Detergent-free biotin switch combined with liquid chromatography/tandem mass spectrometry in the analysis of S-nitrosylated proteins.
High-throughput proteomic analysis based on a biotin switch combined with liquid chromatography/tandem mass spectrometry (LC/MS/MS) enables simultaneous identification of S-nitrosylated sites and their cognate proteins in complex biological mixtures, which is a great help in elucidating the functions and mechanisms of this redox-based post-translational modification. However, detergents such as sodium dodecyl sulfate (SDS) and Triton X-100 adopted in these systems, which are hard to fully remove in the subsequent MS-based analyses, can suppress the peptide signals and influence the SNO-Cys site identification and the reproducibility of the experiments. Here we developed a detergent-free biotin-switch method, which applied urea to replace detergents, and successfully combined it with LC/MS/MS in the analysis of S-nitrosylated proteins. With this approach, 44 SNO-Cys sites were specified on 35 distinct proteins in S-nitrosoglutathione (GSNO)-treated HeLa cell extracts of proteins with good reproducibility. The LC/MS performance was greatly improved as analyzed with Pep3D and the amount of samples for analysis reduced from 40 mg used in the literature to 3-5 mg. For S-nitrosylated targets detected both in the control sample and in the GSNO-treated sample, extracted ion chromatography (XIC) was employed to estimate the quantitative change of S-nitrosylation (S-nitrosation), which facilitates the judgment on 'accept or reject' of the identified targets.
Han
Peiwei
P
National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China.
Chen
Chang
C
D000595:Amino Acid Sequence; D001710:Biotin / Q000737:chemistry*; D002457:Cell Extracts / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003902:Detergents / Q000737:chemistry; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D009569:Nitric Oxide / Q000737:chemistry; D010449:Peptide Mapping; D011506:Proteins / Q000737:chemistry*; D040901:Proteomics; D015203:Reproducibility of Results; D026422:S-Nitrosoglutathione / Q000737:chemistry*; D026403:S-Nitrosothiols / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D053719:Tandem Mass Spectrometry
D002457:Cell Extracts; D003902:Detergents; D011506:Proteins; D026403:S-Nitrosothiols; D009569:Nitric Oxide; D026422:S-Nitrosoglutathione; D001710:Biotin