Since its introduction, Orbitrap-based-MS have and are still playing a pivotal role in many different research areas such as proteomics, metabolomics, biopharma, etc. Each of these applications comes with different challenges to mass spectrometers. To address some of these challenges, new technological developments, as well as improvements on existing mass spectrometers is a necessity. Here we evaluated the Orbitrap Exploris 480 mass spectrometer for proteomics applications, with focus on data dependent acquisition and data independent acquisition. Additionally, we assess the use of the PHiSDM processing algorithm on TMT11plex labeled samples.
The initial results from the comparison of DDA methods between the Q Exactive HF-X and the Orbitrap Exploris 480 MS gives a yield of approximately 10% more peptide and protein identifications when using the same sample and experimental conditions. For the DIA experiments, we were able to reduce the sample amount by a factor of 2 and still achieve the same results as on the Q Exactive HF-X. Furthermore, to demonstrate the qualitative capability of the quadrupole Orbitrap, a two-proteome mixture was analyzed. We determined the precision and accuracy for LFQ using yeast spiked into a constant HeLa background on the Orbitrap Exploris 480 MS and found the highest deviation in accuracy to be only around 10% with high precision. On the Orbitrap Exploris 480 MS a dedicated algorithm, phase-constrained spectrum deconvolution method(SDM), has been implemented to reach higher resolution in shorter times. This is especially beneficial when using reporter ion quantitation such as TMT11plex, as it enables the use of shorter transients to achieve the same mass resolution relative to conventional FT based approaches without sacrificing data quality. With the PHiSDM algorithm activated we were able to boost the total number of quantified proteins in a HeLa digest labeled with TMT 11plex, by 25 %.