Introduction
Bottom-up proteomics has proven to be the most suitable technology for high-throughput analysis of very complex biological samples, such as cell lysates or blood. As the obtained data become more and more employed in biomedical research, the challenge of analyzing smallest amounts of samples in the shortest time remains.
An integrated workflow for label-free quantitative proteomic studies has been developed, to meet all these challenges. A multi-organism dataset of proteomes mixed in known ratios was analyzed in DIA mode using an Orbitrap Astral and compared to a similar dataset acquired on an Orbitrap Exploris™ 480 (OE480).
Methods
Yeast and E. coli proteomes were spiked into a human proteome background at different ratios, yielding three proteome mixtures with total protein loads from 100 to 500 ng/injection. Peptides were separated on a 5.5 cm long µPAC Neo High Throughput column in a Trap-and-Elute workflow, using a Vanquish™ Neo UHPLC under sub-microflow conditions. Different gradient lengths were employed, ensuring throughput from 24 to 180 samples per day, depending on the sample amount and complexity. DIA experiments were run on the Orbitrap Astral and on an OE480. Data were analyzed using Proteome Discoverer 3.1 software with Chimerys™ search algorithm.
Preliminary Data
In this work, we performed ultra-fast (up to 200Hz), narrow windows (2 Th) DIA experiments using an Orbitrap Astral. MS1 scans were collected at 240k resolution using the Orbitrap analyzer in parallel with MS2 scans at a resolution of 80k using the new analyzer, Astral. The multi-organism datasets revealed that the Orbitrap Astral enabled the quantification of a total number of 12,763 protein groups (7,683 human, 4,134 yeast and 946 E. Coli) and 126,626 unique peptides (80,394 human, 41,057 yeast, and 5,175 E. Coli) compared to 5,609 protein groups (4,291 human, 923 yeast, and 395 E. Coli) and 34,381 unique peptides (30,162 human, 2,762 yeast, and 1,457 E. Coli) observed using OE480. This represents 2-3-fold increase in the number of protein groups and unique peptides quantified, compared to the OE480. This increase was achieved using ~2-fold shorter gradient (24 min for the new mass spectrometer and 45 min for the OE480). Median absolute deviation values for yeast peptide and protein ratios were 0.17 and 0.06, respectively, for the Orbitrap Astral, and 0.17 and 0.13 for the OE480. In summary, the workflow developed for DIA label-free quantification using the Orbitrap Astral enables wide proteome coverage at high throughput, while maintaining high quantification accuracy.