Home » Abstracts » PTM & Glycoproteomics » Towards a high throughput sample preparation process for proteomic analysis of S-nitrosylation.

Towards a high throughput sample preparation process for proteomic analysis of S-nitrosylation.

PTM & Glycoproteomics

Abstract #332 Posted on29 March 202510 April 2025 10 April 2025 22h52

Abstract

S-nitrosylation is the result of the reaction between nitric oxide (NO) and the sulfhydryl cysteine of a protein plays a central biological role in the propagation of NO signals within a cell, tissue and its microenvironment. Nowadays accumulative evidences show the implication of protein S-nitrosylation in cellular physiology and pathophysiology.
The aim of this work was the development of a novel optimized sample preparation process capable of managing the major challenges of SNO analyses, namely reliability, throughput (cost) and detection from low quantity of sample.
The optimized method combines a biotin-like based SNO derivatization, single pot solid-phase enhanced sample preparation and a magnetic- based enrichment. The samples were analyzed using a nano-LC system coupled to Q-Exactive HF.
The results of optimizations performed using Hela extracts treated with 100µM GSNO are presented. The methodoly was then applied to analyze endogenous SNO modification from rat heart samples. The SNOs identified were validated by cross-referencing and an artificial intelligence tool. AlLtogether, these results highlight interesting performance gains brought by the novel procedure in terms of speed and efficiency, false-positive management, throughput, and ability to analyze low sample amount. The method will now be applied to identify endogenous SNO proteins and their sites to access the molecular mechanisms and regulatory roles of S-nitrosylation.

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S-nitrosylation is the result of the reaction between nitric oxide (NO) and the sulfhydryl cysteine of a protein plays a central biological role in the propagation of NO signals within a cell, tissue and its microenvironment. Nowadays accumulative evidences show the implication of protein S-nitrosylation in cellular physiology and pathophysiology.
The aim of this work was the development of a novel optimized sample preparation process capable of managing the major challenges of SNO analyses, namely reliability, throughput (cost) and detection from low quantity of sample.
The optimized method combines a biotin-like based SNO derivatization, single pot solid-phase enhanced sample preparation and a magnetic- based enrichment. The samples were analyzed   using a nano-LC system coupled to Q-Exactive HF.
The results of optimizations performed using Hela extracts  treated with 100µM GSNO are presented. The methodoly was then applied to analyze endogenous SNO modification from rat heart samples. The SNOs identified were validated by cross-referencing and an artificial intelligence tool. AlLtogether, these results highlight interesting performance gains brought by the novel procedure in terms of speed and efficiency, false-positive management, throughput, and ability to analyze low sample amount. The method will now be applied to identify endogenous SNO proteins and their sites to access the molecular mechanisms and regulatory roles of S-nitrosylation.

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