Studies on peptide acetylation for stable-isotope labeling after 1-D PAGE separation in quantitative proteomics.
Acetylation is a single labeling process to label peptides in control and experimental samples universally, and is independent of amino acid composition or post-translational modification. Here, we propose a new strategy especially useful to quantify either hydrophobic or extremely acidic and basic proteins involved in acetylation of tryptic peptides after sodium dodecyl sulfate polyarcylamide gel electrophoresis (SDS-PAGE) separation. We studied some essential parameters of acetylation labeling reactions in either in-solution tryptic peptides or in-gel digested extracts systematically. We have found that the acetylation efficiency varies markedly on account of different reactive systems, and demonstrated that stable isotope labeling can be steadily obtained with in-gel digested peptides under optimized conditions. We use this protocol to quantify some proteins of two kinds of hepatocellular carcinoma cell line, non-metastatic hepatocellular carcinoma cells, Hep3B, and metastatic hepatocellular carcinoma cells, MHCC97-H. The experimental results provide positive evidence for the potential application of an acetylation labeling strategy in quantitative proteomics, and an efficient way for global proteome quantification.
Yu
Yanling
Y
Chemistry Department, Fudan University, Shanghai, China.
Cui
Jiefeng
J
Wang
Xiaoyan
X
Liu
Yinkun
Y
Yang
Pengyuan
P
D000107:Acetylation; D002460:Cell Line; D045744:Cell Line, Tumor; D002845:Chromatography; D002851:Chromatography, High Pressure Liquid; D003902:Detergents / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D013058:Mass Spectrometry; D009211:Myoglobin / Q000737:chemistry; D010449:Peptide Mapping / Q000379:methods; D010455:Peptides / Q000737:chemistry*; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000737:chemistry; D020543:Proteome; D040901:Proteomics / Q000379:methods*; D013997:Time Factors; D014357:Trypsin / Q000494:pharmacology
D003902:Detergents; D009211:Myoglobin; D010455:Peptides; D011506:Proteins; D020543:Proteome; D014357:Trypsin