Mapping N-terminus phosphorylation sites and quantitation by stable isotope dimethyl labeling.
We have previously coupled stable isotope dimethyl labeling with IMAC enrichment for quantifying the extent of protein phosphorylation in vivo. The enhanced a(1) signal of dimethylated peptides served as a unique mass tag for unequivocal identification of the N-terminal amino acids. In this study, we demonstrate that the a(1) ion could further assist in mapping the precise phosphorylation site near the N-terminal region and allow the determination of the exact site and level of phosphorylation in one step by stable isotope dimethyl labeling. We show that the a(1) ion signal was suppressed for dimethylated peptides with a phosphorylation site at the N-terminus Ser/Thr residue (N-p*Ser/Thr) but was still enhanced for N-terminus Tyr residue (N-p*Tyr) or internal Ser/Thr residues (-p*Ser/Thr). Based on the dominant de-phosphorylated molecular ions and b-H(3)PO(4) ions for N-p*Ser/Thr, we propose that dimethyl labeling increases the basicity of the N-terminus and accelerates the de-phosphorylation for N-p*Ser/Thr precursors, which, however, suppresses the a(1) ion enhancement due to the resulting unsaturated covalent bond on C(alpha) of the N-terminus amino acid. Using this method, we excluded three Ser/Thr phosphorylation sites in A431 cells, two of which, however, were previously reported to be phosphorylation sites; we confirmed three known phosphorylation sites in A431 cells and quantified their ratios upon EGF treatment. Notably, we identified a novel phosphorylation site on Ser43 residue at N-terminus of the tryptic peptide derived from SVH protein in pregnant rat uteri. SVH protein has not been reported or implied with any phosphorylation event, and our data show that the Ser43 of SVH is an intrinsic phosphorylation site in pregnant rat uteri and that its phosphorylation level was slightly decreased upon c-AMP treatment.
Wu
Chin-Jen
CJ
Department of Chemistry, National Cheng Kung University, Tainan, Taiwan.
Hsu
Jue-Liang
JL
Huang
Sheng-Yu
SY
Chen
Shu-Hui
SH
D001665:Binding Sites; D007553:Isotope Labeling / Q000379:methods*; D013058:Mass Spectrometry / Q000379:methods*; D008745:Methylation; D010449:Peptide Mapping / Q000379:methods*; D010455:Peptides / Q000737:chemistry*; D010766:Phosphorylation; D011485:Protein Binding
D010455:Peptides