Proteome-wide profiling of S-nitrosylated proteins using the SNOTRAP probe and mass spectrometry-based detection.
Publication Year:
2025
PubMed ID:
41258014
Funding Grants:
Public Summary:
This publication reports a robust, novel chemical approach to characterize the effects of redox (chemical transfer of electrons) due to air pollutants like PM2.5 and other environmental toxins on the brain and stem cell-based models of the human brain by analyzing proteins for S-nitrosylation (addition of SNO to proteins to affect their function). This new approach will allow us to characterized Autism Spectrum Disorder and Intellectual Disability (ASD/ID) and other neurological diseases and how they are affected by this redox modification of proteins.
Scientific Abstract:
Protein S-nitrosylation (SNO) is a ubiquitous post-translational modification, which regulates a broad range of functional parameters, including protein stability; enzymatic, transcriptional and ion channel activity; and cellular signal transduction. Aberrant protein SNO is associated with diverse pathophysiology, from cardiovascular, metabolic and respiratory disorders to neurodegeneration and cancer. Drugs that enhance or inhibit specific SNO reactions are being developed as potential disease-modifying therapeutics. However, owing to a lack of suitable approaches to monitor SNO proteins, which often exist at low abundance with ephemeral expression, a systematic understanding of their roles in disease remains elusive. Here we report a robust and proteome-wide approach for the exploration of the S-nitrosoproteome in human and mouse tissues, using the brain as an example, with a probe named SNOTRAP (a triphenylphosphine thioester linked to a biotin molecule through a polyethylene glycol spacer group) in conjunction with mass spectrometry (MS)-based detection. In this Protocol, we detail tissue sample preparation, synthesis of SNOTRAP under an argon atmosphere and subsequent MS-based identification and analysis of SNO proteins. In situ labeling of SNO proteins is achieved by the SNOTRAP probe, concomitantly yielding a disulfide-iminophosphorane as a labeling tag. The chemically tagged proteins can be digested, followed by streptavidin capture, release by triscarboxyethylphosphine and relabeling of the liberated free Cys with N-ethylmaleimide. This approach selectively enriches SNO-containing peptides at specific sites for label-free quantification by Orbitrap MS. It requires about 5 d for synthesis of the SNOTRAP probe, 2-2.5 d for sample preparation and about 5 d for nano-liquid chromatography-tandem MS measurement and analysis.
