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Detection of Protein Ubiquitination Sites by Peptide Enrichment and Mass Spectrometry

Detection of Protein Ubiquitination Sites by Peptide Enrichment and Mass Spectrometry

Published on: March 23, 2020

An extra dimension in protein tagging by quantifying universal proteotypic peptides using targeted proteomics.

Giel Vandemoortele^1,2, An Staes^1,2, Giulia Gonnelli^1,2

¹VIB Medical Biotechnology Center, B-9000 Ghent, Belgium.

Scientific Reports

|June 7, 2016

Summary

This study introduces novel proteotypic peptides for quantifying tagged proteins using targeted proteomics. These peptides enable sensitive and accurate analysis of endogenous proteins, advancing cell biology research.

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Area of Science:

Proteomics
Molecular Biology
Biochemistry

Background:

Protein tagging revolutionized cell biology and biochemical analysis by enabling efficient recovery of protein complexes.
Endogenous protein tagging is common in lower organisms but now feasible in mammalian systems due to genome engineering advances.
Targeted proteomics requires peptides with ideal mass spectrometry characteristics for accurate protein quantification.

Purpose of the Study:

To select peptides with ideal mass spectrometry characteristics for quantifying tagged proteins.
To develop a method for sensitive quantification of endogenous proteins using novel peptides.
To demonstrate the utility of these peptides in analyzing protein complexes via co-immunoprecipitation.

Main Methods:

Mining the proteome of Pyrococcus furiosus to identify unique proteotypic peptides.
Developing 'Proteotypic peptides for Quantification by SRM' (PQS peptides).
Combining PQS peptides with epitope tags for co-immunoprecipitation experiments in transfected and genome-engineered systems.

Main Results:

Identified two unique proteotypic peptides from Pyrococcus furiosus.
Demonstrated sensitive quantification of tagged proteins in complex backgrounds using PQS peptides.
Successfully applied PQS peptides with epitope tags in co-immunoprecipitation for protein pair analysis.

Conclusions:

Endogenous protein tagging combined with PQS peptides enables absolute quantification.
This approach provides a powerful tool for detailed characterization of endogenous proteins.
Advances in genome engineering facilitate widespread application of endogenous protein tagging.