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Spike-In Proteome Enhances Data-Independent Acquisition for Thermal Proteome Profiling.

Qiqi Wang1,2, Qiufen Chen1,2, Yue Lin1,2

  • 1Department of Chemistry and Research Center for Chemical Biology and Omics Analysis, College of Science, Southern University of Science and Technology, Shenzhen, Guangdong 518055, P.R. China.

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|December 2, 2024
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A new spike-in proteome strategy enhances data-independent acquisition with thermal proteome profiling (DIA-TPP), improving drug-target identification. This method overcomes limitations of label-free approaches, offering better protein coverage and precision for drug discovery.

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Area of Science:

  • Proteomics
  • Chemical Biology
  • Drug Discovery

Background:

  • Target deconvolution is crucial for understanding small-molecule drugs, including their efficacy and toxicity.
  • Thermal proteome profiling (TPP) is a key method for identifying drug-protein interactions.
  • Traditional TPP methods using isobaric labeling are laborious and expensive, while label-free approaches compromise data quality.

Purpose of the Study:

  • To develop an improved label-free method for TPP that enhances protein coverage and quantification precision.
  • To address the limitations of existing label-free data-independent acquisition (DIA) TPP methods.
  • To enable more efficient and accurate identification of drug-protein interactions.

Main Methods:

  • A novel spike-in proteome strategy was developed for DIA-TPP to compensate for protein loss during heating.
  • A calibration algorithm was created to correct for potential biases introduced by the spike-in proteome.
  • The enhanced DIA-TPP method was integrated with the matrix-augmented pooling strategy (MAPS) for increased throughput.

Main Results:

  • The spike-in proteome strategy significantly improved protein coverage, data completeness, and quantification precision in DIA-TPP.
  • The developed calibration algorithm effectively corrected for spike-in effects on fold-change measurements.
  • The integrated DIA-TPP-MAPS approach demonstrated performance comparable to established TMT-TPP-MAPS methods.
  • The method successfully identified specific drug-target interactions, including dorzolamide hydrochloride with CA13 and opicapone with GSTZ1 and tyrosyl-DNA phosphodiesterase 1, which were missed by conventional methods.

Conclusions:

  • The spike-in proteome strategy represents a significant advancement for DIA-TPP, enhancing its utility in drug discovery.
  • This improved method provides a more robust, precise, and efficient approach for identifying drug-protein interactions.
  • The strategy enables the detection of previously elusive drug targets, facilitating a deeper understanding of drug mechanisms and potential applications.