Leaders in Pharmaceutical Business Intelligence (LPBI) Group

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,

1. can suppress the peptide signals and
2. influence the SNO-Cys site identification and
3. the reproducibility of the experiments.

Here we developed a detergent-free biotin-switch method, which applied

1. urea to replace detergents, and successfully
2. 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.