ABSTRACTProteinS-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 ofS-acylated proteins, has the potential of capturing the entireS-acylproteome in any types of biological samples. Here, we showed that current ABE methods suffer from high background arising from the co-isolation of non-S-acylated proteins. The background can be substantially reduced by an additional blockage of residual free cysteine residues with 2,2’-dithiodipyridine prior to biotin-HPDP reaction. Coupling the low-background ABE (LB-ABE) method with label-free quantitative proteomics, 2,895 high-confidence candidateS-acylated proteins (including 1,591 knownS-acylated proteins) were identified from human prostate cancer LNCaP cells, representing so-far the largestS-acylproteome dataset identified in a single study. Immunoblotting analysis confirmed theS-acylation of five known and five novel prostate cancer-relatedS-acylated proteins in LNCaP cells and suggested that theirS-acylation levels were about 0.6-1.8%. In summary, the LB-ABE method largely eliminates the co-isolation of non-S-acylated proteins and enables deepS-acylproteomic analysis. It is expected to facilitate much more comprehensive and accurate quantification ofS-acylproteomes than previous ABE methods.