Increased Depth and Breadth of Plasma Protein Quantitation via Two-Dimensional Liquid Chromatography/Multiple Reaction Monitoring-Mass Spectrometry with Labeled Peptide Standards
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Absolute quantitative strategies are emerging as a powerful and preferable means of deriving concentrations in biological samples for systems biology applications. Method development is driven by the need to establish new—and validate current—protein biomarkers of high-to-low abundance for clinical utility. In this chapter, we describe a methodology involving two-dimensional (2D) reversed-phase liquid chromatography (RPLC), operated under alkaline and acidic pH conditions, combined with multiple reaction monitoring (MRM)-mass spectrometry (MS) (also called selected reaction monitoring (SRM)-MS) and a complex mixture of stable isotope-labeled standard (SIS) peptides, to quantify a broad and diverse panel of 253 proteins in human blood plasma. The quantitation range spans 8 orders of magnitude—from 15 mg/mL (for vitamin D-binding protein) to 450 pg/mL (for protein S100-B)—and includes 31 low-abundance proteins (defined as being <10 ng/mL) of potential disease relevance. The method is designed to assess candidates at the discovery and/or verification phases of the biomarker pipeline and can be adapted to examine smaller or alternate panels of proteins for higher sample throughput. Also detailed here is the application of our recently developed software tool—Qualis-SIS—for protein quantitation (via regression analysis of standard curves) and quality assessment of the resulting data. Overall, this chapter provides the blueprint for the replication of this quantitative proteomic method by proteomic scientists of all skill levels.
Key wordsMultidimensional liquid chromatography Multiple reaction monitoring Plasma Protein Proteomics Quantitation Sensitive Stable isotope-labeled standard
We wish to thank Genome Canada and Genome BC for STIC (Science and Technology Innovation Centre) funding and support. Carol Parker (UVic-Genome BC Proteomics Centre) is acknowledged for assisting in the manuscript editing process.
Competing Interests: Christoph Borchers is the director of the Centre and the Chief Scientific Officer of MRM Proteomics, which has commercialized the performance kits noted above for system/platform assessment.