Low-background Acyl-biotinyl Exchange Largely Eliminates the Co-isolation of Non-S-acylated Proteins and Enables Deep S-acylproteomic Analysis

Zhou, B.; Wang, Y.; Yan, Y.; Mariscal, J.; Di Vizio, D.; Freeman, M. R.; Yang, W.

doi:10.1101/588988

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bioRxiv · 10.1101/588988

Low-background Acyl-biotinyl Exchange Largely Eliminates the Co-isolation of Non-S-acylated Proteins and Enables Deep S-acylproteomic Analysis

Zhou, B.·Wang, Y.·Yan, Y.·Mariscal, J.·Di Vizio, D.·Freeman, M. R.·Yang, W.

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Abstract

Protein S-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 S-acylated proteins, has the potential of capturing the entire S-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 candidate S-acylated proteins (including 1,591 known S-acylated proteins) were identified from human prostate cancer LNCaP cells, representing so-far the largest S-acylproteome dataset identified in a single study. Immunoblotting analysis confirmed the S-acylation of five known and five novel prostate cancer-related S-acylated proteins in LNCaP cells and suggested that their S-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 deep S-acylproteomic analysis. It is expected to facilitate much more comprehensive and accurate quantification of S-acylproteomes than previous ABE methods.

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Zhou, B., Wang, Y., Yan, Y., Mariscal, J., Di Vizio, D., Freeman, M. R., Yang, W.. 2019-03-26. Low-background Acyl-biotinyl Exchange Largely Eliminates the Co-isolation of Non-S-acylated Proteins and Enables Deep S-acylproteomic Analysis. https://doi.org/10.1101/588988

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