Session: Session 3

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

Introduction: Nowadays accumulative evidences show the implication of protein S-nitrosylation in cellular physiology and pathophysiology. Global identification of S-nitrosylated (SNO) proteins and their modification sites in a wide variety of context are therefore highly needed for this theme.  

Objective: 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 small quantity of sample.

Methods: The optimized method combines a biotin-like based SNO derivatization, single pot solid-phase enhanced sample preparation used initially just for proteolytic digestion and later also used for the sample clean-up steps during SNO derivatization.  and a magnetic- based enrichment. The samples were analyzed  with a nano-LC system coupled to Q-Exactive HF, a tandem mass spectrometer with Orbitrap ultra-high-field analyzer

Results:  The results of optimizations performed using protein extracts  treated with 100µM GSNO are presented. The SNOs identified were validated by cross-referencing with data published in the literature. 

Conclusions : Altogether, these results highlight huge 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 detect endogenous S-nitrosylation in complex biological mixtures.