MSACL 2026 Abstract

Robust and High-Throughput Targeted Peptide Quantitation Using a Next-Generation Triple Quadrupole and Automated Ion Source platform

Qin Fu (1), Katherine L. Walker (1), Runsheng Zheng (2), Neloni R. Wijeratne (1), Charles E. Maxey (1), Alec Valenta (2), Huguet, Romain (1), Jennifer E. Van Eyk (3) and Kerry M. Hassell (1)
(1) Thermo Fisher Scientific, San Jose, CA, (2) Thermo Fisher Scientific, Germering, Germany (3) Cedars-Sinai Medical Center, Los Angeles, CA

Qin Fu, PhD (Presenter) Thermo Fisher Scientific

Presenter Bio: Qin Fu joined the Chromatography and Mass Spectrometry group at Thermo Fisher since 2024. Before that, she was a member of Jennifer Van Eyk’s research group at Cedars Sinai and Johns Hopkins, where she developed workflows for automated MS sample preparation, multiplexed targeted MS assays, quantitative DIA analysis, and biomarker discovery and validation. She received her PhD from the University of Minnesota and post-doctoral training at UCSF.

Relevant Financial Disclosures (within past 24 months, reported on Jun 30, 2026) Other Potential Conflicts Thermo Fisher Scientific / Employee Using Generic Terms? YES Conflict mitigation NOT REQUIRED.

BACKGROUND:
Targeted peptide quantitation by LC–MS/MS is widely used in proteomics, biomarker verification, and translational research due to its high sensitivity and quantitative precision. As requirements for multiplexing and sample throughput increase, maintaining reproducibility, retention time stability, and sensitivity becomes increasingly challenging. Advances in acquisition speed, ion transmission efficiency, and workflow operability are required to support large-cohort studies and routine analytical use. In this study, we evaluate the quantitative performance and robustness of a next-generation triple quadrupole mass spectrometer equipped with a novel capillary-flow electrospray ionization interface. A 57 plasma protein biomarkers panel health surveillance panel was used to assess sensitivity, linearity, reproducibility, and long-term stability under high-throughput analytical conditions.

METHODS:
Peptide analyses were performed using a Thermo Scientific™ Vanquish™ Neo UHPLC system coupled to a Thermo Scientific™ TSQ Certis™ triple quadrupole mass spectrometer with the plug-and-play Thermo Scientific™ OptiSpray™ ion source. Ionization was achieved using a Thermo Scientific™ Optispray™ PepMap™ Neo 150µm×15cm Cartridge which contains a 15 µm tapered emitter. Stable isotope–labeled peptides were spiked into pooled human plasma digest and analyzed using a multiplexed 57 protein Health Surveillance Panel. An 11-point calibration curve spanning 0–120 fmol on-column was evaluated. Samples were analyzed in a trap-and-elute configuration with a 14.4-minute run time, enabling a throughput of 100 samples per day. Data were acquired using scheduled selected reaction monitoring transitions and processed using two independent software workflows (Skyline and TraceFinderTM).

RESULTS:
An automated capillary flow emitter routine was performed with the OptiSpray ion source to position the emitter in the optimal position and sheath gas was applied to obtain more stable spray. Quantitative performance was evaluated across 1,080 scheduled SRM transitions using replicate injections and extended analytical runs. More than 96% of transitions achieved over eight data points per chromatographic peak, with a median of 16 points, supporting accurate quantitation in a highly multiplexed assay. Median limits of detection and quantification were 0.019 fmol and 0.164 fmol on-column, respectively. Greater than 99% of peptides demonstrated excellent linearity across the calibration range, with coefficients of determination (R²) exceeding 0.99.

Peak area reproducibility across six replicate injections showed an average relative standard deviation (RSD) of 3.2%, with over 98% of peptides exhibiting RSDs below 10%. Long-term robustness was assessed over 71 consecutive injections of plasma digest spiked with internal standards. Approximately 89% of endogenous peptide signals showed RSDs below 10%, and 95% were below 20%, with higher variability attributed to low-abundant peptides. Retention time stability was exceptional, with RSDs below 0.6% for FDA-relevant peptides across the extended run.

Independent data processing using Skyline and TraceFinder produced consistent quantitative results, confirming robustness across analysis platforms. These data demonstrate high sensitivity, reproducibility, and chromatographic stability in a high-throughput, highly multiplexed targeted peptide workflow suitable for large-cohort and translational studies.

CONCLUSION:
Demonstration of robust, high-throughput multiplexed peptide quantitation with exceptional reproducibility using next-generation triple quadrupole and automated ion source instrumentation.