Absolute Quantification of Proteins by Mass Spectrometry
Peptides and Proteins
Published Online: 18 SEP 2013
Copyright © 2000 John Wiley & Sons, Ltd. All rights reserved.
Encyclopedia of Analytical Chemistry
How to Cite
Shuford, C. M. and Muddiman, D. C. 2013. Absolute Quantification of Proteins by Mass Spectrometry. Encyclopedia of Analytical Chemistry. 1–26.
- Published Online: 18 SEP 2013
Mass spectrometry (MS) provides unambiguous molecular specificity relative to all other analytical and immunological techniques and, consequently, has great potential to provide unambiguous estimates of absolute protein quantities within complex biological systems. Here, we provide a critical review and discussion of the predominant MS-based technology used for absolute protein quantification, referred to as protein cleavage-isotope dilution mass spectrometry (PC-IDMS). This technique requires proteolytic conversion of the analyte protein into its quantitative surrogate peptide, which is subsequently quantified using a stable isotope-labeled (SIL) peptide analog as an internal standard (IS). All phases of the PC-IDMS workflow, from the SIL peptide production platforms to data analysis, are described and characterized in detail. An emphasis is placed on the practical quantitative aspects for each step of the workflow and their implication on quantitative accuracy.