Protein phosphorylation analysis by site-specific arginine-mimic labeling in gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.
Although recent advances in gel electrophoresis and mass spectrometry have greatly facilitated separation, purification, and identification of proteins, significant challenges remain in relation to phosphoprotein analysis. Here we introduce a powerful method for analysis of protein phosphorylation in which phosphorylation sites are labeled with guanidinoethanethiol (GET) by beta-elimination/Michael addition prior to proteolysis and mass spectrometry (MS) analysis. This technique is especially useful in conjunction with gel-based technology in that all of the processes involved, including GET labeling, washing, and phosphospecific enzymatic hydrolysis, can be carried out in excised gel slices, thereby minimizing sample loss and contamination. The novel GET tag, which has a highly basic guanidine group, increases the peak intensities for the GET-labeled tryptic peptides by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS. In addition, phosphospecific proteolytic cleavage occurs at guanidinoethylcysteine (Gec) residue, which is arginine-mimic formed by GET tagging of phosphorylated serine residues. Thus, GET tagging is especially useful in analysis of long tryptic phosphopeptides. To illustrate the utility of the in-gel GET tagging and digestion approach, we used it to precisely analyze the phosphorylation sites of human glutathione S-transferase P1 (GSTP1), an enzyme involved in phase II metabolism of many carcinogens and anticancer drugs. The in-gel GET tagging/digestion technique significantly enhances the analytical potential of gel electrophoresis/MS in studies of proteome phosphorylation.
Ahn
Yeong Hee
YH
Division of Instrument Development, Korea Basic Science Institute, Daejeon 305-333, Republic of Korea.
Ji
Eun Sun
ES
Kwon
Kyung Hoon
KH
Lee
Jae Yong
JY
Cho
Kun
K
Kim
Jin Young
JY
Kang
Hyun Ju
HJ
Kim
Ho Guen
HG
Yoo
Jong Shin
JS
D000970:Antineoplastic Agents / Q000378:metabolism; D001120:Arginine / Q000032:analysis*; D002273:Carcinogens / Q000378:metabolism; D003110:Colonic Neoplasms / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D005982:Glutathione Transferase / Q000032:analysis* / Q000378:metabolism; D006801:Humans; D009363:Neoplasm Proteins / Q000032:analysis* / Q000378:metabolism; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D000970:Antineoplastic Agents; D002273:Carcinogens; D009363:Neoplasm Proteins; D001120:Arginine; D005982:Glutathione Transferase