Mapping N-terminus phosphorylation sites and quantitation by stable isotope dimethyl labeling

Abstract

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 dephosphorylation for N-p*Ser/Thr precursors, which, however, suppresses the a 1 ion enhancement due to the resulting unsaturated covalent bond on C ␣ 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. (J Am Soc Mass Spectrom 2010, 21, 460 -471)

Stable isotope dimethyl labelling for quantitative proteomics and beyond

“…Wu et al reported that the a 1 signal was suppressed for precursor ions with phosphorylated Ser/Thr N-termini (N-p*Ser/Thr) but no suppression was observed for the precursor ion with phosphorylated Tyr N-terminus [36]. Although somehow negative, this provides a useful hint for mapping phosphorylation sites at the N-termini [106]. Despite diverse usefulness, a 1 ion is not detectable for ion trap instruments due to the '1/3 rule', which also occurs for other tandem mass tags like iTRAQ.…”

Shotgun Proteomics Analysis of Differentially Expressed Urinary Proteins Involved in the Hepatocellular Carcinoma

“…Quantitative proteome analysis by shotgun approach coupled with stable isotope dimethyl labeling has been used in identifying candidate biomarkers or target factors in different types of samples on account of the fact that this approach can detect differentially released proteins at relatively low abundance [28][29][30][31]. In this study, we conducted a comparative proteomics investigation of urine between HCC patients and control group by a bottom-up shotgun proteomic approach.…”

Quantitative Proteomics Analysis of Differentially Expressed Proteins Involved in Renal Clear Cell Carcinoma by Shotgun Approach Coupled with Stable Isotope Dimethyl Labeling

“…In this study we aim to conduct the comparative and quantitative proteome analysis between RCC and their nearby normal portion of the same tissue by means of gel-free shotgun proteomic approach coupled with stable isotope dimethyl labeling and nanoLC-MS/MS [26][27][28][29]. The differentially expressed proteins identified by the quantitative approach will be employed to delineate the biosignaling pathways involved in the tumorigenesis of RCC.…”