An optimized protocol for isolation of S-nitrosylated proteins from C. elegans

Abstract

Post-translational modification by S-nitrosylation regulates numerous cellular functions and impacts most proteins across phylogeny. We describe a protocol for isolating S-nitrosylated proteins (SNO-proteins) from C. elegans, suitable for assessing SNO levels of individual proteins and of the global proteome. This protocol features efficient nematode lysis and SNO capture, while protection of SNO proteins from degradation is the major challenge. This protocol can be adapted to mammalian tissues. For complete information on the generation and use of this protocol, please refer to Seth et al. (2019).

Keywords: Model Organisms; Protein Biochemistry; Proteomics.

Graphical abstract

Figure 1

Robust S-nitrosylation is observed in C. elegans A silver-stained SDS-PAGE gel showing abundant C. elegans SNO-proteins isolated using the above protocol, from wildtype young adult worms under standard growth conditions using wildtype E. coli as the food source. Many SNO-proteins are visible in the plus Ascorbate (+ Ascorbate) lane but not in the minus Ascorbate (−Ascorbate) lane, verifying that the thiol-blocking step was effective. M= molecular weight marker.

Figure 2

Relative abundance of C. elegans SNO-proteome and specific SNO-proteins (Figure reprinted with permission, from (Seth et al., 2019)). C. elegans lysates were prepared and SNO-RAC was performed from the young adult N2 worms using the protocol detailed here. (A) Left-panel shows a silver stained gel where the SNO-proteins have been isolated from C. elegans grown either on wild-type B. subtilis 1A1 (WT) or B. subtilis mutants lacking bacterial nitric oxide synthase (Δnos). Middle panel shows the “minus-ascorbate” controls and the right panel shows the Coomassie blue staining of the C. elegans total proteome loading controls. (B) Immunoblot of the exemplary C. elegans protein ALG-1 after SNO-RAC was performed on lysates prepared from C. elegans grown as in (A).