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TMT Sample Preparation for Proteomics Facility Submission and Subsequent Data Analysis
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Multiplexed Data-Independent Acquisition-Based Proteomics Enabled by TMTpro Complementary Ions.

Zicong Wang1, Peng-Kai Liu2, Haiyan Lu1

  • 1School of Pharmacy, University of Wisconsin─Madison, Madison, Wisconsin 53705, United States.

Analytical Chemistry
|November 26, 2025
PubMed
Summary
This summary is machine-generated.

We developed a new 3-plex TMTpro complementary ion (TMTproC)-based DIA strategy for accurate quantitative proteomics. This method overcomes ratio distortion in data-independent acquisition (DIA) and enhances proteome coverage.

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Area of Science:

  • Proteomics
  • Analytical Chemistry
  • Biochemistry

Background:

  • Data-independent acquisition (DIA) offers superior proteome coverage and reproducibility compared to data-dependent acquisition (DDA).
  • Multiplexed isobaric labeling in DIA is hindered by ratio distortion from coisolation and cofragmentation interference.

Purpose of the Study:

  • To introduce a novel 3-plex TMTpro complementary ion (TMTproC)-based DIA strategy.
  • To achieve accurate quantification in DIA without increasing spectral complexity by utilizing complementary ions.

Main Methods:

  • Implemented TMTproC labeling with 4-Da spacing between complementary ions to minimize spectral overlap.
  • Optimized higher-energy collisional dissociation (HCD) settings for complementary ion generation.
  • Validated the method using tryptic bovine serum albumin (BSA) peptides and yeast proteome samples.

Main Results:

  • Achieved median peptide-level ratios within 10% of expected values for BSA peptides.
  • Demonstrated median coefficients of variation (CVs) below 4% across triplicates.
  • Showcased high quantification precision and minimal ratio distortion across a 10-fold dynamic range in yeast proteome analysis.

Conclusions:

  • TMTproC-DIA is a robust and versatile strategy for accurate quantitative proteomics.
  • This method effectively reduces ratio distortion and enhances proteome coverage in DIA.
  • The TMTproC-DIA approach is scalable for high-throughput proteomic studies.