Strategies for Incorporating Novel Post-Translational Modification Enrichment Methods in a Quantitative Proteomics Environment
Year: 2012
Type: article
Abstract: Low-stoichiometry post-translational modifications (PTMs) such as phosphorylation, acetylation, s-nitrosylation (SNO), and s-acylation are integral to intracellular signaling and are thought to capture the most immediate response of a cell upon external stimulus. These modifications, among others, have been the subject of intense focus in the mass spectrometry world because of the ability of the mass spectrometer to identify the peptide and many times the site of modification with a much higher degree of specificity than antibody-based approaches. However, PTMs present a distinct analytical challenge because they exist at low levels in biological samples and therefore must be enriched in order to be measured with the mass spectrometer in a high-throughput manner from a complex system. Three categorical methods exist to enrich PTMs, including chemical enrichment, antibody enrichment, and PTM-switch methods. In a biomedical research environment, one of the most challenging aspects to implementing any enrichment strategy is to properly qualify a technique so that it can be performed with high specificity and reproducibility. Each of the three categories of enrichments has unique analytical concerns and challenges. Recently, our laboratory has sought to extend the study of PTMs from primarily a qualitative environment to a quantitative one, in order to study selected PTMs in systems where isotopic labeling is not possible, such as animal models or clinical studies.[1-4] This presentation will focus on the key steps in the development and deployment of label-free quantitation using three of our recently-published PTM-switch enrichment approaches, S-nitrosothiol resin-assisted capture (SNO-RAC), S-acylation resin-assisted capture (Acyl-RAC), and the acetyl biotin-switch technique (Acetyl-BST).
Source: Europe PMC (PubMed Central)
Language: English
Subfield: Spectroscopy
Field: Chemistry
Domain: Physical Sciences
Open Access status: green
OpenAlex ID: W1625459468