Immuno-Spin Trapping Method for the Analysis of S-Nitrosylated Proteins

Esha Sircar, Detcho A. Stoyanovsky, Timothy R. Billiar, Rajib Sengupta*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

1 Scopus citations

Abstract

The dynamic and unstable nature of protein nitrosothiols (PSNOs) derived from complex biological matrices (like cell lysates) make them unsuitable for proteomic/biochemical analysis in vitro. In an attempt to increase the stability of cell-derived PSNOs, scientists have devised methods to derivatize thiols undergoing nitrosylation, with a suitable molecule, to yield a stable adduct that could easily be detected using appropriate antibodies. The Biotin Switch Assay (BTSA) is currently the most widely used method for tagging PSNOs; however, the error-prone and cumbersome nature of the BTSA protocol prompted the development of alternative mechanisms of tagging cell-derived PSNOs. One such method is the immuno-spin trapping method using 5,5-dimethyl-1-pyrroline N-oxide (DMPO), which effectively overcomes the shortcomings of the BTSA and proves to be a promising alternative. Here we describe the protocol for DMPO-based PSNO labeling and subsequent proteomic analysis by western blotting with an anti-DMPO antibody.

Original languageEnglish
Article numbere262
JournalCurrent Protocols
Volume1
Issue number9
DOIs
StatePublished - Sep 2021
Externally publishedYes

Keywords

  • DMPO
  • S-nitrosylation
  • immune-spin trapping
  • thiols
  • thiyl radical

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