From Discovery to Process Control: Positioning DIA Proteomics in Biomanufacturing Pipelines

Abstract Gaining control of existing biomanufacturing chassis organisms, such as Escherichia coli K12, and novel isolates, such as the salt tolerant Halomonas bluephagenesis sp TD01 studied here may be facilitated by the investigation and monitoring of their metabolic and regulatry processes, particularly through proteomics. Here we consider the performance of a range of typically available proteomics platforms across a range of price points to map chasis organisms’ metabolic pathways. A set of model bacterial samples was prepared from E. coli and H. bluephagenesis sp. TD01 in 1:2 and 2:1 ratios and analysed using five LC-MS systems. Data from the timsTOF HT, Exploris 480, ZenoTOF 7600 and Select Series MRT were processed through DIANN and MSStats. Data from the Vion was processed through Skyline then MSstats. Of the 8,222 proteins identified across all samples analysed (4,401 proteins from E. coli; 3,821 from Halomonas sp. TD01), the TimsTOF and Exploris were able to achieve extensive proteome coverage quantifying 5.5k and 5k proteins respectively, with the ZenoTOF, Waters MRT and the legacy Waters Vion respectively quantifying 3.5k, 1.3k, and ∼850 proteins at 1% FDR. Proteins comprising the pathways of chassis’ organisms core metabolism critical to biomanufacturing were quantified with all instruments, demonstrating suitability of these platforms to explore their manipulation in the context of biomanufacturing. Competing Interest Statement The authors have declared no competing interest. Footnotes To correct the spelling of one author name.