Luiz Fernando Arruda Santos¹, Till Reinhardt², Tabiwang Arrey², Anna Pashkova², Eduard Denisov², Johannes Petzoldt², Bernd Hagedorn², Yannick Mueller², Immo Colonius², Arne Kreutzmann², Hamish Stewart², Christian Hock², Eugen Damoc²

Introduction:

The recently introduced Thermo Scientific™ Orbitrap™ Astral™ mass spectrometer has significantly expanded the scale and scope of proteomics experiments, advancing discovery and translational research. Building on this innovation, we demonstrate the performance of a modified Orbitrap Astral mass spectrometer at throughputs ranging from 30 to 300 samples per day (SPD) using HeLa digest standard. The modified instrument features a higher acquisition rate (270 Hz vs. 200 Hz) achieved through improved ion optics settling times and faster ion transfers, increased sensitivity via parallel accumulation, and deeper coverage through enhanced internal spectral processing. Comparative analysis with the classic Orbitrap Astral MS reveals significant improvements in peptide and protein group identifications enabling even deeper and higher quality coverage at high throughput whilst maintaining precise quantitation.

Methods:

Chromatographic separation of the Thermo Scientific™ Pierce™ HeLa Digest standard was performed using an IonOpticks Aurora Ultimate XT 25x75 C18 column (30 SPD) in a direct injection setup or an Thermo Scientific™ EASY-Spray™ ES906 150 mm x 150 µm C18 column (300, 180, 100, and 60 SPD) in a Trap&Elute setup, connected to a Thermo Scientific™ Vanquish™ Neo UHPLC system. Data-independent acquisition (DIA) with narrow isolation windows (2-10 Th) was conducted using both the classical and modified Thermo Scientific™ Orbitrap™ Astral™ MS and a Thermo Scientific™ EASY-Spray™ source. Data processing was carried out with Thermo Scientific™ Proteome Discoverer™ software using the CHIMERYS™ algorithm and LPGF protein validator node, as well as Biognosys Spectronaut 19 using human FASTA (42k sequences) and contaminants FASTA file.

Preliminary data:

High-throughput LC-MS methods using the modified Orbitrap Astral enabled the quantification of >90,000 peptides and >8,000 protein groups at 200 ng and a throughput of 300 SPD (3 files analyzed together). Longer gradient times of 30 SPD quantified >11,000 protein groups and >200,000 peptides at 200 ng load, providing even more comprehensive and deeper coverage of complex biological samples. Depending on throughput and injection load, the modified instrument consistently generated an increase in precursor and peptides (10-25 %), as well as protein group IDs (5-15%),compared to the classical Orbitrap Astral MS, while maintaining high quantitative precision. Notably, the highest gains were observed with the shortest gradients and in general identifications are of higher quality. This enhanced capability allows researchers to further streamline their high-throughput proteomic pipelines.

Novel aspect:

High-throughput proteomics performance analysis on a novel, improved generation of the Thermo Scientific™ Orbitrap™ Astral™ mass spectrometer platform.

Agradecimentos: