Multiple Reaction Monitoring-Mass Spectrometry Enables Robust Quantitation of Plasma Proteins Regardless of Whole Blood Processing Delays That May Occur in the Clinic.

Plasma is an important biofluid for clinical research and diagnostics. In the clinic, unpredictable delays-from minutes to hours-between blood collection and plasma generation are often unavoidable. These delays can potentially lead to protein degradation and modification and might considerably affect intact protein measurement methods such as sandwich enzyme-linked immunosorbent assays that bind proteins on two epitopes to increase specificity, thus requiring largely intact protein structures. Here, we investigated, using multiple reaction monitoring mass spectrometry (MRM-MS), how delays in plasma processing affect peptide-centric "bottom-up" proteomics. We used validated assays for proteotypic peptide surrogates of 270 human proteins to analyze plasma generated after whole blood had been kept at room temperature from 0 to 40 h to mimic delays that occur in the clinic. Moreover, we evaluated the impact of different plasma-thawing conditions on MRM-based plasma protein quantitation. We demonstrate that >90% of protein concentration measurements were unaffected by the thawing procedure and by up to 40-h delayed plasma generation, reflected by relative standard deviations (RSDs) of <30%. Of the 159 MRM assays that yielded quantitative results in 60% of the measured time points, 139 enabled a stable protein quantitation (RSD <20%), 14 showed a slight variation (RSD 20-30%), and 6 appeared unstable/irreproducible (RSD > 30%). These results demonstrate the high robustness and thus the potential for MRM-based plasma-protein quantitation to be used in a clinical setting. In contrast to enzyme-linked immunosorbent assay, peptide-based MRM assays do not require intact three-dimensional protein structures for an accurate and precise quantitation of protein concentrations in the original sample.