Here we investigate the qualitative and quantitative capabilities of the Orbitrap Astral mass spectrometer for single-shot based LC-MS analysis of different proteome samples with different complexity and dynamic range such as yeast cells, mammalian cells or microbiome.
Yeast and mammalian cell line digests were resuspended in 0.1 % Formic acid to a concentration of 200 ng/µl. Microbiome proteins were extracted and digested using AccelerOme platform. Peptides were separated on a 50 cm µPAC Neo reversed phase column using direct injection mode on a Vanquish Neo UHPLC system. The LC was coupled to the Orbitrap Astral mass spectrometer, operated in either DIA or DDA mode. Based on the complexity of the proteome, different LC gradients (18 and 90 min) and MS methods were used for the data acquisition. The raw data acquired were processed with Proteome Discoverer 3.1.
Due to high ion transmission and extremely high MS2 scan speed and sensitivity of the Orbitrap Astral mass spectrometer, DIA acquisition can be performed with narrow isolation widths. To evaluate this unique platform, we analyzed 3 samples of different complexity: (1) low complexity yeast digest, (2) medium complexity mammalian cell line digest, and (3) high complexity Gut microbiome digest. First results from both DIA and DDA runs show that with 50 ng yeast digest on column, an average of 4,400 protein groups were identified using 18 min gradient (60 SPD). These results show that complete yeast proteome can now be analyzed 3 times faster and with less sample compared to previous published data [A. S.Hebert & A. L. Richards at al. 2014]. A quick comparison of the yeast proteome results shows that DIA method identifies 5% more protein groups and 15% more unique peptides compared to DDA method.
From the mammalian cell line, an average of 8,000 protein groups and > 60,000 unique peptides were identified with 18min gradient (60 SPD) and 50 ng sample on column. Increasing the concentration to 200 ng, the average number of protein groups and unique peptides increased to > 9,000 and 70,000, respectively. The Gut Microbiome standard contains 21 different organisms mixed in different ratios. Preliminary results show that >18,000 protein groups and >100,000 unique peptides were identified from all 21 organisms using 90 min LC gradient representing an increase of 46% protein groups and 25% unique peptides compared to the Orbitrap Exploris 480 leading to higher proteome coverage and higher protein sequence coverage for each organism.