Phosphopeptide sequencing

Songyang Z, Shoelson SE, Chaudhuri M, Gish G, Pawson T, Haser WG, King F, Roberts T, Ratnofsky S, Lechleider RJ, Neel BG, Birge RB, Fajardo JE, Chou MM, Hanafusa H, Schaffhausen B, Cantley LC. SH2 domains recognize specific phosphopeptide sequences. Cell 1993 72 767-778. 

By compiling data from numerous runs, we have attempted to estimate the efficiency of phosphopeptide sequencing (Fig. 4). Although the repetitive yields cannot be estimated accurately due to variability, using data from the longer runs, we have nevertheless estimated a repetitive yield of 85%. This estimate... 

Songyang Z, Shoelson SE, Chaudhuri M et al (1993) Sh2 domains recognize specific phosphopeptide sequences. Cell 72 767-778... 

Schroeder, M.J. Shabanowitz, J. Schwartz, J.C. Hunt, D.F. Coon, J.J. A neutral loss activation method for improved phosphopeptide sequence analysis by quadrupole ion trap mass spectrometry. Anal. Chem. 2004, 76, 3590-3598. 

Johnson T, Packman LC, Hyde CB, Owen D, Quibell M (1996) Backbone protection and its application to the synthesis of a difficult phosphopeptide sequence. J Chem Soc 1 719-728... 

The specificity profile of individual SH2 domains was determined in a series of studies employing degenerate phosphopeptide library screens [53, 98] from which two consensus motifs emerged for SH2 domain binding [52-55,99,100]. One group of SH2 domains preferentially binds to a pTyr-Glu-Glu-Ile motif that defines a generic recognition sequence ... 

Identification of associated protein kinases Based on phosphopeptide analyses it became clear that associated kinases modify principally serine and threonine residues. Moreover, the analysis of putative phosphorylation-speciflc consensus sequences of p53, c-Jun, p27, ICSBP and I/cBa revealed that the protein kinase CK2 and a member of the protein kinase C family might be associated with the CSN. 

Human Proteinpedia provides a list of phosphopeptides identified in mass spectrometry-based phosphoproteomic studies. In addition, phosphorylation and dephosphorylation data curated from the literature are mapped to corresponding site and residue of sequences provided in HPRD. Using PhosphoMotif Finder one can analyze the presence of phosphorylation-based motifs, which are derived from the literature, in any protein of interest. This is a valuable data for biomedical investigators in the development of phospho-spedfic antibodies and peptide arrays. Availability of many raw MS datasets deposited in Human Proteinpedia... 

Kulkarni, G., Rao, G.S., Srinivasan, N.G., Hofer, H.W., Yuan, P.M. and Harris, B.G. (1987) Ascaris suum phosphofructokinase. Phosphorylation by protein kinase and sequence of the phosphopeptide. Journal of Biological Chemistry 262, 32-34. 

If polar sequences or short phosphopeptides fail to bind to the stationary phase upon loading, the acetonitrile content of the eluent could be reduced to 3%. A further decrease is not possible for standard Ci8-columns, as the stationary phase could collapse reducing the loading capacity and the reproducibility. Instead, TFA could be replaced by the more hydrophobic ion-pair reagent HFBA (heptafluorobutyric acid), or a stationary phase with a special coating compatible with pure aqueous eluents could be used (e.g., Aqua-column from Phenomenex). 

SH2 domains recognize peptide sequences bearing phos-photyrosine (pTyr) residues. They bind to compatible phosphopeptides with an affinity in the 10-100 nM range and bind to phosphopeptides of random sequence with a 1000-fbld lower affinity. They have practically no affinity for unphosphorylated peptides. xhe topography of a tyrosyl-binding SH2 domain is well known, thanks to structural studies (Plate 2).21.22... 

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