High-throughput proteomics holds great promise, however, faces several challenges, including limited sample acquisition, slow and cumbersome sample preparation, and long acquisition times. Our Heat and Beat (HnB) method1, an end-to-end high-throughput proteomic sample preparation method, has successfully addressed sample preparation challenges, allowing the processing of numerous samples within an hour. However, current-generation mass spectrometers still face a bottleneck due to extended mass spectrometry (MS) acquisition times. The next-generation Thermo Orbitrap Astral Mass Spectrometer is expected to address this issue, significantly reducing sample analysis time from 90 minutes, as seen in existing workflows, to as little as 8 minutes
To test the sensitivity, speed, and quantitation accuracy of the Orbitrap Astral mass spectrometer, we used the HnB protocol to prepare E. coli, HEK-293, and yeast digests. Three pools of the 3-proteome mixed sample were generated using these digests, with varying ratios of E. coli and yeast across the pools while maintaining a constant 50% ratio of HEK. Subsequently, the three pools underwent analysis using 60 SPD (24 minutes), 100 SPD (14.4 minutes), and 180 SPD (8 minutes) workflows. Additionally, the Sciex TripleTOF 6600 and Sciex ZenoTof 7600 were used to analyse the pools with 15-minute, 30-minute, and 90-minute runs, and their performance was used to benchmark that of the Orbitrap Astral.
While the Sciex TripleTOF 6600 identified just over 5000 protein groups across a 90-minute run, the Zenotof 7600 enhanced proteome coverage further, identifying well over 6000 proteins across the three pools in just 15 minutes. The Orbitrap Astral demonstrated superior depth of coverage, identifying over 15000 proteins in a 24-minute run (60 SPD), with even the shortest workflow of 180 SPD identifying just under 14000 protein groups.
Quantitative accuracy assessment on the Orbitrap Astral revealed an average error of less than 0.1% for human proteins and less than 2% for yeast. Furthermore, 90% of proteins exhibited a coefficient of variation (CV) of less than 20%, while 80% had a CV of less than 10%. Both MS1 and MS2 level quantitation displayed a median CV of 5% or less.
The Orbitrap Astral Mass Spectrometer, when combined with the Heat and Beat sample preparative technique, facilitates high-throughput proteomic sample analysis with remarkable coverage and sensitivity, offering rapid results within a condensed timeframe.