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Related Experiment Video

Updated: Jul 8, 2025

Selected Reaction Monitoring Mass Spectrometry for Absolute Protein Quantification
09:04

Selected Reaction Monitoring Mass Spectrometry for Absolute Protein Quantification

Published on: August 17, 2015

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Cheaper, faster, simpler trypsin digestion for high-throughput targeted protein quantification.

Christopher M Shuford1, Russell P Grant1

  • 1Labcorp, Center for Esoteric Testing, Burlington, NC, USA.

Journal of Mass Spectrometry and Advances in the Clinical Lab
|December 14, 2023
PubMed
Summary

Optimizing trypsin digestion speeds up protein quantification for clinical use. Standard tryptic digestion with optimized additives and concentration offers a fast, cost-effective solution for serum biomarker analysis.

Keywords:
Bottom-up proteomicsLiquid chromatographyMass spectrometryProtein quantificationTrypsin

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Area of Science:

  • Biochemistry
  • Analytical Chemistry

Background:

  • Liquid chromatography-mass spectrometry (LC-MS) protein quantification is often perceived as costly and slow.
  • Trypsin digestion is a key bottleneck, particularly for clinical biomarker quantification requiring efficient surrogate peptide recovery.

Purpose of the Study:

  • To enhance tryptic digestion efficiency for analyzing serum protein biomarkers like thyroglobulin.
  • To identify methods for improving the yield and speed of surrogate peptide formation.

Main Methods:

  • Evaluated various methodologies including different additives, trypsin types, microwave/pressure-assisted systems, and enzyme concentrations.
  • Assessed the impact of these variables on digestion yield and speed for surrogate peptides.
  • Optimized digestion time and additive use.

Main Results:

  • Digestion additives/denaturants significantly impacted surrogate peptide yield and speed.
  • Increased trypsin concentration accelerated digestion without compromising yield.
  • Standard-grade trypsin with optimized additives and conventional incubation proved effective, negating advantages of advanced systems.

Conclusions:

  • Trypsin digestion for targeted serum protein quantification can be fast and cost-effective.
  • Established a framework for developing efficient trypsin digestion protocols.
  • Aims to facilitate wider adoption of LC-MS assays in clinical settings.