MSACL 2019 EU Abstract

Comparison of MRM- and DIA-based Protein Quantification Using a 500 Protein Blood Panel in Plasma Samples Demonstrates Linearity between Methods

Sebastian Muller (1), Jakob Vowinckel (1), Nicholas Dupuis (1), Tobias Treiber (1), Lukas Reiter (1), Oliver Rinner (1), Claudia Escher (1)
(1) Biognosys AG, Schlieren, Switzerland

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Claudia Escher (Presenter) Biognosys AG

Presenter Bio: Claudia is Biognosys' Chief Operating Officer. She graduated from the ETH Zurich in Environmental Sciences and obtained her PhD from the University of Basel, Department of Neurobiology (group of Markus A. Rüegg) and the University of Applied Sciences Northwestern Switzerland for her work in biomarker discovery for muscular dystrophies using mass spectrometry. Claudia joined Biognosys in 2008 and is heading the contract research department.

Relevant Financial Disclosures (within past 24 months) Salary Biognosys AG Discussing Products? YES, presenter will present educational content related to products/services of one or more ineligible company(ies) listed above. Conflict mitigation IN PROCESS.

Background
Monitoring of circulating protein biomarkers in plasma has utility for early detection, differential diagnosis, predicting response to therapy and treatment monitoring. SRM/MRM based methods have been the mass spectrometric (MS) platform of choice for low to mid multiplexed protein quantification in biological samples. Recently, data-independent acquisition (DIA) MS has emerged as technology for label-free quantification of very high numbers of proteins. To evaluate the quantification of proteins from DIA data with respect to the comparator method (MRM), we evaluate a mixed plasma sample set (lung cancer, pancreatic cancer and normal controls) with a panel of stable-isotope standard (SIS) peptides for linearity and reproducibility.
Methods
Plasma samples from subjects with non-small cell lung cancer (NSCLC, n = 15), pancreatic cancer (PC, n = 6) and healthy donors (n = 15) were processed to peptides and prepared for mass spectrometry. Prior to analysis, PQ500, a panel of SIS peptides covering 582 plasma proteins (804 peptides), was spiked into each sample enabling absolute quantification of detectable peptide species. The entire sample set was analyzed twice. DIA data were acquired using 40-minute gradients on a C18 capillary-flow liquid chromatography column (5 µl/min) coupled to a Thermo Scientific Orbitrap Fusion Lumos mass spectrometer operating in DIA acquisition mode. MRM data were acquired using similar capillary-flow liquid chromatography (5 µl/min) with a one-hour linear gradient coupled to a Thermo Scientific TSQ Altis triple quadrupole mass spectrometer. Target to reference ratios for the data acquired with the two methods were compared and evaluated for linearity over the sample set.
Results
Due to the sample pathology, multiple acute phase reactants (e.g. CRP and SAA1) have significant dynamic ranges across the sample set (1.5 – 2.5 orders of magnitude). When evaluated for linearity CRP displayed excellent concordance for two unique peptides which were quantified with R2 values of >0.91. A similar evaluation was performed for all peptides that were quantified.
Conclusions
For plasma proteins with significant dynamic ranges of expression in the plasma sample set, a high degree of correlation is observed between DIA and MRM based quantification. The data suggests that the quantitative properties of DIA are comparable to targeted MS methods while providing a high degree of multiplexing for unbiased exploratory studies in clinical samples.