Tools and Troubles: Automation of Meso-Scale Proteomics With 3D-Printed Hardware and Investigation of the ESX-4 Secretion System in Mycobacteria

Bottom-up (shotgun) proteomics enables the global identification and quantification of proteins in complex biological samples. This is achieved by subjecting protein extracts from cells to proteolytic digestion and analyzing them via LC-MS/MS. Preparing samples for MS analysis is often tedious and error-prone. Automation can reduce this burden; however, proteomic research often operates at a lower throughput than is needed to justify the cost of automation. Here, I present my novel work on adapting a lower-cost liquid handling robot for bottom-up proteomic sample preparation. In order to address the lack of automation in lower throughput work, I adapted a commercial lab robot, an Andrew+, to perform bottom-up proteomics. I generated custom protocols and 3D-printed adapters to achieve this. I automated preparation, solid-phase extraction, and MALDI target analyte and matrix deposition. I evaluated the performance of the automated methods by comparing samples prepared with the automated method to samples prepared manually in parallel. The automated methods showed a trend of lower recovery but greater reproducibility. I concluded that the most appropriate use case for the automated methods would be in applications that are not limited by sample availability, where the time saved would justify the use of additional sample material. M. abscessus is one of the non-tuberculous bacteria causing growing concern due to its rising prevalence, multidrug resistance, and deadliness in patients with complicating conditions like cystic fibrosis and previous mycobacterial infections. The type VII secretion systems help bacteria survive within the host and perpetuate disease. The type VII secretion system 4 (ESX-4) is regulated through the RNA polymerase sigma factor, SigM. Here, I demonstrate the novel application of a proteo-genetic approach to examine the regulation of the ESX-4 system in M. abscessus. I subjected wildtype, ?sigM, and ?sigM+sigM M. abscessus strains to bottom-up proteomics analysis. I found that ESX-4 is regulated in a sigM-dependent manner and identified additional potential protein targets of sigM regulation.<p></p>