Standardized Protocols for Quality Control of MRM-based Plasma Proteomic Workflows

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Abstract

Mass spectrometry (MS)-based proteomics is rapidly emerging as a viable technology for the identification and quantitation of biological samples, such as human plasma--the most complex yet commonly employed biofluid in clinical analyses. The transition from a qualitative to quantitative science is required if proteomics is going to successfully make the transition to a clinically useful technique. MS, however, has been criticized for a lack of reproducibility and interlaboratory transferability. Currently, the MS and plasma proteomics communities lack standardized protocols and reagents to ensure that high-quality quantitative data can be accurately and precisely reproduced by laboratories across the world using different MS technologies. Toward addressing this issue, we have developed standard protocols for multiple reaction monitoring (MRM)-based assays with customized isotopically labeled internal standards for quality control of the sample preparation workflow and the MS platform in quantitative plasma proteomic analyses. The development of reference standards and their application to a single MS platform is discussed herein, along with the results from intralaboratory tests. The tests highlighted the importance of the reference standards in assessing the efficiency and reproducibility of the entire bottom-up proteomic workflow and revealed errors related to the sample preparation and performance quality and deficits of the MS and LC systems. Such evaluations are necessary if MRM-based quantitative plasma proteomics is to be used in verifying and validating putative disease biomarkers across different research laboratories and eventually in clinical laboratories.

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