Proteome profiling with precision on a clinical cohort: Pioneering a large-scale ultra-high throughput validation study employing a novel high-speed hybrid nominal mass platform

Protein and peptide quantification methods, such as Multiple Reaction Monitoring (MRM) and Parallel Reaction Monitoring (PRM), utilize LC-MS for the targeted, bottom-up quantification of protein biomarkers by monitoring signature peptides representative of candidate biomarkers. The challenges are balancing throughput with assay/study variables, peptide parameters that define precision, ease for establishing quantifiability (linearity, sensitivity, reproducibly) and data acquisition across large cohort. The recent development of a novel high-speed hybrid nominal mass platform has enabled us to perform large-scale, high-throughput proteomic profiling of 83 Stable Isotope Labeled (SIL) peptides representing 53 Health Surveillance Panel (HSP) proteins (containing 21 FDA-approved proteins assay) in an inflammatory bowel disease (IBD) cohort (n=498) and age/sex-matched healthy individuals (n=547), totaling 1,005 plasma samples. 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 four different throughput levels: 100 samples per day (SPD), 144 SPD, 180 SPD, and 300 SPD. Excellent reproducibility was observed at all four throughputs, with median CV% ranging from 5% to 10% for all 83 SILs. The median points per peak were 10 (100 SPD), 11 (144 SPD), 8 (180 SPD), and 5 (300 SPD). The observed sensitivity for the 83 SIL peptides across the four throughputs was sub-fmol (LOQ < 0.5 fmol), with 93% of the peptides achieving this for 100 SPD, 87% for 144 SPD, 48% for 180 SPD, and 53% for 300 SPD. For LLOQ (>5 fmol), the percentages were 99% for 100 SPD, 97% for 144 SPD, 49% for 180 SPD, and 72% for 300 SPD. For linearity, more than 95% of all peptides had an R² > 0.9. The 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 1,005 samples within 14 days (180 SPD). The study included 2,000 injections when accounting for system suitability tests, low, mid, and high-quality controls, washes, and blanks, performed robustly and reproducibly. In 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 by increasing the ease to establishing quantifiability (establishing linearity, sensitivity, reproducibly) and ease for consistency data acquisition across large cohort is highlighted. This approach allows us to profile a large IBD cohort (n=1,005) with precision and robustness, offering insights into biologically important plasma proteins, including 21 FDA-approved plasma protein markers.