MSACL 2025 Abstract

Development and Clinical Evaluation of a Multiplexed Health Surveillance Panel Using Ultra-High Throughput PRM-MS in an Inflammatory Bowel Disease Cohort

Qin Fu (1,2), Philip M. Remes (2), Jihyeon Lee (1), Cristina Jacob (2), Dalin Li (1), Manasa Vegesnas (1), Koen Raedschelders (1), Ali Haghani (1), Sandy Joung (1), Susan Cheng (1), Scott Peterman (2), Justyna Fert-Bober (1), Dermot McGovern (1), Murray Christopher (1), Jennifer E. Van Eyk (1)
(1) Cedars-Sinai Medical Center, (2) ThermoFisher

Jennifer van Eyk, PhD (Presenter) Cedars-Sinai Heart Institute

Presenter Bio: Jennifer Van Eyk, PhD, is an international leader in the area of clinical proteomics and her lab has focused on developing technical pipelines for de novo discovery and larger scale quantitative mass spectrometry methods. This includes multiple reaction monitoring (MRM, also known as SRM) and most recently data independent acquisition. Dr. Van Eyk's laboratory is well known for the extreme technical quality of the data generated, rigorous quality control with tight %CV while applying these to key clinical questions. The aim is to maximize throughput and reproducibility in order to move targeted and robust discovery methods into large population healthy continuous assessment and clinical grade assays focusing on brain and cardiovascular diseases.

Relevant Financial Disclosures (within past 24 months, reported on Apr 21, 2026) No relevant financial relationship(s) to disclose.

INTRODUCTION:
Plasma is a readily accessible biofluid reflecting an individual's physiological state, making it invaluable source for clinical biomarkers of disease. Despite the advances in clinical proteomics, translating protein biomarker discoveries into clinical use remains challenging due to the technical complexity of the validation process. Targeted MS-based proteomics approaches, Parallel Reaction Monitoring (PRM) offer sensitive and specific assays for biomarker translation. Personalized medicine, particularly in chronic diseases like inflammatory bowel disease (IBD), requires assay for patient stratification and treatment optimization. In this study, we developed a PRM assay to quantify 57 plasma proteins (including 21 FDA-approved proteins) and analyzed in 1007 IBD and control patients on the Stellar MS platform, demonstrating high-throughput capacity and reproducibility.

METHOD:
A PRM-MS assay was developed on a plasma pool comprising 100 healthy males and 100 females on the Stellar MS platform. Sample preparation (denaturation, reduction, alkylation, digestion) was carried out an automated Beckman i7 workstation. Loading curves were conducted at 100, 144, 180, and 300 samples per day (SPD) using four independent, optimized, and scheduled PRM methods across 11 SIL load (120 to 0.006097 fmol on column). 300 ng digested control plasma pool was added as the matrix for all load levels (n=5 injections/load). Following optimization, IBD cohort plasma sample (496 matched controls and 511 IBD subjects) were analyzed at a throughput of 180 SPD in order to quantify the 53 protein panel (83 SIL peptides).

RESULTS
Using a pooled human plasma sample (100 healthy males and 100 healthy females), we evaluated the targeted peptides' sensitivity, linearity, LLOD, LLOQ, and reproducibility across all SPD. Mean CV% for all SPD ranged from 5% to 10% for all 83 SILs. The median points per peak were 10, 11, 8 and 5 (100, 144,180 and 300 SPD, respectively). The observed sensitivity for the 83 SIL peptides across the four throughputs was sub-fmol (LOQ < 0.5 fmol), with ,87,48 and 53, 42, 45 and 37% for 100, 144, 180 SPD, and 300 SPD, respectively. For LLOQ (<5 fmol), the percentages were 99, 98, 99 and 76% for 100, 144, 180 and 300 SPD, respectively. For linearity, > 95% of all peptides had an R2>0.9. Total median CV% based on injections each day for 5 days for the 83 SIL peptides was less than 10% at all throughputs. The implementation of the Adaptive Retention Time (ART) algorithm, which allows real-time retention time adjustments, proved advantageous in achieving robustness and enhancing reproducibility when acquiring the IBD cohort of 1,007 samples within 14 days (180 SPD). The study included 2,000 injections, accounting for system suitability tests, low, mid, and high-quality controls, washes, and blanks, performed robustly and reproducibly.
Conclusion. The combination of a novel high-speed hybrid nominal mass platform and the ART algorithm provides a powerful solution to the challenges of large-scale, high-throughput, targeted, and scheduled MS-based quantification. This approach increases the ease of establishing quantifiability (linearity, sensitivity, reproducibility) and consistency in data acquisition across large cohorts. This method allowed us to profile a large IBD cohort (n=1,007) with precision and robustness, offering insights into biologically important plasma proteins, including 21 FDA-approved plasma protein markers. The PRM-MS validated IBD markers observed from discovery, in particular, CRP and A1AG1.

NOVELTY:
High-throughput (180 SPD) and large-scale (n=1,007) PRM-MS analysis validated known IBD biomarkers.