Dynamic and reversible protein S-acylation, most commonly occurring as S-palmitoylation, plays an important role in protein/membrane association and the regulation of intracellular signaling via cycles of palmitoylation and depalmitoylation. Direct analysis of protein S-acylation by mass spectrometry (MS) offers several benefits over indirect detection methods in that it can definitively determine the location and nature of the acyl modification, and is not prone to false discoveries. However, characterization of acyl proteins is challenging because of the tendency of acyl loss during sample preparation and tandem MS analysis. In this chapter, we present a sample preparation protocol that preserves labile acyl modifications and an LC-MS/MS workflow for detection of S-acylation with high confidence and sensitivity.
Keywords: Reversed phase liquid chromatography–mass spectrometry (RPLC-MS); S-palmitoylation; Tandem mass spectrometry (MS/MS).