Unlocking biological insights with the Stellar mass spectrometer for Adaptive RT-enhanced quantitative proteomics for plasma biomarker analysis

Stellar mass spectrometer for Adaptive RT-enhanced quantitative proteomics of plasma biomarkers — technical summary

Importance of the topic

Targeted, multiplexed proteomic assays are critical for moving discovery-phase protein candidates into large-scale clinical studies and routine translational workflows. Plasma is a challenging matrix due to high dynamic range and interferences; scalable targeted methods that maintain sensitivity, reproducibility and throughput are therefore essential for biomarker verification and longitudinal clinical studies. The described approach combines an ultrafast mass spectrometer with real-time retention time alignment and optional MS3 fragmentation to increase multiplexing capacity while preserving sensitivity for low-abundance plasma peptides.

Goals and study overview

This work aimed to develop and demonstrate a large-scale, multiplexed targeted proteomics workflow that leverages Adaptive Retention Time (Adaptive RT) scheduling on the Stellar mass spectrometer. The objectives were to: build a broad PRM panel (derived from the PQ500 reference peptide kit), minimize scheduled retention time windows to maximize concurrent targets, improve signal-to-noise for low-abundance peptides via MS3 where necessary, and apply the method to quantify candidate biomarkers in human plasma from disease cohorts (lung cancer, Alzheimer’s disease, colorectal cancer).

Methodology and workflow

Sample preparation and standards:

• Disease and healthy human plasma were obtained from BioIVT and digested using an automated AccelerOme sample-preparation platform.
• Biognosys PQ500 heavy peptide reference standards were used to create scheduled PRM assays and for quantitative reference.

Chromatography and acquisition strategy:

• Vanquish Neo UHPLC with an EASY-Spray 2 µm C18 column; 30-minute peptide gradient; column temperature 55 °C; autosampler 7 °C.
• Adaptive RT (real-time retention time alignment) was used to shrink scheduled windows (reported down to ~0.65 min or 0.6 min) compared to conventional ~2 min windows, enabling higher multiplexing without missing eluting peaks.

Mass spectrometry and targeted design:

• Stellar mass spectrometer operated in PRM (tMS2) mode for the main targeted panel; MS3 workflows were created (via PRM Conductor) for low-abundance peptides with suspected interferences to improve S/N by fragmenting MS2 product ions.
• Acquisition settings included HCD fragmentation (approx. 30% CE), narrow isolation windows for PRM (1 m/z reported for tMS2), dynamic injection times or auto modes and standard AGC targets; adaptive scheduling used a reference file generated from the heavy peptide list or Acquire Reference functionality.
• Skyline-daily was used for method generation, scheduled RT export, and downstream quantitation and calibration analyses.

Data analysis and performance metrics:

• Skyline performed calibration curve fitting, quantitation and peptide-level group comparisons.
• Key performance indicators reported: low attomole sensitivity for certain peptides (example LOQ 13.6 amol on-column), high linearity (majority of calibration curves R2 > 0.9), and strong reproducibility (over 94% of 804 targeted peptides exhibited CV < 25% across replicates).
• Retention-time stability was excellent: mean RT CV ~0.36% (median 0.35%), and spike-in SIL peptides showed median CV ~3% across a large study example cited.

Used instrumentation

• Thermo Scientific Vanquish Neo UHPLC system with EASY-Spray HPLC column (150 µm × 15 cm, 2 µm C18).
• Stellar mass spectrometer (featuring a linear ion trap capable of MS3 acquisition) with Thermo Scientific Easy-Spray source.
• Biognosys PQ500 reference peptide kit (heavy peptides) used as quantitative standards.
• Thermo Scientific AccelerOme automated digestion/sample-prep platform.
• Software: PRM Conductor for MS3 method creation and Skyline-daily for targeted method generation and quantitative analysis.

Main results and discussion

• Panel scale and coverage: A scheduled PRM panel for 804 peptides (PQ500-derived) was implemented and chromatographically separated in a 30-minute gradient. Combining MS2 and MS3 approaches, the study identified 322 endogenous proteins and 507 endogenous peptides in plasma, including 57 proteins represented among FDA-approved biomarkers.
• Sensitivity and quantitation: The platform demonstrated ultrasensitive detection down to low attomole levels for certain peptides with calibration linearity (R2 > 0.9 for ~90% of peptides) and low LOQs (example 13.6 amol). Using higher on-column loads (1 µg vs 25 ng) increased detected proteins by ~73%.
• Reproducibility and retention-time control: Adaptive RT reduced the required scheduled window to ~0.65 min, enabling capture of all targets without frequent method rescheduling. Retention times were highly stable across runs, supporting longitudinal studies and large cohorts.
• MS3 benefits: For peptides affected by matrix interferences, MS3 improved signal-to-noise and enabled recovery of targets otherwise masked in MS2. The combined MS2/MS3 strategy yielded ~10.3% more protein identifications and ~7.3% more peptides than MS2-only in this dataset.
• Biological application — CRC case study: A comparative analysis of colorectal cancer (CRC) patient plasma vs healthy controls identified 29 proteins with adjusted p < 0.05 and >2-fold changes. Examples with increased levels in CRC included SAA2, A2GL, and CO9, consistent with prior literature on CRC-associated plasma changes.

Practical benefits and applicability

• High multiplexing density: Adaptive RT dramatically increases the number of concurrent targets per run by tightening scheduled windows, making large targeted panels feasible within short gradients.
• Clinical and longitudinal suitability: Stable RT, good reproducibility, and sensitivity across a wide concentration range support applications in longitudinal biomarker studies, cohort screening and verification studies.
• Enhanced specificity for low-abundance targets: MS3 capability on the Stellar instrument provides an orthogonal fragmentation step that reduces matrix interference and improves quantitation for challenging peptides.
• Workflow compatibility: Use of commercial heavy peptide kits and established software (Skyline) facilitates transferability and method deployment across laboratories.

Future trends and applications

• Scaling to larger cohorts: Real-time RT alignment and rapid PRM acquisition enable application to very large longitudinal cohorts with minimal method maintenance, supporting population-scale proteomic surveillance and biomarker validation.
• Integration with clinical pipelines: The combination of automated sample prep, reference standards and robust targeted acquisition paves the way for higher-throughput proteomic assays in translational and clinical research labs.
• Hybrid acquisition strategies: Combining adaptive-scheduled PRM with intelligent MS3 selection or data-independent acquisition overlays could further increase depth and quantitative confidence for low-abundance proteins.
• Panel expansion and standardization: Wider adoption of standardized heavy peptide panels and inter-lab ring trials will be important to translate such targeted assays toward regulated clinical use.

Conclusions

The Stellar mass spectrometer platform, when combined with Adaptive RT scheduling and an MS2/MS3 targeted strategy, enables high-density PRM assays in short gradients with excellent sensitivity, reproducibility and retention-time stability. The approach successfully quantified hundreds of peptides and proteins from plasma, recovered additional identifications through MS3, and identified biologically relevant changes in disease cohorts such as CRC. This platform and workflow represent a practical path to scale targeted proteomics for biomarker verification and larger translational studies.

Reference(s)

1. Q. Li et al., TN003861: New paradigm for plasma proteomics biomarker research: large-scale targeted PRM proteomics assays enabled by Thermo Scientific Stellar mass spectrometer.
2. Q. Fu et al., Development and Clinical Evaluation of a Multiplexed Health Surveillance Panel Using Ultra High-Throughput PRM-MS in an Inflammatory Bowel Disease Cohort. Angew. Chem. Int. Ed. Engl. (2025). doi:10.1002/anie.202507610.
3. J. Chantaraamporn et al., Glycoproteomic Analysis Reveals Aberrant Expression of Complement C9 and Fibronectin in the Plasma of Patients with Colorectal Cancer. Proteomes. 2020;8(3):26.
4. B. Geary et al., Discovery and Evaluation of Protein Biomarkers as a Signature of Wellness in Late-Stage Cancer Patients in Early Phase Clinical Trials. Cancers. 2021;13(10):2443.
5. T. A. Davis et al., Serum Amyloid A Promotes Inflammation-Associated Damage and Tumorigenesis in a Mouse Model of Colitis-Associated Cancer. Cell Mol. Gastroenterol. Hepatol. 2021;12(4):1329–1341.
6. M. du Plessis et al., A functional role for Serum Amyloid A in the molecular regulation of autophagy in breast cancer. Front. Oncol. (Year) 12:1000925.
7. J. D. McFadyen et al., Feasibility, Effectiveness and Safety of Elastomeric Pumps for Delivery of Antibiotics to Adult Hospital Inpatients—A Systematic Review. Front. Immunol. 2018;9:1351.
8. A. Skesters et al., Selenium, selenoprotein P, and oxidative stress levels in SARS-CoV-2 patients during illness and recovery. Inflammopharmacology. 2022;30:499–503.
9. M. Rathore et al., Leucine-Rich Alpha-2-Glycoprotein 1 Promotes Metastatic Colorectal Cancer Growth Through HER3 Signaling. Gastroenterology. 2025;68(2):300–315.