S-nitrosoproteome in endothelial cells revealed by a modified biotin switch approach coupled with Western blot-based two-dimensional gel electrophoresis.
NO-mediated S-nitrosation of cysteine residues has been recognized as a fundamental post-translational modification. S-Nitrosation of endothelial cell (EC) proteins can alter function and affect vascular homeostasis. Trace amounts of S-nitrosoproteins in endothelial cells (ECs) in vivo coupled with lability of the S-nitroso bond have hindered a comprehensive characterization. We demonstrate a convenient and reliable method, requiring minimal sample, for the screening and identification of S-nitrosoproteins. ECs treated with the NO donor S-nitroso-N-acetylpenicillamine (SNAP) were subjected to the biotin switch method of labeling, then detected by analytical Western blot-based two-dimensional gel electrophoresis (2-DE). More than 89 SNAP-increased S-nitrosoproteins were detected and 28 of these were successfully excised from preparative 2-DE gel and identified by LC-MS/MS. Moreover, the nitrosocysteine residue for each protein (HSPA9/368, beta-actin/16, TMP3/170, vimentin/328) was also determined, and the relative ratio of S-nitrosation/non-S-nitrosation for Cys328 of vimentin was estimated using MASIC software. By the combination of the biotin switch method with 2-DE and Western blot analysis, S-nitrosoproteins can be screened and characterized by MS, providing a basis for further study of the physiological significance of each S-nitrosoproteins.
Huang
Bin
B
Institute of Biomedical Sciences, Academia Sinica, Taipei 11529, Taiwan.
Liao
Chung Ling
CL
Lin
Ya Ping
YP
Chen
Shih Chung
SC
Wang
Danny Ling
DL
D001360:Avidin / Q000378:metabolism; D001710:Biotin / Q000378:metabolism*; D015153:Blotting, Western / Q000379:methods*; D002460:Cell Line; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D042783:Endothelial Cells / Q000378:metabolism*; D006801:Humans; D013058:Mass Spectrometry; D009569:Nitric Oxide / Q000378:metabolism; D015538:Nitrosation; D010446:Peptide Fragments / Q000032:analysis; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D015203:Reproducibility of Results; D026403:S-Nitrosothiols / Q000737:chemistry / Q000378:metabolism*
D010446:Peptide Fragments; D011506:Proteins; D026403:S-Nitrosothiols; D001360:Avidin; D009569:Nitric Oxide; D001710:Biotin