S-Nitrosylation

Introduction
S-Nitrosylation refers to a covalent attachment of an NO moiety to sulfhydryl residues of
proteins. The sulfhydryl residues belongs to a subset of specific cysteine residues in proteins,
the resulting SNO is an S-nitrosoprotein. SNOs have a short half-life in the cytoplasm
because of the host of reducing enzymes, including glutathione (GSH) and thioredoxin, that
denitrosylate proteins. Therefore, SNOs are often stored in membranes, vesicles, the
interstitial space and lipophilic protein folds to protect them from denitrosylation. For example,
caspases, which mediate apoptosis, are stored in the mitochondrial intermembrane space as
SNOs.

Similar to phosphorylation, S-Nitrosylation is a reversible precess. The denitrosylation is an

enzymatic catalyzing process that reverses the S-Nitrosylation process. However, S-
nitrosylation is not a random event, and only specific cysteine residues are S-nitrosylated.
Under physiologic conditions, protein S>-nitrosylation and SNOs provide protection
preventing further cellular oxidative and nitrosative stress. Aberrant S-Nitrosylation may lead
to protein misfolding, synaptic damage, and apoptosis. Dysfunction of the SNO signaling has
been implicated in the pathogenesis of many diseases, such as Alzheimer’s disease
cardiovascular diseases.

Because proteins may contain multiple cysteines and due to the labile nature of SNOs, S-
nitrosylated cysteines can be difficult to detect and distinguish from non-S-nitrosylated amino
acids. Creative Proteomics has established a highly sensitive HPLC-MS/MS pipeline that can
analyze S-nitrosylated cysteines in both eukaryotic and prokaryotic organisms. In addition, we
have optimized our protocol, to enable more fast and sensitive site mapping service for S-
nitrosylated cysteines.

Workflow of our S-Nitrosylation analysis service:

 In gel or in solution digestion of proteins
 Enrichment of S-nitrosocysteine using anti-S-nitrosocysteine antibody
 HPLC separation, followed by ESI-TOF MS/MS analysis
 Mass spectrometry data interpretation
Technology platform:
 Ion Chromatography
 High Performance Liquid Chromatography (HPLC)
 Matrix Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS)