Low-input label-free proteomics using the Orbitrap Astral MS and μPAC Neo Plus Columns

Summary — Low-input label-free proteomics using the Orbitrap Astral MS and μPAC Neo Plus Columns

Significance of the topic

The ability to identify and quantify proteins from very small sample amounts (sub-ng to low-ng) is critical for emerging applications such as single-cell proteomics, limited clinical biopsies, and rare cell populations. Combining highly efficient nano‑LC separations with high‑sensitivity, high‑throughput mass analysis and gas‑phase fractionation reduces missing values and improves depth and precision for label‑free quantitation (LFQ). This technical note demonstrates an integrated workflow that addresses sensitivity, throughput, and reproducibility tradeoffs important in practical laboratory settings.

Goals and overview of the study

The primary objective was to demonstrate peptide and protein identification depth and quantitative precision at low input levels using data‑independent acquisition (DIA) on the Orbitrap Astral Mass Spectrometer coupled to the μPAC Neo Plus 50 cm column and Vanquish Neo UHPLC. Specific aims included: evaluating identification depth across a HeLa digest dilution series (50 pg–20 ng), comparing FAIMS compensation strategies, testing multiple throughputs (20–100 samples per day, SPD), and comparing direct injection and trap‑and‑elute workflows.

Methodology

- Sample: Thermo Scientific Pierce HeLa Protein Digest Standard prepared in 0.02% n‑dodecyl‑β‑D‑maltoside (DDM) in 0.1% formic acid; dilution series prepared at 50 pg to 20 ng load per injection; replicate triplicates for each level.
- Chromatography: Thermo Scientific μPAC Neo Plus 50 cm, 75 μm ID column; temperature 50 °C; direct injection and trap‑and‑elute backward‑flush workflows evaluated; throughput ranges from 20–100 SPD with method‑specific gradient lengths and flow rates. Trapping column: μPAC trapping column (300 μm × 1 cm) used for trap workflows.
- Mass spectrometry: Orbitrap Astral Mass Spectrometer operated in peptide application mode with parallel Orbitrap and Astral analyzers; FAIMS Pro Duo used for gas‑phase fractionation and background ion removal. MS1 quantitation used for 50 pg–1 ng loads; MS2 quantitation used for ≥2.5 ng loads. Load‑dependent DIA windows and maximum injection times were optimized (see method table summary).
- Data analysis: Spectronaut 21.1 directDIA+ analysis with 1% FDR; additional tools mentioned include Proteome Discoverer and DIA‑NN for complementary analysis.

Used instrumentation

• Orbitrap Astral Mass Spectrometer (Thermo Scientific)
• FAIMS Pro Duo Interface
• Vanquish Neo UHPLC System with Vanquish User Interface
• μPAC Neo Plus 50 cm HPLC Column and μPAC trapping column
• μPAC Neo Plus voltage spacer, grounding cable and EASY‑Spray emitter (10 μm I.D.)
• EASY‑Spray Source and Sonation column oven for μPAC Neo columns
• Standard LC‑MS solvents and consumables (LC‑MS grade water, ACN, formic acid, isopropanol)
• Software: Spectronaut 21.1 (Biognosys), Proteome Discoverer, DIA‑NN (Aptila)

