trypsin (i.e. Endoproteinase AspN, or LysC) have produced better ionizing peptides representing the sites of phosphorylation. Once phosphopeptides have been identified their phosphorylated residues can be determined by examination of their CID (MS/MS) spectra. The MALDI QIT TOF instrument has been particularly successful in analyzing a broad range phosphopeptides which tend to ionize very well in the DHB (2, 5 -Dihydroxybenzoic acid) matrix used.
2347.00 (193- DFQEYVEPGEDFPASPQRR -211)
1582.72 (386- QPPTAAGRPVDASPR -400)
1297.55 (401- VPGGSPRTPNR -411)
Three phosphopeptides representing four phosphorylation sites were identified after TiO2 enrichment of a tryptic digest of Dynein light intermediate chain 1 (LIC1). The phosphopeptides were not evident in the whole tryptic digest before enrichment. Details of this study can be found in (Sivaram et. al. EMBO 2009 doi:10.1038/emboj.2009.38).