FluoroTRAQ: Quantitative Analysis of Protein S‑Nitrosylation through Fluorous Solid-Phase Extraction Combining with iTRAQ by Mass Spectrometry

S-Nitrosylation
is an important post-translational modification
that occurs on cysteine amino acid and regulates signal transduction
in diverse cell processes. Dysregulation of protein nitrosylation
has shown close association with cardiovascular and neurological diseases,
thus demanding further precise and in-depth understanding. Mass spectrometry-based
proteomics has been the method of choice for analyzing S-nitrosylated
(SNO-) proteins. However, due to their extremely low expression level
and rapid turnover rate, quantitative analysis of the S-nitrosylation
at the proteomic level remains challenging. Herein, we developed a
novel approach termed FluoroTRAQ, which combined the fluorous solid-phase
extraction of SNO-peptides and iTRAQ labeling for the quantitative
analysis of the SNO-proteome with high sensitivity and specificity.
This new analytical strategy was subsequently applied to examine the
dynamic SNO-proteome changes of human umbilical vein endothelial cells
upon in vitro S-nitrosoglutathione induction. Our data identified
a number of novel SNO-proteins and revealed their temporal modulation
as validated by biotin switch assay. Our study offered a practical
approach for quantitative analysis of protein S-nitrosylation.