Abstract
Post-translational modifications (PTMs) occur dynamically, allowing cells to quickly respond to changes in the environment. Lysine residues can be targeted by several modifications including acylations (acetylation, succinylation, malonylation, glutarylation, and others), methylation, ubiquitination, and other modifications. One of the most efficient methods for the identification of post-translational modifications is utilizing immunoaffinity enrichment followed by high-resolution mass spectrometry. This workflow can be coupled with comprehensive data-independent acquisition (DIA) mass spectrometry to be a high-throughput, label-free PTM quantification approach. Below we describe a detailed protocol to process tissue by homogenization and proteolytically digest proteins, followed by immunoaffinity enrichment of lysine-acetylated peptides to identify and quantify relative changes of acetylation comparing different conditions.
Highlights
Proteomics techniques utilizing mass spectrometry (MS) have quickly become a preferred method to measure relative changes in protein abundance as well as significant changes in posttranslational modifications (PTMs)
Post-translational modifications have been studied for decades; recent studies utilizing mass spectrometry have revolutionized the analysis of PTM profiles and identified thousands of novel PTM sites
We present a detailed protocol to allow for a routine workflow of robust identification and quantification of lysine acetylation sites by affinity enrichment of PTM-containing peptides followed by mass spectrometric analysis
Summary
Proteomics techniques utilizing mass spectrometry (MS) have quickly become a preferred method to measure relative changes in protein abundance as well as significant changes in posttranslational modifications (PTMs) (see Chaps. 8, 13–15). We present a detailed protocol to allow for a routine workflow of robust identification and quantification of lysine acetylation sites by affinity enrichment of PTM-containing peptides followed by mass spectrometric analysis. This will provide a standardized protocol for the study of lysine acetylation sites to streamline the identification and quantification of PTMs. This will provide a standardized protocol for the study of lysine acetylation sites to streamline the identification and quantification of PTMs This method provides a strategy to study PTMs with relatively low starting material, even with capabilities for multiplexing and enriching for multiple PTMs simultaneously [9] in an unbiased approach using label-free DIA-MS [10–12] This method provides a strategy to study PTMs with relatively low starting material, even with capabilities for multiplexing and enriching for multiple PTMs simultaneously [9] in an unbiased approach using label-free DIA-MS [10–12] (see Chaps. 8, 22–24) in combination with multiple different software programs, such as Skyline and Spectronaut [13, 14] or others (see Chap. 31)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
