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-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.
Highlights
Diagnostic development and public health surveillance require technologies that provide specific identification and absolute quantification of protein biomarkers
Selection of Staphylococcal Superantigenic Toxin Marker Peptides—SEA and TSST-1 recombinant staphylococcal toxins were submitted to SDS-PAGE and in-gel digestion with trypsin
Quantification of Staphylococcal Superantigenic Toxins in Drinking Water Using Isotope-labeled AQUA Peptides—Commercial SEA and TSST-1 staphylococcal toxins were added in defined amounts into drinking water
Summary
Diagnostic development and public health surveillance require technologies that provide specific identification and absolute quantification of protein biomarkers. Staphylococcal toxic shock syndrome is related to the ability of enterotoxins and TSST-1 to polyclonally and extensively activate T cells at picomolar concentrations, a property referred to as superantigenicity. Besides their superantigenic activity, staphylococcal enterotoxins display specific emetic properties and constitute a major cause of food poisoning [15]. Isotope-labeled Proteins for Absolute Quantitative Proteomics enterotoxin B (SEB) as a potential warfare contaminant of food and water supplies. No assay is referenced for the identification and quantification of these toxins in accidentally or deliberately contaminated food or water supplies In this context, MS, which circumvents the need for antibodies, offers great potential for staphylococcal superantigenic toxin-specific detection and quantification. Comparisons with the current state-of-the-art methodologies demonstrate the great potential of isotope-labeled full-length proteins as standards for MS-based absolute quantification
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