Protocol for Standardizing High-to-Moderate Abundance Protein Biomarker Assessments Through an MRM-with-Standard-Peptides Quantitative Approach

Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 919)


Quantitative mass spectrometry (MS)-based approaches are emerging as a core technology for addressing health-related queries in systems biology and in the biomedical and clinical fields. In several ‘omics disciplines (proteomics included), an approach centered on selected or multiple reaction monitoring (SRM or MRM)-MS with stable isotope-labeled standards (SIS), at the protein or peptide level, has emerged as the most precise technique for quantifying and screening putative analytes in biological samples. To enable the widespread use of MRM-based protein quantitation for disease biomarker assessment studies and its ultimate acceptance for clinical analysis, the technique must be standardized to facilitate precise and accurate protein quantitation. To that end, we have developed a number of kits for assessing method/platform performance, as well as for screening proposed candidate protein biomarkers in various human biofluids. Collectively, these kits utilize a bottom-up LC-MS methodology with SIS peptides as internal standards and quantify proteins using regression analysis of standard curves. This chapter details the methodology used to quantify 192 plasma proteins of high-to-moderate abundance (covers a 6 order of magnitude range from 31 mg/mL for albumin to 18 ng/mL for peroxidredoxin-2), and a 21-protein subset thereof. We also describe the application of this method to patient samples for biomarker discovery and verification studies. Additionally, we introduce our recently developed Qualis-SIS software, which is used to expedite the analysis and assessment of protein quantitation data in control and patient samples.


Biomarker Internal standards MRM Plasma Proteomics Quantitation Standardization 



We wish to thank Genome Canada 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

CHB is the director of the Centre and the Chief Scientific Officer of MRM Proteomics, which has commercialized the performance kits (namely the PeptiQuant LC-MS Platform and PeptiQuant MRM/MS Workflow kits) and the assessment kits (PeptiQuant Human Discovery Assay kit, or BAK-192, and BAK-21) described here.


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Copyright information

© Springer International Publishing Switzerland 2016

Authors and Affiliations

  1. 1.University of Victoria – Genome British Columbia Proteomics CentreVictoriaCanada
  2. 2.Center for Proteomics and MetabolomicsLeiden University Medical CenterLeidenNetherlands
  3. 3.AstraZeneca R&D, Innovative MedicinesMölndalSweden
  4. 4.Department of Biochemistry and MicrobiologyUniversity of VictoriaVictoriaCanada

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