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Updated: Nov 12, 2025

Deep Proteome Profiling by Isobaric Labeling, Extensive Liquid Chromatography, Mass Spectrometry, and Software-assisted Quantification
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Quantitative Top-Down Proteomics in Complex Samples Using Protein-Level Tandem Mass Tag Labeling.

Dahang Yu1, Zhe Wang1, Kellye A Cupp-Sutton1

  • 1Department of Chemistry and Biochemistry, University of Oklahoma, 101 Stephenson Parkway, Norman, Oklahoma 73019, United States.

Journal of the American Society for Mass Spectrometry
|March 16, 2021
PubMed
Summary
This summary is machine-generated.

This study introduces a novel top-down TMT MS platform for quantifying intact proteoforms in complex samples. A new filter-SEC technique prevents protein precipitation, enabling high-throughput analysis of low molecular weight proteoforms.

Keywords:
isobaric labelingliquid chromatographymass spectrometryquantitative proteomicstandem mass tags (TMT)top-down proteomics

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Area of Science:

  • Proteomics
  • Mass Spectrometry
  • Biochemistry

Background:

  • Isobaric chemical tags like tandem mass tag (TMT) are vital for quantitative proteomics.
  • Previous top-down proteomics applications were limited by protein precipitation during TMT labeling of complex samples.

Purpose of the Study:

  • To develop a robust top-down TMT MS platform for identifying and quantifying low molecular weight intact proteoforms in complex biological samples.
  • To overcome challenges associated with protein precipitation in top-down TMT workflows.

Main Methods:

  • Developed a filter-SEC technique combining molecular weight cutoff filtration and size exclusion chromatography (SEC) to reduce sample complexity and remove large proteins.
  • Applied intact protein-level TMT labeling to filtered samples.
  • Utilized a top-down MS platform for proteoform analysis.

Main Results:

  • Successfully identified and quantified hundreds of intact proteoforms from *Escherichia coli* cell lysates.
  • Observed no protein precipitation under the developed intact protein-level TMT labeling conditions.
  • Demonstrated a high-throughput capability for top-down proteoform analysis.

Conclusions:

  • The developed filter-SEC technique effectively prevents protein precipitation in top-down TMT workflows.
  • This novel platform enables high-throughput, quantitative, top-down MS analysis of intact proteoforms in complex biological samples.
  • Represents a significant advancement for top-down quantitative proteomics.