MSACL 2023 Abstract

Comparative Quantitation of 270 Human Plasma Proteins on Agilent High Flow vs. Evosep Micro- and Nanoflow by Bottom-up MRM on Agilent 6495C Triple Quadrupole

Claudia Gaither (1,2), Robert Popp(1), Adeline Shanker (1,3), Christoph H. Borchers (1,3,4,5)
(1) MRM Proteomics Inc., Montréal, Canada (2) Département de Biomédecine Vétérinaire, Faculté de Médecine Vétérinaire, Université de Montréal, Saint-Hyacinthe, Canada (3) Division of Experimental Medicine, McGill University, Montréal, Canada (4) Segal Cancer Proteomics Centre, Lady Davis Institute, Jewish General Hospital, McGill University, Montreal, Canada (5) Gerald Bronfman Department of Oncology, Jewish General Hospital, McGill University, Montréal, Canada

Christoph Borchers, PhD (Presenter) Jewish General Hospital, McGill University Montreal, QC, Canada

Presenter Bio: Dr. Christoph Borchers is recognized as a pioneer and leading figure in the development of mass spectrometry-based methods for protein quantification using Multiple Reaction Monitoring (MRM). He has also published more than 300 peer-reviewed papers in scientific journals, and is the founder and director of the McGill-Lady Davis Institute Integrated Proteomics Program at the Jewish General Hospital, McGill University, where he is currently a full professor in the Department of Oncology. His research is centred around the improvement, development, and application of proteomics and metabolomics technologies, with a major focus on quantitative targeted proteomics for clinical diagnostics, as well as new mass-spectrometry-based techniques for structural proteomics.
Dr. Borchers received his BS, MSc, and PhD degrees from the University of Konstanz, Germany in 1996. After his post-doctoral training and employment as a staff scientist at NIEHS/NIH/RTP, NC and he became the director of the Duke–UNC Proteomics Facility and held a faculty position at the UNC Medical School in Chapel Hill, NC (2001-2006). From 2006 to 2019, he was a Professor in the Department of Biochemistry and Microbiology, and Director of the University-Genome British Columbia Proteomics Centre at the University of Victoria, British Columbia, Canada, where he held the Rix BC Leadership Chair in Biomedical and Environmental Proteomics.
Dr. Borchers is also involved in promoting proteomics research and education through his involvement with HUPO (International Council Member), the British Columbia Proteomics Network (Executive Committee Member, past Scientific Director) and the Canadian National Proteomics Network (Member, past VP External and Chair of the Board of Directors). He is also a Fellow of the Canadian Academy of Health Sciences.

Relevant Financial Disclosures (within past 24 months, reported on Feb 25, 2022) Honorarium/Expenses MRM Proteomics Salary MRM Proteomics Conflict mitigation NOT REQUIRED.

INTRODUCTION:
Quantitation of proteins by multiple reaction monitoring (MRM) based on proteotypic peptides as protein surrogates is an increasingly popular approach over antibody-based, photometric approaches such as enzyme-linked immunosorbent assays (ELISA) due to its high multiplexicity, specificity (retention times, light/heavy peptide pair, precursor-fragment transition), large dynamic range of approximately 6 orders of magnitude, and sensitivity in the low ng/mL range. Due to its robustness, high flow, ultra-high performance liquid chromatography (UHPLC) coupled to MRM is of great interest to clinical labs. While UHPLC-MRM is commonly performed at flow rates of 0.3 to 0.5 µg/µL, loading 10-20 µg of digested protein, recent improvements in LC systems such as the highly robust Evosep One, have allowed coupling nano-to-microflow systems to triple quadrupoles, reducing the flow rate and loading amounts to <1 µL/min and <1 µg, respectively, while increasing sensitivity.
Here we compared the performance of 274 MRM assays to quantify 270 human plasma proteins utilizing a 6495C triple quadrupole (Agilent), connected to three different LC setups representing varying flow rates and loading amounts: Agilent 1290 Infinity II (reference method), Evosep One (60 samples per day, SPD), and Evosep One Whisper (40 SPD).

OBJECTIVES:
A comparison of sensitivity and quantitative performance between three different LC-MS approaches.

METHODS:
Five plasma lots were reduced and denatured (urea/dithiothreitol – DTT), alkylated (iodoacetamide – IAA), digested (Worthington trypsin), then quenched with formic acid (FA). Calibration curve standards and quality control (QC) samples were prepared by spiking digested BSA background matrix with varying levels of a 274-light peptide mix, then normalized (along with the plasma samples) with constant amounts of a 274-stable isotope-labelled standard peptide mix acting as internal standards.
All standards, samples and QCs were prepared in parallel, then split into three (one set for each system/method). Samples designated for the Agilent 1290 Infinity II – 6495C method were cleaned up by solid phase extraction using Waters HLB µElution plates, then dried down, and resuspended in aqueous 0.1% FA. 10 µg worth of material was injected onto a Zorbax Eclipse Plus RP-UHPLC column (2.1 x 150 mm, 1.8 µm particle diameter; Agilent) maintained at 50 °C. Peptide separations were achieved at 0.4 mL∕min over 60 min, via a multi-step LC gradient of 2-80% mobile phase B, and a post-gradient column re-equilibration of 4 min. Mobile phase compositions were A: 0.1% FA/H2O and B: 0.1% FA/acetonitrile.
The samples designated for Evosep One 60 SPD analysis and Evosep One Whisper analysis were loaded onto Evotips following the manufacturer’s instructions. For the 60 SPD analysis (21 min gradient), 1 µg per tip was loaded and samples were injected onto an EV-1109 column (Evosep; 8 cm x 150 µm, 1.5 µm; 1 µL/min). For the Whisper analysis, 20 ng material were loaded per tip and injected onto an EV-1112 column (Evosep; 15 cm x 75 µm,1.9 μm; 100 nL/min) with the 40 SPD method (36 min gradient).
Data were acquired on an Agilent 6495C triple quadrupole with either a Jet Stream ESI source when coupled to the 1290 Infinity II UHPLC, or with an Agilent nano source when coupled to the Evosep One HPLC, in both cases, using dynamic MRM.

PRELIMINARY RESULTS:
Preliminary results show improved Lower Limit of Quantitation values (LLOQs) for 196 of the 274 peptide targets using the Evosep 60 SPD method compared to the 1290 Infinity II reference method. The Evosep 60 SPD approach was up to 92-fold more sensitive, with a median 3-fold sensitivity improvement.

Moreover, while the reference method resulted in a median of 148 of the 274 endogenous proteotypic target peptides being within their respective linear ranges, and thus quantifiable across the five-plasma sample lots, the Evosep 60 SPD method resulted in a median of 178 endogenous peptides being quantifiable, an increase of 20% at half the gradient length, using only 1/10th of the material.
The Evosep One system with its reduced flow rates allows for significant sensitivity improvements, and although data analysis for the 20 ng/mL/Whisper method is still underway, we expect even larger sensitivity improvements.

CONCLUSION:
Moving from a robust high flow system to a robust nano-/microflow Evosep One system, coupled with a 6495C triple quadrupole, allows for significant sensitivity improvements while maintaining robust protein quantitation by highly multiplexed MRM.