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Quantitation of protein phosphorylation in pregnant rat uteri using stable isotope dimethyl labeling coupled with IMAC
Author(s) -
Huang ShengYu,
Tsai MeiLing,
Wu ChinJen,
Hsu JueLiang,
Ho ShihHsin,
Chen ShuHui
Publication year - 2006
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/pmic.200500507
Subject(s) - phosphorylation , phosphopeptide , chemistry , phosphoprotein , protein phosphorylation , quantitative proteomics , threonine , western blot , phosphoproteomics , proteomics , stable isotope labeling by amino acids in cell culture , casein kinase 2 , protein kinase a , biochemistry , chromatography , cyclin dependent kinase 2 , serine , gene
Quantitative analysis of protein phosphorylation provides important insights into molecular signaling mechanisms and a better understanding of many cellular processes. In this study, we coupled stable isotope dimethyl labeling with immobilized metal affinity chromatography (IMAC) enrichment to quantify protein phosphorylation at MS‐determined phosphorylation sites. The proposed method was first characterized using α‐ and β‐casein as two model phosphoproteins, and further applied to the analysis of pregnant rat uteri with and without treatment with 8‐bromo‐cGMP. Dimethyl labeling has several significant advantages: global, fast (within 5 min) and complete (near 100%). Our results indicate that the labeling has no adverse effect on the IMAC enrichment for tryptic peptides having single and multiple phosphorylation sites. Moreover, the enhanced a 1 signal and the complete reaction by dimethyl labeling provide unequivocal identification of both the N‐terminal amino acid and the number of the labeling site. Using these two criteria in data validation, which is particularly important for identifying phosphoproteins, we found that the confidence in interpreting dimethyl‐labeled peptides had greatly increased. In the analysis of late gestation rat uteri, the abundance ratio between treated and un‐treated phosphopeptide signals ranged from 0.51 to 1.69 with an average of around 1.01 ± 0.25. The obtained ratio of the phosphorylation levels at Ser 15 of HSP27 was further confirmed by the consistent results obtained from Western blot analyses. Based on the analysis of the results, it is interesting to note that the activated cGMP dependentt protein kinase G (PKG) seems to affect the phosphorylation of proteins associated with the inhibition of cell migration and proliferation, redistribution of actin‐associated proteins, and the increase of protein synthesis in late‐gestation uteri. These observations provide important evidence suggesting that activated PKG may play a critical role in the shift of pregnant uteri from proliferative to hypertrophic states.