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

Importance of the topic

Proteomics by LC-MS is essential for deep characterization of complex protein mixtures across research, pharmaceutical, and industrial workflows. High-throughput, robust, and reproducible nano/low-flow LC-MS implementations are critical to scale proteome profiling to large sample cohorts while preserving sensitivity and identification depth. The OptiSpray ion source combined with the µPAC Neo High Throughput (HT) cartridge and the Orbitrap Astral Zoom mass spectrometer was developed to address practical bottlenecks of emitter positioning, inter-run variability, and throughput without sacrificing proteomic depth.

Study objectives and overview

This evaluation aimed to assess the analytical performance, robustness, and throughput capabilities of the Thermo Scientific OptiSpray Ion Source with the integrated µPAC Neo HT (5.5 cm) cartridge for bottom-up proteome profiling on an Orbitrap Astral Zoom MS. Key goals included: demonstrating reliable emitter positioning and repositioning, measuring identification depth across a range of HeLa digest loads (20–200 ng) and LC throughputs (300, 180, 100, 60 samples per day, SPD), and evaluating long-term stability and run-to-run reproducibility.

Methodology

• Chromatography: Vanquish Neo UHPLC in trap-and-elute mode with a µPAC Neo HT 5.5 cm analytical cartridge and PepMap Neo 5 µm C18 trap (300 µm × 5 mm). Gradients were tuned to four throughput regimes: 2.5 min (300 SPD), 5.8 min (180 SPD), 10.8 min (100 SPD), and 20.5 min (60 SPD). Flow rates ranged from 3 µL/min down to 0.75 µL/min; the 300 SPD method used a constant 3 µL/min while other methods used higher initial flow followed by rapid decrease to concentrate peptide elution and improve productivity.
• Ion source and emitter handling: µPAC Neo HT cartridge contains an integrated tapered emitter (15 µm). An automated emitter-position optimization procedure in the instrument control software set an optimal emitter position; the cartridge stores this position enabling reproducible repositioning after removal and reinstallation.
• Mass spectrometry: Orbitrap Astral Zoom MS operated in data-independent acquisition (DIA) mode with narrow isolation windows tailored for the throughput methods. Full-scan and DIA settings were chosen to maximize identifications while maintaining short cycle times appropriate for the faster gradients.
• Sample preparation: Thermo Scientific Pierce HeLa digest reconstituted with 0.015% DDM and 0.1% TFA to produce a 100 ng/µL stock; injections were prepared to deliver 20, 50, 100, and 200 ng loads. Transfer line: 55 cm × 20 µm connecting cartridge to source.
• Data processing: Proteome Discoverer 3.3 SP1 with DIA-NN Enterprise 2.5.2. Searches used the Human UniProt database (approx. 20,607 entries) and standard settings (carbamidomethylation static, oxidation dynamic). Results were filtered at 1% FDR (high-confidence identifications). Triplicates per condition were generally processed together; reproducibility experiments were processed per-file (no match-between-runs).

Used instrumentation

• Thermo Scientific OptiSpray Ion Source with automated emitter-position optimization and cartridge memory.
• Thermo Scientific µPAC Neo High Throughput (HT) analytical cartridge, 5.5 cm, integrated replaceable tapered emitter (15 µm).
• Thermo Scientific Vanquish Neo UHPLC System configured in trap-and-elute mode with PepMap Neo trap cartridge.
• Thermo Scientific Orbitrap Astral Zoom mass spectrometer operated in DIA mode.
• Software: Proteome Discoverer 3.3 SP1 with DIA-NN Enterprise 2.5.2 for DIA data processing.

Main results and discussion

• Identification depth: Across HeLa loadings (20–200 ng) and throughput methods (300–60 SPD), protein group identifications ranged roughly from 6,232 to 9,363 and peptide identifications from approximately 70,643 to 175,129, demonstrating that even very short gradients (2.5 min; 300 SPD) can yield thousands of protein identifications from low-ng loads.
• Representative performance: At 200 ng and the longest gradient (20.5 min, 60 SPD) the study reported ~9,363 protein groups and ~175,129 peptides; at 20 ng using the fastest 2.5 min gradient (300 SPD) several thousand protein groups (>7,800) were still identified, illustrating sensitivity retention across throughputs.
• Chromatography and productivity: The use of higher initial flow followed by rapid flow reduction prior to peptide elution produced a more even peptide elution profile and LC–MS productivity above 86% for the evaluated methods. Base peak chromatograms showed consistent, narrow elution windows adapted to each method.
• Emitter reproducibility and cartridge memory: The automated emitter position optimization and cartridge-stored position reduced variability after removal/reinstallation. After nearly two weeks stored at room temperature and then reinstalled, the cartridge returned to the optimized position automatically and produced highly consistent chromatograms after conditioning.
• Stability and reproducibility: Over 100–200 injections of 200 ng HeLa digest (100 and 180 SPD methods), the system produced highly consistent base peak chromatograms and identification counts with low coefficients of variation: ~1.6% CV for protein groups and ~1.8% CV for peptides in extended runs. After two weeks of cartridge storage, protein identifications varied by ≤1.9% and peptides by ≤6.7% relative to initial experiments.

Benefits and practical applications

• High throughput: The µPAC Neo HT cartridge enabled analysis up to 300 samples per day while retaining substantial proteomic depth, supporting large-scale discovery or QC workflows where throughput is a priority.
• Robustness and ease-of-use: Automated emitter optimization and cartridge memory reduce operator-dependent variability, simplify cartridge replacement, and shorten time-to-data in high-throughput environments.
• Reproducibility: Low inter-run and long-term variability make the configuration suitable for longitudinal studies, large cohorts, or workflows requiring consistent quantitation across many injections.
• Flexibility: Adjustable flow profiles and multiple gradient lengths allow balancing throughput and depth to match experimental requirements—from rapid screening to deeper profiling.

Future trends and applications

• Integration with laboratory automation and sample preparation pipelines to further scale throughput and reduce manual handling errors.
• Continued optimization of DIA acquisition strategies and software (e.g., advanced neural-network driven deconvolution) to increase identification and quantitation fidelity on fast gradients.
• Expansion into regulated and clinical research environments following dedicated validation studies (note: current products are designated for general laboratory use, not for in vitro diagnostics without further validation).
• Adaptation of cartridge and emitter designs for even higher robustness, longer lifetime, and suitability for diverse sample matrices, including challenging clinical specimens.
• Potential application to single-cell or very low-input proteomics by combining high-transmission ion sources with optimized trapping/enrichment and sensitive MS instrumentation.

Conclusions

The OptiSpray Ion Source combined with the µPAC Neo HT cartridge and the Orbitrap Astral Zoom MS provides a high-throughput, robust, and reproducible solution for bottom-up proteomics. Automated emitter optimization with stored position information minimizes variability and enables reliable cartridge interchangeability. The platform achieves significant proteome coverage across a wide range of sample loads and gradient lengths while maintaining excellent run-to-run stability and long-term performance after storage, making it attractive for large-scale proteomic studies and high-throughput screening workflows.

Reference

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.