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Molecular & Cellular Proteomics 6:2139-2149, 2007.
© 2007 by The American Society for Biochemistry and Molecular Biology, Inc.

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Research

Isotope-labeled Protein Standards

Toward Absolute Quantitative Proteomics^*,S

Virginie Brun^,,¶,, Alain Dupuis^,,¶, Annie Adrait^,,¶, Marlène Marcellin^,,¶, Damien Thomas^||,**, Magali Court^,,¶, François Vandenesch^||,** and Jérôme Garin^,,¶

From the Commissariat à l’Energie Atomique, DSV, iRTSV, Laboratoire d’Etude de la Dynamique des Protéomes, Grenoble, F-38054, France, INSERM, U880, Grenoble, F-38054, France, ^¶ Université Joseph Fourier, Grenoble, F-38054, France, ^|| INSERM, U851, Immunité, Infection, Vaccination, Lyon, F-69008, France, and ^** Faculté de Médecine Laennec, Université de Lyon, Lyon, F-69008, France

Diagnostic development and public health surveillance require technologies that provide specific identification and absolute quantification of protein biomarkers. Beside immunologically related techniques (e.g. enzyme-linked immunosorbent assay), MS is gaining increasing interest due to its high sensitivity and specificity. Furthermore, MS-based analyses are extremely accurate quantitatively, provided that suitable reference standards are available. Recently, the use of chemically synthesized isotope-labeled marker peptides for MS-based absolute quantification of proteins has led to major advances. However, we show here that the use of such peptides can lead to severe biases. In this work, we present an innovative strategy (Protein Standard Absolute Quantification) that uses in vitro-synthesized isotope-labeled full-length proteins as standards for absolute quantification. As those protein standards perfectly match the biochemical properties of the target proteins, they can be directly added into the samples to be analyzed, allowing a highly accurate quantification of proteins even in prefractionated complex samples. The power of our Protein Standard Absolute Quantification methodology for accurate absolute quantification of biomarkers was demonstrated both on water and urine samples contaminated with Staphylococcus aureus superantigenic toxins as typical biomarkers of public health interest.

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