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Purpose: To examine temporal fluctuations in selected plasma protein levels over a period of nearly 4 months and assess biological variation of these proteins across a healthy population cohort., Experimental Design: Plasma was collected from ten healthy volunteers over two time-courses: (i) weekly for 4 weeks; (ii) bimonthly over 4 months and depleted of albumin and IgG. SRM MS was used to determine the relative quantitation of 31 plasma proteins commonly observed in biomarker studies over these time courses. Estimates of between-subject and within-subject biological variances determined by SRM were calculated for each protein., Results: Replicate analysis demonstrated the high precision of SRM assays of plasma proteins. Statistical analysis indicated that none of the measured proteins exhibited significant temporal fluctuations over either time course. Overall, time-based intraindividual quantitative variation of plasma protein levels was considerably lower than biological variation occurring between individual volunteers., Conclusions and Clinical Relevance: This study is the first to show robust temporal stability of the plasma proteome in healthy individuals using SRM-based peptide quantitation. This is important as it provides a strong basis for reliable detection of disease/treatment-related changes of these plasma proteins and others using SRM., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The widespread clinical adoption of protein biomarkers with diagnostic, prognostic and/or predictive value remains a formidable challenge for the biomedical community. From discovery to validation, the path to biomarkers of clinical relevance abounds with many protein candidates, yet so few concrete examples have been substantiated. In this review, we focus on the recent adoption of selected reaction monitoring (SRM) of plasma proteins in the path to clinical use for a broad range of diseases including cancer, cardiovascular disease, genetic disorders and various metabolic disorders. Recent progress reveals a promising outlook for clinical applications using SRM, which now provides the routine analysis of clinically relevant protein markers at low nanogram per millilitre in plasma., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

An emphasis of current proteomic research is the validation of plasma protein biomarkers. The process of blood collection itself is critical to the accuracy and reproducibility of quantitative biomarker assays. We have developed selected reaction monitoring (SRM) assays to analyse thirteen abundant plasma proteins and evaluated the impact of three different blood collection tubes on the levels of these proteins. We also assessed the implications of the time taken to analyse plasma samples by evaluating the recovery of these proteins. We showed that SRM detects minor differences in the levels of some proteins which can be attributed to collection tube type. The average recovery for 12 of 18 assays was higher for proteins that were collected in tubes containing protease inhibitors compared to conventional collection tubes. For five of the assays, the differential recovery was statistically significant. Delaying MS analysis of a freeze-thawed sample for 1 hour showed greatly reduced recovery of these analytes; however differences attributed to tube type were only evident at the baseline timepoint. Finally, we assessed the natural variation of circulating levels of these proteins in a cohort of seven healthy individuals. This study provides useful information for researchers contemplating blood collection for undertaking protein biomarker studies.