Main results and discussion

- Identification depth: At 250 pg HeLa digest and 50 SPD (1 FAIMS CV) the workflow yielded over 55,000 peptide identifications and more than 6,900 protein groups. Highest peptide counts for 250 pg were obtained at 30–40 SPD (>53,600 peptides and >6,800 proteins).
- FAIMS effect: Using two FAIMS compensation voltages (longer gradient, 32 SPD) increased peptide identifications by ~33% on average and increased protein group identifications by ~4% compared to a single CV setup, indicating gas‑phase fractionation benefits for coverage.
- Load‑dependent DIA optimization: Adapting DIA isolation widths and maximum injection times to sample load maintained linearity and improved sensitivity across the wide dynamic range (50 pg–20 ng). Wider windows and longer injection times benefited low‑input samples by improving ion statistics, while narrower windows and shorter times reduced spectral complexity at higher loads.
- Quantitation strategy: MS1 quantitation applied for 50 pg–1 ng; MS2 quantitation used for higher loads, aligning quantitation strategy to signal quality across loads.
- Throughput vs depth tradeoff: For a fixed 250 pg sample, a tradeoff curve showed highest identifications near 30–40 SPD. The workflow remained robust across 20–100 SPD with median protein group CVs between 5.0% and 6.9%.
- Reproducibility and robustness: Replicate experiments separated by 50 hours with intervening runs showed stable performance; peptide counts varied within ±10% and protein counts within ±5% across runs.
- Trap‑and‑elute findings: Trap‑and‑elute enabled rapid loading/desalting of larger volumes (enabling up to 7.5 μL injections), with modest chromatographic broadening relative to direct injection but largely preserved peptide/protein identification performance. Use of 0.1% TFA for loading slightly narrowed peaks but produced a small decrease (4–6%) in identifications, likely from ion suppression. Larger injection volumes with lower concentration produced only minor declines in IDs (≈5% peptides, ≈2% proteins), suggesting acceptable performance for dilute single‑cell digests.

Benefits and practical applications

• High sensitivity: Astral analyzer plus μPAC separations enable deep proteome coverage from sub‑ng inputs, suitable for single‑cell and low‑input studies.
• Flexible throughput: Workflows support 20–100 SPD with predictable tradeoffs between speed and depth, allowing labs to tailor runs to study needs.
• Improved coverage via FAIMS: Gas‑phase fractionation reduces background and complexity, enhancing peptide identification especially when using multiple CVs.
• Robustness and reproducibility: Demonstrated stability over time and across throughputs supports routine use for LFQ applications.
• Trap‑and‑elute option: Practical for dilute samples and applications requiring rapid desalting or larger injection volumes while maintaining identification depth.

Future trends and potential applications

- Integration with single‑cell sample prep and microfluidic platforms to push sensitivity and throughput for single‑cell proteomics.
- Further algorithmic advances (machine learning‑based DIA deconvolution, spectral prediction) to extract more signal from complex Astral DIA spectra and reduce false positives at very low loads.
- Wider adoption of gas‑phase fractionation strategies (multi‑CV FAIMS) combined with optimized gradients to further boost coverage for targeted biological questions.
- Automation and standardized protocols for clinical small‑sample workflows (biopsies, circulating tumor cells) where sample amount is limiting.
- Continued column and emitter engineering to reduce adsorption and sample loss at ultralow concentrations and to further improve peak shape in trap workflows.

Conclusion

This technical note demonstrates that coupling μPAC Neo Plus 50 cm micro‑pillar array chromatography with the Orbitrap Astral Mass Spectrometer and FAIMS Pro Duo provides a sensitive, reproducible LFQ DIA workflow for very low input proteomics. Load‑dependent DIA optimization, appropriate use of FAIMS, and the option of trap‑and‑elute enable deep proteome coverage (tens of thousands of peptides; several thousand proteins) from sub‑ng inputs while retaining acceptable throughput and quantitative precision for routine laboratory use.

References

1. Hebert AS, Prasad S, Belford MW, Bailey DJ, McAlister GC, Abbatiello SE, Huguet R, Wouters ER, Dunyach J‑J, Brademan DR, Westphall MS, Coon JJ. Comprehensive single‑shot proteomics with FAIMS on a hybrid Orbitrap mass spectrometer. Analytical Chemistry. 2018;90(15):9529–9537.
2. Thermo Fisher Scientific. μPAC Neo Plus HPLC columns: Use and care instructions (Instruction Sheet DOC018). 2025.
3. Arrey TN, Pashkova A, Delanghe B, Jansons M, Damoc E. Enhanced sensitivity of the Orbitrap Astral Zoom mass spectrometer for deeper proteome coverage in single‑cell proteomics applications (Technical Note TN004019). Thermo Fisher Scientific. 2025.