With increased attention toward the quantitation of proteins in biological samples, targeted methodologies such as stable isotope dilution (SID) and multiple reaction monitoring (MRM) mass spectrometry (MS) have shown increased frequency of use in both the academic and clinical settings. However, much progress must still be made to minimize the sources of variation in these experiments before protein-based SID-MRM-MS assays become standard in clinical analyses. The National Cancer Institute has initiated the Clinical Proteomics Technology Assessment for Cancer (CPTAC) consortium, specifically the Experimental Design and Statistics Working Group, to shed light on the variable aspects of protein biomarker verification using SID-MRM-MS.
The reproducibility and transferability of SID-MRM-MS-based assays for candidate biomarker quantification in plasma was assessed by eight separate laboratories using two different triple quadrupole instrument platforms. Ten peptides representing seven proteins were selected for monitoring. The experiment was designed in three parts to incrementally introduce sources of variability related to protein digestion and sample handling. For the most basic of the three studies, the median inter-laboratory CVs near the limit of quantitation (LOQ) ranged from 4-15% across the eights sites, with eight out of ten peptides having inter-laboratory CVs of ≤10%. Median intra-laboratory CV values for the ten peptides were generally ≤ 15% across the concentration range of peptides (1-500 fmol/uL in 1 ug digested plasma). As sample complexity and handling was increased, a concomitant increase in both intra- and inter-laboratory CVs was observed across the sites, but only minimally.
The wealth of data generated from this study has provided a springboard for deeper investigation into sources of variability and which of these factors can be controlled in the laboratory setting. Reproducibility of nanoflow chromatography coupled to electrospray ionization, column configuration, and chromatographic hardware set-up can introduce inconsistencies within a site, causing intra-laboratory CV to inflate. This presentation will focus on aspects of controllable conditions that can be implemented across several labs to increase reproducibility, and ease of transferability in MRM-based analyses. Specifically, efforts to produce a system suitability standard operating procedure that is implemented at each site and at various points throughout a SID-MRM-MS study to help tease apart some of the between site variation that was previously observed.
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