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Proteomics

Proteomics

A proteome is the entire set of proteins that a cell type produces. We can study proteomes using the knowledge of genomes because genes code for mRNAs, and the mRNAs encode proteins. Although mRNA analysis is a step in the right direction, not all mRNAs are translated into proteins.
Proteomics is the study of proteomes' function. It involves the large-scale systematic study of the proteome to denote the protein complement expressed by a genome. Scientist Mark Wilkins coined the term...

Peptide Identification Using Tandem Mass Spectrometry

Peptide Identification Using Tandem Mass Spectrometry

Tandem mass spectrometry, also known as MS/MS or MS2, is an analytical technique that employs two mass analyzers. Essentially it is a series of mass spectrometers that helps isolate a particular biomolecule and then helps study its chemical properties.
This technique helps gather information regarding the protein from which the peptide was obtained and to study the peptides’ amino acid sequence. Identifying peptides from a complex mixture is an important component of the growing field of...

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Related Experiment Video

Updated: Jul 1, 2025

Deep Proteome Profiling by Isobaric Labeling, Extensive Liquid Chromatography, Mass Spectrometry, and Software-assisted Quantification

Deep Proteome Profiling by Isobaric Labeling, Extensive Liquid Chromatography, Mass Spectrometry, and Software-assisted Quantification

Published on: November 15, 2017

Accelerating Proteomics Using Broad Specificity Proteases.

Xuehui Jiang¹, Darien Yeung^1,2, Yang Liu³

¹Manitoba Centre for Proteomics and Systems Biology, Health Science Centre, Winnipeg, Manitoba R3E 3P4, Canada.

Journal of Proteome Research

|March 8, 2024

Summary

This summary is machine-generated.

Broad-specificity proteases like subtilisin, proteinase K, and thermolysin offer fast, cost-effective, and comprehensive protein digestion for proteomics. These enzymes unlock previously inaccessible proteome regions, enhancing protein identification and quantification.

Keywords:

PTM bottom-up proteomics chymotrypsin elastin high throughput nonspecific pepsin phosphorylation proteases unspecific

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Published on: August 6, 2018

Area of Science:

Proteomics
Biochemistry
Mass Spectrometry

Background:

Trypsin is the standard protease for bottom-up proteomics but has limitations in accessing certain protein regions.
Multienzyme strategies are employed to increase sequence coverage, especially for post-translational modification profiling.

Purpose of the Study:

To develop and validate fast, cost-effective protocols using broad-specificity proteases (subtilisin, proteinase K, thermolysin) for enhanced proteome coverage.
To assess the utility of these proteases in protein identification, sequence coverage, and quantitative proteomics.

Main Methods:

Development of SP3- and STRAP-based protocols for subtilisin, proteinase K, and thermolysin.
Analysis of Jurkat cell digests using FragPipe software for rapid data processing.
In-depth proteomic analysis including protein identification, sequence coverage assessment, and label-free quantification.

Main Results:

Subtilisin, proteinase K, and thermolysin achieved near-complete protein digests in 1-5 minutes, at significantly lower costs than trypsin.
These proteases identified thousands of unique proteins with substantial sequence coverage, including previously unreported amino acid sequences.
Machine learning could differentiate true protease products from random cleavages, suggesting potential for predictive modeling.
Label-free quantification comparable to trypsin was achieved, demonstrating their utility in quantitative proteomics.

Conclusions:

Broad-specificity proteases provide a powerful and economical alternative to trypsin for deep and quantitative proteomic analyses.
Their rapid digestion kinetics open possibilities for 'on-the-fly' sample processing in future proteomics workflows.
These enzymes significantly expand the accessible regions of the proteome for comprehensive profiling.