Mapping sites of aspirin-induced acetylations in live cells by quantitative acid-cleavable activity-based protein profiling (QA-ABPP)

Abstract

Target-identification and understanding of mechanism-of-action (MOA) are challenging for development of small-molecule probes and their application in biology and drug discovery. For example, although aspirin has been widely used for more than 100 years, its molecular targets have not been fully characterized. To cope with this challenge, we developed a novel technique called quantitative acid-cleavable activity-based protein profiling (QA-ABPP) with combination of the following two parts: (i) activity-based protein profiling (ABPP) and iTRAQ™ quantitative proteomics for identification of target proteins and (ii) acid-cleavable linker-based ABPP for identification of peptides with specific binding sites. It is known that reaction of aspirin with its target proteins leads to acetylation. We thus applied the above technique using aspirin-based probes in human cancer HCT116 cells. We identified 1110 target proteins and 2775 peptides with exact acetylation sites. By correlating these two sets of data, 523 proteins were identified as targets of aspirin. We used various biological assays to validate the effects of aspirin on inhibition of protein synthesis and induction of autophagy which were elicited from the pathway analysis of Aspirin target profile. This technique is widely applicable for target identification in the field of drug discovery and biology, especially for the covalent drugs.

Cyclin A2 and CDK2 as Novel Targets of Aspirin and Salicylic Acid: A Potential Role in Cancer Prevention

“…Aspirin contains acetyl and salicylate groups both of which have their own targets, and are believed to contribute to its chemopreventive actions. Studies from our laboratory [12-14, 17], and others [15, 16] showed that, besides COX, it can acetylate numerous other proteins. While the identification of the aspirin-mediated acetylation targets has recently gained momentum [15, 16] after our initial reports [12] [14], identification of direct binding targets for salicylic acid has been under-explored [13].…”

Quantitative chemical proteomics profiling of de novo protein synthesis during starvation-mediated autophagy

“…An iTRAQ quantitative proteomics approach was used in tandem with BONCAT to differentiate any potential background contamination from selective enrichment, 29,30 to enhance the reliability of the results obtained. The background contamination includes the pre-existing proteins nonspecifically binding to the beads, as well as the endogenously biotinylated proteins, since streptavidin beads were used for the protein enrichment.…”

“…The iTRAQ labeling technique 29,30,48 was employed to investigate the changes in the proteomic profiles upon autophagy induction, per the manufacturer's instructions with minor modifications. Briefly, the dried digested peptides were resuspended in equal amounts of dissolution buffer (0.5 M TEAB; Sigma-Aldrich, T7408).…”

“…MS/MS spectra were measured in high sensitivity mode, with rolling collision energy and iTRAQ reagent collision adjustments accounted for. 30,49 Peptide identification and quantification Peptide quantification and identification were performed using ProteinPilot TM Software 4.5 (SCIEX) with the Paragon algorithm (4.5.0.0, 1654). Each MS/MS spectrum was searched against SwissProt database (v2015.9) with a total of 40,406 entries.…”

“…We have previously utilized iTRAQ to differentiate the specific binding proteins from the nonspecific ones during affinity enrichment. 29,30 In the current study, via the above approach, we identified and quantified a total of 1176 proteins and among them 711 proteins were found to meet our defined criteria as de novo synthesized proteins during starvation-mediated autophagy. The functions of these proteins were deduced based on subsequent bioinformatics and pathway analysis and the trends of alterations in them (promoting or inhibiting) were predicted.…”