Combined workflow for XL-MS data processing: Application to the FtsH-HflK-HflC membrane protein complex

Abstract

ABSTRACTRecent advances in mass spectrometry (MS) yielding sensitive and accurate measurements along with developments in software tools have enabled the characterization of complex systems routinely. Thus, structural proteomics and cross-linking mass spectrometry (XL-MS) have become a useful method for structural modeling of protein complexes. Here we assessed the utility of commonly used XL-MS software tools, XlinkX, MS Annika, MeroX, and MaxLynx to elucidate the protein interactions within a membrane protein complex containing FtsH, HflK, and HflC, over-expressed inE.coli. The MS data were processed using various XL-MS software-tools. The comparison involved both software performance criteria such as processing time, computer usage, and number of identified inter and intra-protein interactions. Each interaction was manually checked using the raw MS/MSMS data to verify inter- and intra-protein interactions. Total 106 interactions of HflC, 96 interactions of FtsH, and 23 interactions of HflK, including the FtsH-HflK-HflC complex, with other proteins were determined. Approximately 93% of the interactions were found in the purified complex sample, which can be combined with molecular docking for single or complex protein structural modeling. In addition to XL-MS, according to protein mapping and network studies, genes encoding proteins were in 60 and 108 different categories for the purified protein complex and the solubilized membrane sample. The top 15 biological processes were determined, 60% of which are involved in protein translation and quality control. Our study will provide a complete workflow for XL-MS studies of protein complexes, an overview of XL-MS software.