Abstract
BackgroundThe development of commercially available panels for human blood plasma screening via selected reaction monitoring (SRM) offers reliable, cost-efficient and highly-standardized discovery and validation of protein biomarkers. However, protein detection by SRM can be hampered by interfering peptide fragment ions. To estimate the influence of interference on protein detection, we performed different types of sample preparation and implemented SRM measurements for well-characterized protein targets approved by the US Food and Drug Administration.MethodsWe used the PlasmaDeepDive™ SRM assay from BiognoSYS AG for absolute quantification of 18 proteins in 19 samples of human plasma using three different protocols for sample preparation. SRM measurements were performed using iRT standards for retention time normalization and isotopically-labeled reference peptides for absolute quantification. SpectroDive™ software was used for automated detection of reliable peak groups.ResultsFourteen targeted proteins were quantitatively measured in more than half of the samples. Depletion of highly-abundant plasma proteins and peptide fraction clean-up on centrifuge plates resulted in detection of all 18 targeted proteins in femtomolar to picomolar concentrations.ConclusionsIt was shown that commercially designed SRM kits are suitable for SRM detection of well-established plasma/serum biomarkers.Electronic supplementary materialThe online version of this article (doi:10.1186/s40169-015-0071-4) contains supplementary material, which is available to authorized users.
Highlights
The development of commercially available panels for human blood plasma screening via selected reaction monitoring (SRM) offers reliable, cost-efficient and highly-standardized discovery and validation of protein biomarkers
Selected Reaction Monitoring (SRM) is a targeted mass spectrometry method which has emerged as a promising challenger to antibody techniques of protein analysis in biological samples [1]
We studied proteins in blood plasma samples from patients aged 50–70 years
Summary
The development of commercially available panels for human blood plasma screening via selected reaction monitoring (SRM) offers reliable, cost-efficient and highly-standardized discovery and validation of protein biomarkers. Methods: We used the PlasmaDeepDiveTM SRM assay from BiognoSYS AG for absolute quantification of proteins in samples of human plasma using three different protocols for sample preparation. Selected Reaction Monitoring (SRM) is a targeted mass spectrometry method which has emerged as a promising challenger to antibody techniques of protein analysis in biological samples [1]. The detection method, based on triple quadrupole mass spectrometry, isolates the proteotypic peptide by its m/z and quantifies the content of fragments in a sample. This physical principle of signal registration of characteristic ions’ pairs ensures high selectivity and sensitivity of SRM when analyzing low-abundance proteins [3]. Due to its technical limitations, the quadrupole mass spectrometer cannot precisely isolate charged particles of particular m/z, which results in overlapping flows of charged particles originating from different analytes within the given
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