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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This is very insightful. There is no doubt that there is the bias you refer to. 42 years ago, when I was postdocing in biochemistry/enzymology before completing my residency in pathology, I knew that there were very influential mambers of the faculty, who also had large programs, and attracted exceptional students. My mentor, it was said (although he was a great writer), could draft a project on toilet paper and call the NIH. It can’t be true, but it was a time in our history preceding a great explosion. It is bizarre for me to read now about eNOS and iNOS, and about CaMKII-á, â, ã, ä – isoenzymes. They were overlooked during the search for the genome, so intermediary metabolism took a back seat. But the work on protein conformation, and on the mechanism of action of enzymes and ligand and coenzyme was just out there, and became more important with the research on signaling pathways. The work on the mechanism of pyridine nucleotide isoenzymes preceded the work by Burton Sobel on the MB isoenzyme in heart. The Vietnam War cut into the funding, and it has actually declined linearly since.
A few years later, I was an Associate Professor at a new Medical School and I submitted a proposal that was reviewed by the Chairman of Pharmacology, who was a former Director of NSF. He thought it was good enough. I was a pathologist and it went to a Biochemistry Review Committee. It was approved, but not funded. The verdict was that I would not be able to carry out the studies needed, and they would have approached it differently. A thousand young investigators are out there now with similar letters. I was told that the Department Chairmen have to build up their faculty. It’s harder now than then. So I filed for and received 3 patents based on my work at the suggestion of my brother-in-law. When I took it to Boehringer-Mannheim, they were actually clueless.