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Realised by ALMS™
developer of the AIDS-HIV Reference project
Abstract No.: ThP-248
Session: Proteomics: Quantification
Presentation date: Thu, Aug 31, 2006
Presentation time: 14:30 – 16:00

The Feasibility of Accurate Protein Quantification by Isotope Dilution Mass Spectrometry of Proteolytic Peptides

Gavin O'Connor1, William Burkitt1, Cristian Arsene2, Andre Henrion2, David Bunk3

1 LGC, Teddington, United Kingdom
2 Physikalisch Technische Bundesanstalt, Braunschweig, Germany
3 National Institute of Standards and Technology, Gaithersburg, United States

Correspondence address: Gavin O'Connor, LGC, Bio-Organic Mass Spectrometry, Queens Road, Teddington, TW110LY United Kingdom.

Keywords: Peptide; Protein; Quantitative Analysis; Reference Compound/Material.

Novel aspect: Absolute protein quantification for the provision of protein standards.


One of the many roles of a national measurement institute (NMI) is in the provision of traceable measurements for the value assignment of reference materials. Isotope dilution mass spectrometry (IDMS) has been the method of choice for determining the amount of substance, in matrix and primary reference materials. The approach has been well established for the determination of small clinical biomarkers, such as cholesterol, steroid hormones, glucose and creatinine, in a wide variety of biological matrices. However, its application to macromolecules such as proteins and DNA has proven more difficult. The method is reliant on the availability of isotopically labeled analogues, of sufficient isotopic and chemical purity, where the enriched isotope and degree of labeling can be critical. Also, a well-characterized primary standard is required. Arguably such an approach would offer the best chances of traceable quantitative measurement in the long term. However, the production of such standards would be prohibitively time consuming and costly.

One approach that is increasingly being used for the quantification of protein concentrations is the use of isotopically labeled peptides combined with mass spectrometry of the enzymatically digested proteins. The primary application of tryptic digestion has been the qualitative identification of proteins in complex mixtures rather than absolute quantification. Completeness of proteolysis is not necessary in this qualitative context. However, for absolute protein quantification, completeness of the tryptic digestion is required. An approach to protein quantification, using complete enzymatic digestion of the protein mixtures with trypsin and double IDMS of the released peptide will be demonstrated.

A set of model proteins, alone and in mixtures, were used to evaluate the conditions necessary for quantitative proteolytic protein fragmentation, enabling accurate quantification of proteins. The evaluated conditions for the tryptic digestion of the proteins into peptides and examples of the absolute quantification of proteins in complex mixtures will be presented and discussed. Analysis of the digested protein solutions was accomplished using liquid chromatography and a number of mass spectrometers. These included a 4.7 T Bruker BioApex III FTICR mass spectrometer, a Micromass Ultima Triple Quadrupole mass spectrometer and an Applied Biosystems QTRAP mass spectrometer. Complete tryptic digestion of the protein mixture with minimal covalent modifications and non-specific cleavages was achieved. This allowed three iterations of double IDMS of the peptide to be undertaken. The values for the protein concentrations that were calculated using this approach were found to be reproducible.