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bioRxiv · 10.1101/2023.06.07.544024

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

Akkulak, H.·Goc, G.·Ince, H. K.·Kabasakal, B. V.·Ozcan, S.
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Abstract

Recent 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 utilized commonly used XL-MS software tools to elucidate the protein interactions within a membrane protein complex containing FtsH, HflK, and HflC, over-expressed in E.coli. The MS data were processed using MaxLynx, MeroX, MS Annika, xiSEARCH, and XlinkX software tools. The number of identified inter- and intra-protein cross-links varied among software. Each interaction was manually checked using the raw MS and MS/MS data and distance restraints to verify inter- and intra-protein cross-links. A total of 37 inter-protein and 148 intra-protein cross-links were determined in the FtsH-HflK-HflC complex. The 59 of them were new interactions on the lacking region of recently published structures. These newly identified interactions, when combined with molecular docking and structural modeling, present opportunities for further investigation. The results provide valuable information regarding the complex structure and function to decipher the intricate molecular mechanisms underlying the mega FtsH-HflK-HflC complex. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=123 SRC="FIGDIR/small/544024v2_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@8e10f5org.highwire.dtl.DTLVardef@fb809borg.highwire.dtl.DTLVardef@1b84df3org.highwire.dtl.DTLVardef@105c8e6_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical AbstractC_FLOATNO The overall workflow of XL-MS analysis: the steps of XL-MS studies applied to the two samples taken from different stages of purification process of mega membrane protein complex FtsH-HflC-HflK. C_FIG

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Akkulak, H., Goc, G., Ince, H. K., Kabasakal, B. V., Ozcan, S.. 2023-06-07. Combined workflow for XL-MS data processing: Application to the FtsH-HflK-HflC membrane protein complex. https://doi.org/10.1101/2023.06.07.544024

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