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

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Modern Proteomics – Sample Preparation, Analysis and Practical Applications

Part of the book series: Advances in Experimental Medicine and Biology ((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.

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Acknowledgements

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.

Author information

Authors and Affiliations

  1. University of Victoria – Genome British Columbia Proteomics Centre, Vancouver Island Technology Park, #3101 – 4464 Markham St., Victoria, BC, V8Z 7X8, Canada

    Andrew J. Percy, Juncong Yang, Andrew G. Chambers, Yassene Mohammed & Christoph H. Borchers

  2. Center for Proteomics and Metabolomics, Leiden University Medical Center, 2333 ZA, Leiden, Netherlands

    Yassene Mohammed

  3. AstraZeneca R&D, Innovative Medicines, S-431 83, Mölndal, Sweden

    Tasso Miliotis

  4. Department of Biochemistry and Microbiology, University of Victoria, Petch Building Room 207, 3800 Finnerty Rd., Victoria, BC, V8P 5C2, Canada

    Christoph H. Borchers

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  1. Andrew J. Percy
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  2. Juncong Yang
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  3. Andrew G. Chambers
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  4. Yassene Mohammed
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  5. Tasso Miliotis
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  6. Christoph H. Borchers
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Corresponding author

Correspondence to Christoph H. Borchers .

Editor information

Editors and Affiliations

  1. UT Southwestern Medical Center, Dallas, Texas, USA

    Hamid Mirzaei

  2. Biotech Division, Neurophagy Therapeutics, INC, Odessa, Texas, USA

    Martin Carrasco

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Percy, A.J., Yang, J., Chambers, A.G., Mohammed, Y., Miliotis, T., Borchers, C.H. (2016). Protocol for Standardizing High-to-Moderate Abundance Protein Biomarker Assessments Through an MRM-with-Standard-Peptides Quantitative Approach. In: Mirzaei, H., Carrasco, M. (eds) Modern Proteomics – Sample Preparation, Analysis and Practical Applications. Advances in Experimental Medicine and Biology, vol 919. Springer, Cham. https://doi.org/10.1007/978-3-319-41448-5_24

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