Ultra-high throughput proteomics at >500 samples/day using advanced Vanquish Neo UHPLC methods with Orbitrap Astral Zoom MS

Ultra-high-throughput proteomics (>500 samples/day) using Vanquish Neo UHPLC and Orbitrap Astral Zoom — summary

Significance of the topic

High-throughput LC–MS proteomics is essential for large-cohort clinical studies, population-scale research, drug screening, and chemoproteomic target discovery. Increasing sample throughput while retaining depth, quantitative precision, and instrument robustness shortens project timelines and reduces per-sample cost, but poses challenges for chromatographic stability, MS acquisition speed, and reproducibility under ultra-short gradients. The presented workflow addresses these challenges by combining rapid trap-and-elute UHPLC methods with a high-speed DIA-capable Orbitrap Astral Zoom mass spectrometer.

Objectives and overview of the study

• Extend practical LC–MS throughput beyond existing trap-and-elute limits (from ~300 SPD to 500–600 samples per day) while preserving deep proteome coverage and reproducibility.
• Evaluate the impact of progressively shortened chromatographic gradients (3.0 to 1.0 min) on identifications, MS2 data points per peak, and quantitative precision.
• Demonstrate long-term robustness across 1000 injections and assess applicability to chemoproteomic screening (activity-based protein profiling, ABPP).

Methodology

Sample preparation and loading:

• Thermo Scientific Pierce HeLa Digest standard reconstituted to 100–200 ng/µL, aliquoted into 96-well plates. Robustness tests used 50 ng injections with PRTC peptide spike-ins (30 fmol) for retention monitoring.
• Chemoproteomic ABPP samples from Staphylococcus aureus were analyzed to evaluate target-enrichment performance at different throughputs.

Chromatography and rapid cycle workflow:

• Vanquish Neo UHPLC in NanoCap Trap & Elute configuration with prototype 150 µm × 70 mm C18 separation column (not commercial at time of study), operated at 4 µL/min and room temperature.
• PepMap Neo 5 µm C18 300 µm × 5 mm cartridge used as trap; separation column coupled via 15 µm ID EASY-Spray emitter. Prototype methods reduced LC duty cycle to ~1 minute for sample pickup/loading plus integrated trap offline wash; standard gradients were compressed to 1–3 minutes (notably 1.7 min for 500 SPD and 1.2 min for 600 SPD).

Mass spectrometry and acquisition strategy:

• Thermo Scientific Orbitrap Astral Zoom operated in data-independent acquisition (DIA) mode. Acquisition parameters were tailored to ultra-short gradients to maintain sufficient MS2 sampling.
• Typical DIA settings used narrow windows (4 Th in many experiments, with the isolation width and precursor mass range adaptable depending on gradient and load) and maximum MS2 injection times of 3 ms to achieve rapid cycle times. The study reports an approximate DIA cycle of ~320 ms to cover ~80 DIA windows, yielding median MS2 data points per peak (DPPP) of approximately 3–4 for the fastest methods.
• Full-scan Orbitrap resolution was set to 240k in reported methods; full-scan and MS2 scan ranges and injection times are adjustable to balance coverage and DPPP.

Data processing:

• Raw files processed with Spectronaut 20.5, Proteome Discoverer 3.3 SP1 (using CHIMERYS and DIA-NN nodes), and DIA-NN Enterprise. Searches were run against the Homo sapiens SwissProt proteome and contaminant FASTA. Peptide counts refer to unique stripped peptides.

Applied instrumentation

• Vanquish Neo UHPLC system (NanoCap Trap & Elute configuration)
• Prototype 150 µm × 70 mm C18 separation column (prototype)
• Thermo Scientific PepMap Neo C18 trap cartridge (300 µm × 5 mm, 5 µm)
• EASY-Spray emitter (15 µm ID, ES994) and EASY-Spray source
• Orbitrap Astral Zoom mass spectrometer (ICSW AST 2.2), operated in DIA
• Data analysis tools: Spectronaut 20.5, Proteome Discoverer 3.3 SP1 (CHIMERYS, DIA-NN nodes), DIA-NN Enterprise

Main results and discussion

Effect of gradient compression:

• Reducing gradient length from 3.0 to ~1.2–1.7 min compresses the peptide elution window proportionally, but protein-group identifications were largely preserved down to ~1.6 min gradients. Significant loss in identifications became apparent only beyond that point.
• MS2 data points per peak scale with gradient length and DIA cycle time; the very short gradients used here produced a median of ~3–4 MS2 DPPP with the chosen MS settings. MS parameters (window width, injection time, scan range) can be optimized to raise DPPP if required.

Throughput vs. proteome depth and precision:

• With the rapid prototype methods enabled by a software update, throughputs of 500 SPD (1.7 min gradient) and 600 SPD (1.2 min gradient) were achieved. At 500 SPD, triplicate runs identified ~5,600–6,600 protein groups (~54,500–67,600 peptides). At 600 SPD the identifications were ~5,300–5,900 protein groups (~46,600–51,500 peptides).
• Quantitative precision remained strong despite compressed separations: median protein-group coefficients of variation (CVs) ranged ~6.7% to 4.6% and median peptide-level CVs were ~12.5% to 9.2% across the fastest methods.

Robustness over repeated injections:

• Over 1000 consecutive injections of 50 ng HeLa digest at 600 SPD, the workflow showed no systematic performance drift. Retention time and protein identifications remained stable with an overall reported variability of ~1.7% CV, supporting long-term robustness under ultra-short gradient, elevated-flow operation.

Application to chemoproteomic screening (ABPP):

• ABPP samples derived from S. aureus treated with a small-molecule antibiotic probe were analyzed across throughputs from 60–500 SPD. While total proteome coverage decreased moderately at the highest throughputs, the rapid methods reliably recovered the enriched putative targets of the probe, demonstrating suitability for high-throughput target-discovery workflows.

Benefits and practical applications

• Substantially increased sample throughput (up to 600 SPD) without catastrophic loss of proteome depth, enabling population-scale projects, large screening campaigns, and rapid chemoproteomic studies.
• High quantitative precision and reproducibility support comparative and differential analyses even with ultra-short gradients.
• Trap-and-elute architecture with rapid duty-cycle methods minimizes LC idle time and supports extended continuous operation with low drift over many injections.
• Flexible MS parameterization allows users to trade throughput for additional MS2 sampling if specific applications require higher DPPP or deeper coverage per injection.

Future trends and potential applications

• Further optimization of DIA window schemes, injection times, and narrow-window approaches can improve MS2 sampling density on ultra-fast gradients, increasing sensitivity and identifications.
• Automation and integration with sample-handling robotics will further decrease per-sample overhead and enable true population-scale proteomics pipelines.
• Broader deployment of prototype column formats and refinements in trap-and-elute cartridges could enhance chromatographic performance tailored to ultra-short gradients.
• Applications likely to benefit include large clinical cohort studies, high-throughput drug/compound screening, rapid biomarker validation, and expanded chemoproteomic screening workflows.

Conclusion

The combined Vanquish Neo rapid trap-and-elute UHPLC methods and Orbitrap Astral Zoom DIA acquisition extend practical LC–MS throughput to 500–600 samples per day while retaining substantial proteome depth, strong quantitative precision, and robust performance across 1000 injections. The approach offers a viable path to scale proteomic experiments for large-cohort and high-throughput screening applications, with configurable MS settings to balance depth and sampling requirements.

References

• Mostert et al., Chemical Science, 2025, 16, 9472-9483.
• Thermo Fisher Scientific, Technical Note TN003939 — High-throughput proteomics using narrow window DIA on the Orbitrap Astral Zoom mass spectrometer.