MSACL 2025 Abstract

NexProQ: Multiplexed Quantitation of 500+ Proteins in Whole Blood and Dried Blood Spots Using PRM-PASEF and TMTpro

Laleh Ebrahimi Ghahnavieh (1,2), Vincent R. Richard (2), Timon Geib (2), Peter Kubiniok (2), Yassene Mohammed (3), Daniel Hornburg (4), Matt Willetts (5), Gary Krupa (6), Christoph H. Borchers (1,2,7)
(1) Division of Experimental Medicine, McGill University, Montréal, QC, Canada (2) Segal Cancer Proteomics Centre, Jewish General Hospital, Montreal, QC, Canada (3) Center for Proteomics and Metabolomics, Leiden University Medical Center, Leiden, Netherlands (4) Bruker Daltonics GmbH & Co. KG, San Jose, CA, USA (5) Bruker Daltonics GmbH & Co. KG, Billerica, MA, USA (6) Bruker Daltonics GmbH & Co. KG, Bremen, Germany (7) Gerald Bronfman Department of Oncology, McGill University, Montréal, QC, Canada

Laleh Ebrahimi Ghahnavieh, Master of Sciences (Presenter) McGill University / Jewish General Hospital >> POSTER (PDF)

Presenter Bio: Laleh Ebrahimi Ghahnavieh is a PhD student in Experimental Medicine and a Research Assistant at the Segal Cancer Proteomics Centre, affiliated with McGill University and the Jewish General Hospital in Montreal, Canada. Her research focuses on advancing translational proteomics for clinical diagnostics, with an emphasis on multi-omics integration. Her long-term goal is to contribute to precision medicine by developing accessible, standardized proteomic assays for widespread clinical application.

Relevant Financial Disclosures (within past 24 months, reported on Apr 28, 2026) No relevant financial relationship(s) to disclose. Conflict mitigation NOT REQUIRED.

INTRODUCTION:
Quantitative proteomics in clinical samples have remained constrained by limited multiplexing capacity, high reagent costs, and low sample throughput. To address these limitations, we developed NexProQ, a novel platform combining TMTpro multiplexing with PRM-PASEF acquisition on the timsTOF HT. This approach enables us simultaneous absolute quantitation of over 500 protein targets in whole blood (WB) and dried blood spot (DBS) samples in a single run.

METHODS:
Proteotypic peptides were selected from 500+ clinically relevant targets and synthesized as unlabeled NAT standards. Internal calibration curves were prepared using TMT-pro 18-plex labels and peptide dilutions (spanning 1000× to 1× of the lower limit of quantitation (LLOQ)) mixture to reach five-point internal calibration curves. Then multiplexed with plasma and DBS digests preloaded onto EvoTips. Peptides analysis has been done at 30SPD using PRM-PASEF, and assay performance was assessed in terms of linearity, limits detection, intra-assay reproducibility, and matrix effects.

RESULTS:
Assay linearity was robust across most targets (R² > 0.98), with median LLOQs of 0.1–0.5 fmol on-column. Utilizing non-adjacent TMTpro reporter ions boosts quantitation reproducibility and decreases interference due to isotopic overlap. Most targets' intra-assay CVs are below 5%, hence quantitation is reproducible across different protein concentrations and sample types.
Comparison across platforms illustrates the higher mass accuracy and consistency of reporter ion intensities on the QE+, while PRM-PASEF enables superior throughput. The strong concordance for high-abundance proteins has done by validating 274-peptide panel against reference MRM data.

CONCLUSION:
NexProQ offers significant escalating for scalability and accessibility of clinical proteomics. Its capacity to quantify hundreds of proteins across multiple samples within minutes supports its advantages in biomarker discovery, clinical validation, and large-cohort screening studies. This study paves the way for population-scale proteomic screening with performance exceeding current antibody- or aptamer-based methods. Commercial kit development is in progress to support standardization across labs.