The promise of the Orbitrap ASTRAL mass-spectrometer for proteomics analysis

Over the last two decades, the advent of high-throughput sequencing has revolutionized our access to genomic and transcriptomic data, and thus our understanding of biological systems. On the other hand, mass spectrometry (MS)-based proteomics has undergone a slow steadily technology advancements in terms of sensitivity and robustness. However, the last innovations allow complete proteome coverage, with short turnaround time and reduced sample material. They will facilitate the implementation of the proteomics methods in preclinical and clinical routine practice. In that context, the introduction of the Orbitrap asymmetric track lossless (ASTRAL, Thermo Fisher) analyzer is a major technological leap for MS-based analysis with a scan speed up to 200 Hz, combining sensibility, reproducibility, robustness and high throughput.

Following our recent acquisition of the ASTRAL, we performed multiple experiments to assess the performances of the system in terms of identification of proteins at bulk and single cell levels. The results were compared with our gold standard Exploris 480 (E480). Both mass spectrometers were coupled to the Vanquish Neo using an EasySpray source. Data were analyzed using Proteome Discoverer (PD 3.1, Thermo Fisher).

After injection of 250 ng HeLa digest, the ASTRAL identified 10,000 proteins in only 30 minutes, whereas the E480 identified 7,000 proteins in 60 minutes. By increasing both the sample material 500 ng and the gradient 120 minutes, the E480 could identify up to 8,500 proteins.

We then analyzed different mixtures of 3 commercial digests (HeLa (Thermo), yeast (Thermo) and E. coli (Waters)) to measure the accuracy of the quantification with a fixed and variable proportion for HeLa and the two other species, respectively. We used a 9-minute gradient, corresponding to about 160 samples analyzed per day, to monitor the ASTRAL performance in a high-throughput analysis. The three proteomes were each time identified in the different mixtures with a total of 10,000 proteins identified per mixture. The theoretical ratios were also conserved with a minimum precision of 80%.

Last but not least, we tested the ASTRAL coupled to vanquish Neo in single cell conditions against E480 equipped with field asymmetric ion mobility spectrometry (FAIMS) and Loload column. 250 pg of the previous digest mixtures were injected on the ASTRAL, allowing the identification of 3,500 proteins, despite the absence of the FAIMS. On the other hand, E480 identified up to 2,093 proteins but with four times more material (1 ng).

Altogether, our preliminary results showed that the ASTRAL outperformed the performances of the E480 in terms of depth, identification of proteins and coverage of the proteomes, requiring lower amount of material and shorter turnaround time. We consider that the ASTRAL, and preferentially coupled to the FAIMS, will help to discover new proteins of interest that were never seen and to implement the proteomics in clinical routine.