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

  • Andrew J. Percy
  • Juncong Yang
  • Andrew G. Chambers
  • Yassene Mohammed
  • Tasso Miliotis
  • Christoph H. Borchers
Chapter
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 919)

Abstract

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.

Keywords

Biomarker Internal standards MRM Plasma Proteomics Quantitation Standardization 

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Copyright information

© Springer International Publishing Switzerland 2016

Authors and Affiliations

  • Andrew J. Percy
    • 1
  • Juncong Yang
    • 1
  • Andrew G. Chambers
    • 1
  • Yassene Mohammed
    • 1
    • 2
  • Tasso Miliotis
    • 3
  • Christoph H. Borchers
    • 1
    • 4
  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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