Low-Background Acyl-Biotinyl Exchange Largely Eliminates the Coisolation of Non- <i>S</i> -Acylated Proteins and Enables Deep <i>S</i> -Acylproteomic Analysis

Abstract

Protein <i>S</i>-acylation (also called palmitoylation) is a common post-translational modification whose deregulation plays a key role in the pathogenesis of many diseases. Acyl-biotinyl exchange (ABE), a widely used method for the enrichment of <i>S</i>-acylated proteins, has the potential of capturing the entire <i>S</i>-acylproteome in any type of biological sample. Here, we showed that current ABE methods suffer from a high background arising from the coisolation of non-<i>S</i>-acylated proteins. The background can be substantially reduced by an additional blockage of residual free cysteine residues with 2,2'-dithiodipyridine prior to the biotin-HPDP reaction. Coupling the low-background ABE (LB-ABE) method with label-free proteomics, 2 895 high-confidence candidate <i>S</i>-acylated proteins (including 1 591 known <i>S</i>-acylated proteins) were identified from human prostate cancer LNCaP cells, representing so-far the largest <i>S</i>-acylproteome data set identified in a single study. Immunoblotting analysis confirmed the <i>S</i>-acylation of five known and five novel prostate cancer-related <i>S</i>-acylated proteins in LNCaP cells and suggested that their <i>S</i>-acylation levels were about 0.6-1.8%. In summary, the LB-ABE method largely eliminates the coisolation of non-<i>S</i>-acylated proteins and enables deep <i>S</i>-acylproteomic analysis. It is expected to facilitate a much more comprehensive and accurate quantification of <i>S</i>-acylproteomes than previous ABE methods.