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bioRxiv · 10.1101/2023.09.13.557589

Increasing the Throughput and Reproducibility of Activity-Based Proteome Profiling Studies with Hyperplexing and Intelligent Data Acquisition

Budayeva, H. G.·Ma, T. P.·Wang, S.·Choi, M.·Rose, C. M.
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Abstract

Intelligent data acquisition (IDA) strategies, such as real-time database search (RTS), have improved the depth of proteome coverage for experiments that utilize isobaric labels and gas phase purification techniques (i.e., SPS-MS3). While most applications of IDA have been focused on the analysis of protein abundance, these approaches have recently been applied to activity-based proteome profiling (ABPP) studies aimed at characterization of protein site engagement by small molecules. In this work, we extend IDA capabilities offered by vendor software through a program called InSeqAPI. First, we demonstrate robust performance of InSeqAPI in the analysis of biotinylated cysteine peptides from ABPP experiments. Then, we describe PairQuant, a method within InSeqAPI designed for the hyperplexing approach that utilizes protein-level isotopic labeling and peptide-level TMT labeling. PairQuant allows for TMT analysis of 36 conditions in a single sample and achieves [~]98% coverage of both peptide pair partners in a hyperplexed experiment as well as a 40% improvement in the number of quantified cysteine sites compared to non-RTS acquisition. We applied this method in ABPP study of ligandable cysteine sites in the nucleus leading to an identification of additional druggable sites on protein-and DNA-interaction domains of transcription regulators and on nuclear ubiquitin ligases.

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Budayeva, H. G., Ma, T. P., Wang, S., Choi, M., Rose, C. M.. 2023-09-13. Increasing the Throughput and Reproducibility of Activity-Based Proteome Profiling Studies with Hyperplexing and Intelligent Data Acquisition. https://doi.org/10.1101/2023.09.13.557589

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