Increased Sub-Proteome Coverage of Myxococcus xanthus using a combination of 1D and 2D-chromatography incorporating Mobility-enabled Data Independent Acquisition

摘要

1. Proteins were identified from three sub-proteomes of Myxococcus xanthus using conventional and mobility-enabled data acquisition. 2. The use of mobility-enabled HDMS~E data independent acquisition improved the number of protein identifications from an identical sample over 2-fold, with no additional instrument time required, even with as little sample material as 60 ng. 3. The use of 3-step 2D RP/RP HDMS~E acquisition further increased protein identification rates by over 2-fold combined with an increase in the average number of peptides identified per protein and average sequence coverage. 4. Sample loading for the HDMS~E experiments was not optimised for this study and further increases in the number of identifications could be possible. 5. The protein observations agree well with the presumed mechanism of predation by M. xanthus. 6. OMVs and cell supernatants are enriched for hydrolytic enzymes including lipases, proteases and murein hydrolases likely to be important for prey cell lysis. 7. The existing tools for predicting extracellular localisation of proteins in M. xanthus were ineffective furthermore the results did not agree between tools. 8. Metabolic functionalities identified in the M. xanthus secretome suggest that of the molecules released by prey lysis, the most important metabolites for M. xanthus are proteins/peptides, which are partially metabolised outside the cell prior to uptake. 9. Proteins previously rejected by replication filtering of >1 could be included in the final protein output with some confidence in this study if they were identified by >2 peptides and achieved a PLGS score >1500. Further studies would be required to validate this observation on more complex samples from a variety of sources.