A protein microarray-based analysis of S-nitrosylation

Yeast protein microarrays were utilized to investigate determinants of S-nitrosylation by biologically relevant low-mass S-nitrosothiols (SNOs). Large numbers of S-nitrosylated yeast proteins were identified after treatment with SNOs, among which those with active-site Cys thiols residing at N termini of alpha-helices or within catalytic loops were particularly prominent. However, S-nitrosylation varied substantially even within these families of proteins (e.g., papain-related Cys-dependent hydrolases and rhodanese/Cdc25 phosphatases), suggesting that neither secondary structure nor intrinsic nucleophilicity of Cys thiols was sufficient to explain specificity. Further analyses revealed a substantial influence of NO-donor stereochemistry and structure on efficiency of S-nitrosylation as well as an unanticipated and important role for allosteric effectors. Thus, high-throughput screening and unbiased proteome coverage reveal multifactorial determinants of S-nitrosylation (which may be overlooked in alternative proteomic analyses), and support the idea that target specificity can be achieved through rational design of S-nitrosothiols Invitrogen yeast Protoarrays for kinase substrate identification (KSI) were treated with S-nitrosothiols and assayed for protein S-nitrosylation by using a modified biotin switch protocol. Slides were scanned and with a Genepix 4000b scanner (Molecular Devices) using Genepix Pro and analyzed by using Prospector Analyzer (Invitrogen). Results were validated using yeast cell lysates and recombinant, purified yeast proteins.