Adaptation of CysPAT technique to characterize endogenous S-nitrosylation in RAW264.7 cells

ABSTRACT: The aim of the study was to adapt the CysPAT technique to efficiently identify and characterize endogenous S-nitrosylation in cells under physiological conditions of an experiment from a small amount of sample. Using a macrophage cell line RAW 264.7 we compared the efficacy of the method in the identification of total endogenous S-nitrosylation and the corresponding modification of the samples treated with exogenous SNO donor (GSNO) or with DTT. We identified 999 unique formerly S-nitrosylated peptides (SIA peptides) and 965 unique SNO sites from untreated RAW cells which belong to 569 endogenous nitrosylated proteins. We discovered 579 novel S-nitrosylation sites and identified 232 proteins as new S-nitrosylation targets. A total of 1450 S-nitrosylated peptides belonging to 795 proteins were identified in samples treated with GSNO (n = 3). Interestingly, we found a large overlap (>53%) of S-nitrosylated peptides in both subsets of samples, demonstrating a high sensitivity of the method to analyze endogenous S-nitrosylation. The large number of identified endogenous S-nitrosylated peptides allowed the identification of two nitrosylation consensus sites, and to highlight protein translation and redox processes as key S-nitrosylation targets in macrophages.

INSTRUMENT(S):

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Macrophage

SUBMITTER: Maria Ibañez-Vea  

LAB HEAD: David Escors

PROVIDER: PXD008182 | Pride | 2022-02-22

REPOSITORIES: Pride