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

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Published on: August 17, 2015

Recombinant isotope labeled and selenium quantified proteins for absolute protein quantification.

Nico Zinn1, Dominic Winter, Wolf D Lehmann

  • 1Molecular Structure Analysis, German Cancer Research Center, Im Neuenheimer Feld 280, Heidelberg, Germany.

Analytical Chemistry
|February 19, 2010
PubMed
Summary

A new method produces precisely quantified recombinant proteins, called RISQ (recombinant isotope labeled and selenium quantified) proteins, for mass spectrometry. These labeled proteins serve as reliable internal standards in proteomic quantification and analysis of protein modifications.

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Area of Science:

  • Proteomics
  • Analytical Chemistry
  • Biotechnology

Background:

  • Quantitative proteomics relies on accurate internal standards for mass spectrometry-based analysis.
  • Current methods for producing protein standards can be limited by post-translational modifications and labeling complexity.

Purpose of the Study:

  • To develop a novel method for producing absolutely quantified proteins as internal standards for mass spectrometry.
  • To create recombinant isotope-labeled and selenium-quantified (RISQ) proteins for enhanced proteomic studies.

Main Methods:

  • Utilized gateway technology for efficient assembly of expression vectors for cell-free protein synthesis.
  • Expressed proteins in a medium with selenomethionine and stable isotope-labeled amino acids (e.g., lysine, arginine).
  • Quantified proteins using element mass spectrometry (selenium) and mass spectrometry (isotope labels) for absolute quantification.

Main Results:

  • Successfully synthesized Apolipoprotein A1 as a RISQ protein standard.
  • Demonstrated the utility of RISQ proteins as internal standards, achieving accurate quantification of a reference material.
  • Confirmed that cell-free expression yields unmodified proteins, suitable for analyzing specific peptide regions.

Conclusions:

  • RISQ proteins offer a robust and versatile solution for absolute protein quantification in mass spectrometry.
  • These standards are valuable for accurate proteomic analysis, particularly for unmodified peptides.
  • RISQ proteins represent a new tool for the quantitative analysis of covalent protein modifications.