New paradigm for plasma proteomics biomarker research

Significance of the topic

Targeted large-scale plasma proteomics is essential for translating discovery biomarkers into clinically useful assays because plasma contains proteins spanning a huge dynamic range and many low-abundance, disease-relevant analytes. Efficient, scalable targeted workflows that combine high multiplexing, robust retention-time control and attomole-level sensitivity enable rapid biomarker verification and prioritization for downstream validation and clinical studies.

Study goals and overview

This study demonstrates a streamlined, three-day workflow for building and running large-scale PRM (parallel reaction monitoring) assays on the Thermo Scientific Stellar mass spectrometer, using the Biognosys PQ500 heavy reference peptide kit to target >800 peptides (804 heavy peptides). The objectives were to: create unscheduled PRM methods, refine and schedule PRM assays with automated tools, quantify hundreds of peptides in plasma with short LC gradients (14–24 min), and evaluate sensitivity, linearity, precision and throughput for biomarker verification applications.

Methodology

Workflow summary (three days):

1. Day 1 — Build unscheduled PRM methods: import PQ500 transition list into Skyline, generate spectral library and iRT calibration, export unscheduled PRM lists split into fractions for acquisition.
2. Day 2 — Wide-window scheduling and filtering: run unscheduled methods on Stellar MS using neat heavy peptides and plasma; import results to Skyline; install and use PRM Conductor external tool to filter precursors/transitions by S/N, retention time, peak area, charge state and precision criteria; export wide-window PRM methods.
3. Day 3 — Narrow-window scheduled PRM: acquire wide-window results in plasma, refine retention windows (e.g., 0.6 min for 60 SPD and 0.35 min for 100 SPD), add light peptide targets, enable Adaptive RT alignment and export final scheduled PRM methods for routine acquisition.

Key analytical practices:

• Matrix: Pierce neat human plasma digest; heavy-labeled PQ500 peptides spiked and serially diluted in 300 ng/µL digested plasma for calibration curves and LOD/LOQ assessment.
• Injection: 1 µL on-column.
• Assays: two throughput modes — 60 samples per day (SPD) using a 24-minute gradient and 100 SPD using a 14-minute gradient.
• Acquisition strategy: combined Adaptive RT DIA (for alignment), full-scan MS (for TIC normalization) and tMSn (PRM) experiments in sequence to support robust scheduling and quantification.
• Data processing: Skyline (ver. 23.1.1.503) with PRM Conductor for method building and downstream filtering; offline LOD/LOQ computed from dilution curves.

Instrumentation used

• Thermo Scientific Stellar hybrid quadrupole–linear ion trap mass spectrometer (Stellar MS) with Easy‑Spray source.
• Thermo Scientific Vanquish Neo UHPLC system in trap‑and‑elute configuration.
• Thermo Scientific EASY‑Spray ES906A analytical column (2 µm C18, 150 µm × 15 cm) and PepMap Neo trap cartridge (5 µm C18, 300 µm × 5 mm).
• Biognosys PQ500 heavy reference peptide kit and Pierce neat digested human plasma (standardized QC material).

Main results and discussion

Assay composition and scheduling:

• Initial library: 804 PQ500 heavy peptides (plus 14 PRTC peptides) used to create unscheduled methods split into 10 fractions.
• Final methods: both 60 SPD and 100 SPD assays ended with 1,622 precursors and ~13.7k transitions after filtering; narrow RT windows and Adaptive RT scheduling were applied to enable dense multiplexing.

Performance metrics and robustness:

• Throughput: 24‑minute gradients (60 SPD) and 14‑minute gradients (100 SPD) supported the large target load while maintaining data quality.
• Data points per chromatographic peak: median ~7.1 (60 SPD) and ~6.8 (100 SPD), exceeding practical Nyquist criteria for Gaussian peaks and enabling robust peak quantification.
• Precision: 10‑replicate CVs had medians of 3.8% (60 SPD) and 4.8% (100 SPD); >93% of peptides had CV <20% in both methods.
• Sensitivity: many peptides were quantified at attomole levels. Example peptide STVEELHEPIPSLFR showed LOQ = 17 amol and LOD = 5.7 amol on-column. Across the assay, >85% of peptides had LOQ <500 amol, and the majority had LOD <50 amol; LOD/LOQ were ~1.6× lower in the 60 SPD method versus 100 SPD.
• Linearity and accuracy: calibration curves demonstrated broad linear dynamic range and accuracy >90% across tested concentration levels for the example peptide and globally for the assay.
• Endogenous detection: endogenous peptides such as alpha‑2‑macroglobulin (A2MG) were detected in neat plasma, illustrating the ability to quantify disease‑relevant proteins at physiological abundance.

Key enabling features:

• Stellar MS hardware improvements (higher acquisition speed and ion trap capabilities) increased concurrent target capacity and sensitivity.
• Adaptive RT real‑time chromatogram alignment reduced missingness from RT shifts and allowed narrow scheduling windows without extensive manual RT standard spikes or method re‑tuning.
• PRM Conductor integrated into Skyline automated transition selection and filtering, simplifying assay construction and balancing precursor load across fractions.

Benefits and practical applications

The developed workflow offers several practical advantages for biomarker verification in translational research and clinical proteomics:

• Speed: complete from method creation to processed quantitative results within three days, accelerating candidate triage.
• Scalability: dense multiplexing (thousands of potential targets) using short gradients and narrow windows while preserving analytical quality.
• Sensitivity and precision: attomole-level detection and low CVs support confident quantitation of low-abundance biomarkers in plasma.
• Workflow simplification: automated tools (PRM Conductor + Skyline) reduce manual spreadsheet handling and method development time, improving reproducibility and throughput.

Future trends and applications

Anticipated developments and opportunities include:

• Broader adoption of real‑time RT alignment (Adaptive RT) and automated method-building tools to scale targeted assays across multi-site studies with reduced manual calibration effort.
• Integration of deeper isotope-labeled panels and expanded reference libraries to move from verification to clinical validation for prioritized biomarker panels.
• Further hardware and software advances to push sensitivity and multiplexing limits (more targets per run, shorter gradients) enabling routine high-throughput targeted proteomics in larger cohort studies and clinical workflows.
• Application of this workflow to other complex matrices (e.g., CSF, urine) and disease areas to accelerate translational research and biomarker discovery pipelines.

Conclusion

The study demonstrates a rapid, scalable PRM workflow on the Stellar mass spectrometer that accomplishes large‑scale plasma peptide quantification with attomole‑level sensitivity, high precision, and short LC gradients. Combining hardware speed, Adaptive RT alignment and Skyline/PRM Conductor automation permits creation of multiplexed, scheduled PRM assays covering hundreds to thousands of targets within days, making this approach well suited for biomarker verification and translational proteomics workflows.

References

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