MSACL 2025 Abstract

Development of a Ready-To-Use Evotip-Based Workflow for Plasma Protein Quantification

Elodie Logerot (1), Timon Geib (1), Peter Kubiniok (1), Vincent R. Richard (1), Robert Popp (2), René P. Zahedi (3,4,5), Christoph Borchers (1,6,7,8)
(1) Segal Cancer Proteomics Centre, Jewish General Hospital, Montreal, QC, Canada, (2) MRM Proteomics, Inc, Montreal, QC, Canada, (3) Manitoba Centre for Proteomics and Systems Biology, Winnipeg, MB, Canada, (4) Department of Internal Medicine, University of Manitoba, Winnipeg, MB, Canada, (5) Department of Biochemistry and Medical Genetics, University of Manitoba, Winnipeg, MB, Canada, (6) Gerald Bronfman Department of Oncology, Jewish General Hospital, Montreal, QC, Canada, (7) Division of Experimental Medicine, McGill University, Montreal, QC, Canada, (8) Department of Pathology, McGill University, Montreal, QC, Canada

Elodie Logerot, PhD (Presenter) Segal Cancer Proteomics Centre, Jewish General Hospital >> POSTER (PDF)

Presenter Bio: I obtained my Ph.D. in 2022 from the University of Montpellier in France, where I focused on the structural analysis of peptides using tandem mass spectrometry. My research involved MS/MS sequencing using various activation methods (CID, ETD) and different types of mass spectrometers, including Ion Trap, MALDI-TOF/TOF, and Q-TOF instruments. Following my Ph.D., I pursued my first postdoctoral position in Montpellier, specializing in structural proteomics. I applied HDX-MS and native MS approaches on a Q-TOF mass spectrometer to study protein dynamics and interactions. Currently, I am a postdoctoral researcher at the University of McGill in Montreal, where I focus on developing and optimizing quantitative proteomics workflows using both targeted and untargeted MS methods. My specialties include peptide and protein characterization, quantitative method development, and structural mass spectrometry. I'm particularly interested in translating MS data into meaningful biological insights.

Relevant Financial Disclosures (within past 24 months, reported on Jul 15, 2025) No relevant financial relationship(s) to disclose. Conflict mitigation NOT REQUIRED.

INTRODUCTION:
Accurate and reproducible quantification of plasma proteins poses a significant challenge to mass spectrometry-based proteomics due to its high dynamic range and inherent matrix complexity. Highly abundant proteins can mask the detection of biologically relevant, low-abundance species, and matrix effects and batch-to-batch variability further complicate quantitative analyses. As part of the SysQuan project, we use stable isotope-labeled mouse plasma (SILAC) as an internal standard for absolute quantification, which helps us address these limitations and improve consistency between workflows. Our goal is to develop an innovative, solution that will streamline plasma proteomics and enable its routine use in clinical and research environments.

OBJECTIVE:
The long-term goal is to create Evotips preloaded with SILAC mouse plasma, which will enable the direct addition and subsequent digestion of human plasma samples for quantification. This system is designed to be a robust tool for absolute protein quantification in clinical and research applications.

METHODS:
Currently, method development and optimization are based on human plasma samples. A "one-pot" digestion strategy is employed directly on Evotips to ensure compatibility with high-throughput workflows. This digestion protocol eliminates transfer steps and reduces sample handling errors by sequentially adding denaturation, reduction, alkylation, and digestion reagents directly onto the Evotip. Resulting digests were analyzed using an Evosep One LC system coupled with a timsTOF HT mass spectrometer operating in DIA-PASEF mode. Data were analyzed using a spectral library generated from fractionated, depleted and non-depleted plasma, and identification was performed using DIA-NN.

RESULTS:
On average, 450 proteins and 3,500 peptides were consistently identified in all technical replicates per sample. The workflow demonstrated high reproducibility, with coefficients of variation below 15% between replicate analyses. Notably, 91% of the proteins identified using the conventional S-Trap-based workflow were recovered during the "one-pot" digestion on Evotips. This significant overlap underscores the analytical depth of the streamlined protocol while significantly reducing processing times and preparation steps. Furthermore, around 75% of the peptides detected using the Evotip approach overlap with those included in the SysQuan panel (i.e., lysine-containing and shared between mouse and human), underlining the method's compatibility with absolute quantification pipelines.
These initial results validate the digestion strategy on Evotips and support its integration into SILAC-based quantification workflows, including MRM or PRM-PASEF methods developed on a triple quadrupole mass spectrometer such as the Agilent 6495D or timsTOF HT as part of the SysQuan project.

CONCLUSION:
We present a reproducible and simplified method for plasma proteome analysis using Evotips that yields promising results in terms of protein and peptide coverage. The next phase of this project will implement SILAC mouse plasma on the Evotips to serve as a protein-level internal standard, which will enable robust quantification. The ability to integrate an internal standard directly into the Evotip format paves the way for standardized, plasma quantification solutions, with potential applications in translational research and routine workflows.

REFERENCES:
Ye, Z., Sabatier, P., Martin-Gonzalez, J. et al. One-Tip enables comprehensive proteome coverage in minimal cells and single zygotes. Nat Commun 15, 2474 (2024). https://doi.org/10.1038/s41467-024-46777-9.