Isotope-labeled Protein Standards

Abstract

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-la- beled marker peptides for MS-based absolute quantifica- tion 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 pro- teins as standards for absolute quantification. As those protein standards perfectly match the biochemical prop- erties of the target proteins, they can be directly added into the samples to be analyzed, allowing a highly accu- rate quantification of proteins even in prefractionated complex samples. The power of our Protein Standard Absolute Quantification methodology for accurate abso- lute quantification of biomarkers was demonstrated both on water and urine samples contaminated with Staphylo- coccus aureus superantigenic toxins as typical biomark- ers of public health interest.