Evaluation of OptiSpray technology and the Orbitrap Astral Zoom mass spectrometer for proteomics applications

Evaluation of OptiSpray technology and the Orbitrap Astral Zoom MS for proteomics applications

Importance of the topic

LC–MS proteomics balances sensitivity, chromatographic resolution and practical robustness. High-throughput nanoflow/trap-and-elute workflows promise large sample throughput for discovery and large-cohort studies, but they are often limited by emitter positioning, column interchangeability and run-to-run variability. Reliable, easy-to-use ion source designs and integrated cartridge emitters that preserve optimized geometry can markedly improve instrument uptime, reproducibility and quantitative depth—critical factors for clinical research, large-scale biomarker discovery and industrial QA/QC proteomics.

Study objectives and overview

• Assess the performance, robustness and reproducibility of Thermo Scientific OptiSpray technology combined with a µPAC Neo High Throughput (HT) 5.5 cm cartridge in high-throughput bottom-up proteomics.
• Demonstrate throughput scaling (300, 180, 100 and 60 samples per day—SPD) using rapid gradients and a trap-and-elute Vanquish Neo UHPLC setup.
• Measure protein/peptide identification depth and stability using data-independent acquisition (DIA) on an Orbitrap Astral Zoom MS and processing with Proteome Discoverer + DIA-NN.

Materials and sample preparation

• Thermo Scientific Pierce HeLa digest reconstituted into 100 ng/µL stock (with 0.015% DDM, 0.1% TFA) and aliquoted to produce injection amounts of 20, 50, 100 and 200 ng.
• Trap-and-elute setup: PepMap Neo 5 µm C18 300 µm × 5 mm trap cartridge used prior to the µPAC Neo HT analytical cartridge.

Used instrumentation (Použitá instrumentace)

• Orbitrap Astral Zoom mass spectrometer operated in data-independent acquisition (DIA) mode with narrow isolation windows and high-resolution MS1/MS2 sampling.
• OptiSpray Ion Source with integrated recognition and automated emitter-position optimization; µPAC Neo HT (5.5 cm) cartridge with replaceable tapered emitter (15 µm).
• Thermo Scientific Vanquish Neo UHPLC system in trap-and-elute configuration; 55 cm × 20 µm transfer line to the HPLC port.
• Software: Proteome Discoverer 3.3 SP1 with DIA-NN Enterprise 2.5.2 for peptide/protein identification (Human UniProt FASTA, FDR 1%).

Chromatography and throughput design

• µPAC Neo HT (5.5 cm) enabled extremely fast gradients for high throughput: 2.5 min (300 SPD), 5.8 min (180 SPD), 10.8 min (100 SPD) and 20.5 min (60 SPD).
• Flow rates varied between 3 and 0.75 µL/min; for three faster methods flow was increased early in the gradient then rapidly decreased before peptide elution to concentrate elution into a productive window, improving LC–MS productivity (>86%).
• Column temperature: 60 °C for the 300 SPD method and 55 °C for 180, 100 and 60 SPD methods.

Mass spectrometry and data analysis

• DIA acquisition on the Orbitrap Astral Zoom MS with narrow isolation widths and short injection times to support high duty cycles suitable for fast gradients.
• Raw data analyzed with Proteome Discoverer 3.3 SP1 using DIA-NN Enterprise 2.5.2; default search settings (oxidation variable, carbamidomethylation fixed); triplicate raw files processed jointly per condition (except reproducibility runs processed individually); high-confidence identifications filtered at 1% FDR.

Main results and discussion (Hlavní výsledky a diskuse)

• Identification depth: Across HeLa loads (20–200 ng) and four throughput methods, protein identifications ranged approximately from 6,232 to 9,363 groups; peptide identifications ranged from ~70,643 to ~175,129 unique peptides. The highest depth was achieved with the longest gradient (20.5 min, 60 SPD) and 200 ng loading.
• High throughput capability: The 5.5 cm µPAC HT cartridge supported up to 300 SPD (2.5 min gradient) while still achieving substantial proteome depth (for 20 ng: ~7,849 protein groups and ~106,625 peptide groups reported in one experiment).
• Reproducibility and robustness: Over 100 sequential injections of 200 ng HeLa (with both 180 and 100 SPD methods) BPCs and retention times were highly consistent after initial conditioning. Mean identifications averaged ~8,329 and ~8,749 protein groups and ~123,567 and ~147,847 peptides for the 180 and 100 SPD methods, respectively.
• Longer-term stability: After disconnecting and storing the µPAC cartridge at room temperature for nearly two weeks, the cartridge automatically restored the previously optimized emitter position on reinstallation. Protein identifications varied by <1.9% and peptide identifications by <6.7% between the initial and post-storage experiments (200 ng HeLa).
• Run-to-run variability: Across >200 injections, coefficients of variation (CVs) were low (≈1.6% for proteins and ≈1.8% for peptides), demonstrating excellent quantitative and identification stability suitable for large series measurements.

Key methodological points and figure/table synthesis

• Automated emitter-position optimization and cartridge memory reduced manual alignment errors and improved reproducibility following cartridge exchange or storage.
• Gradient shaping with transient higher flow at method start followed by rapid flow reduction before elution produced a more uniform peptide elution profile and improved instrument productivity (>86% of duty cycle productive).
• Base peak chromatograms presented in the study showed nearly identical peak elution times and intensities after two weeks of storage and across many injections, supporting method robustness.

Practical benefits and applications (Přínosy a praktické využití metody)

• High-throughput proteomics for large cohort studies and screening: methods up to 300 SPD with maintained proteome depth allow scaling of sample numbers without sacrificing data quality.
• Routine laboratory robustness: cartridge-based emitter memory and automated positioning simplify source maintenance and reduce variability due to manual assembly—useful for multi-operator or multi-site labs.
• Reliable quantitative performance: low CVs and stable identifications across many injections make the workflow appropriate for comparative proteomics, longitudinal studies and quality-controlled pipelines.

Future trends and possibilities for deployment (Budoucí trendy a možnosti využití)

• Integration with automated sample preparation and plate-based workflows to further increase end-to-end throughput for population-scale studies.
• Broad adoption of cartridge-based emitters with stored geometry could standardize nanoflow setups across platforms and sites, improving reproducibility in multi-center studies.
• Further optimization of DIA window schemes and fast-scan acquisition strategies on instruments like the Astral Zoom will push depth for short gradients, narrowing the gap between throughput and proteome coverage.
• Potential expansion into clinical research pipelines will require additional validation under regulated conditions, but the demonstrated robustness is a favorable foundation.

Conclusions (Závěr)

• The OptiSpray ion source combined with the µPAC Neo HT cartridge and Orbitrap Astral Zoom MS provides a robust, high-throughput proteomics workflow that maintains deep proteome coverage and excellent reproducibility across rapid gradients.
• Automated emitter optimization and cartridge memory significantly reduce setup variability and support consistent performance even after cartridge removal and ambient storage.
• Overall, this combination is well suited for laboratories requiring reliable, high-throughput bottom-up proteome profiling with minimal manual intervention.

References

• Arrey TN, Reinhardt T, Walker K, Silveira J, Boeser C, Zheng R, Valenta A, Op de Beeck J, Wouters ER, Damoc E. Evaluation of OptiSpray technology and the Orbitrap Astral Zoom mass spectrometer for proteomics applications. Thermo Fisher Scientific; 2026.