Abstract
BackgroundPlasma is a potentially rich source of protein biomarkers for disease progression and drug response. Large multi-center studies are often carried out to increase the number of samples analyzed in a given study. This may increase the chances of variation in blood processing and handling, leading to altered proteomic results. This study evaluates the impact of blood processing variation on LC–MS/MS proteomic analysis of plasma.MethodsInitially two batches of patient plasma samples (120 and 204 samples, respectively) were analyzed using LC–MS/MS shotgun proteomics. Follow-up experiments were designed and carried out on healthy donor blood in order to examine the effects of different centrifugation conditions, length of delay until first centrifugation, storage temperature and anticoagulant type on results from shotgun proteomics.ResultsVariable levels of intracellular proteins were observed in subsets of patient plasma samples from the initial batches analyzed. This observation correlated strongly with the site of collection, implicating variability in blood processing procedures. Results from the healthy donor blood analysis did not demonstrate a significant impact of centrifugation conditions to plasma proteome variation. The time delay until first centrifugation had a major impact on variability, while storage temperature and anticoagulant showed less pronounced but still significant effects. The intracellular proteins associated with study site effect in patient plasma samples were significantly altered by delayed processing also.ConclusionsVariable blood processing procedures contribute significantly to plasma proteomic variation and may give rise to increased intracellular proteins in plasma. Accounting for these effects can be important both at study design and data analysis stages. This understanding will be valuable to incorporate in the planning of protein-based biomarker discovery efforts in the future.
Highlights
Plasma is a potentially rich source of protein biomarkers for disease progression and drug response
Observation of intracellular proteins in patient plasma samples As part of a biomarker discovery effort we carried out shotgun proteomic analysis of two separate sample batches from a national patient registry
The median count of proteins detected per sample and corresponding interquartile range (IQR) was 351 (IQR = 20) and 373 (IQR = 28) for the first and second batches respectively
Summary
Plasma is a potentially rich source of protein biomarkers for disease progression and drug response. Large multi-center studies are often carried out to increase the number of samples analyzed in a given study. This may increase the chances of variation in blood processing and handling, leading to altered proteomic results. This study evaluates the impact of blood processing variation on LC–MS/MS proteomic analysis of plasma. Previous studies have established that preanalytical variables can impact both individual protein measurements and proteome-wide measurements. Previous studies have demonstrated the impact on plasma concentrations of selected proteins as measured by ELISA of multiple freeze/thaw cycles [4] and platelet enrichment, implicating centrifugation as a key variable [5]. An evaluation of a broad panel of plasma analytes has demonstrated effects of storage temperature and duration to the first centrifugation on measured levels of several additional proteins in plasma that can be assessed by clinical laboratory instruments [6]
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