Analysis of S-nitrosylated Proteins: Detergent-free biotin switch combined with liquid chromatography/tandem mass spectrometry
Author and Curator: Larry H Bernstein, MD, FACP
Detergent-free biotin switch combined with liquid chromatography/tandem mass spectrometry in the analysis of S-nitrosylated proteins.
Han P; Chen C
National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China.
Rapid Commun Mass Spectrom. 2008 Apr; 22(8):1137-45. http://dx.doi.org/10.1002/rcm.3476. PMID: 18335467
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.
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