Cross-linking mass spectrometry for mapping protein complex topologies in situ

Abstract

Cross-linking mass spectrometry has become an established technology to provide structural information on the topology and dynamics of protein complexes. Readily accessible workflows can provide detailed data on simplified systems, such as purified complexes. However, using this technology to study the structure of protein complexes in situ, such as in organelles, cells, and even tissues, is still a technological frontier. The complexity of these systems remains a considerable challenge, but there have been dramatic improvements in sample handling, data acquisition, and data processing. Here, we summarise these developments and describe the paths towards comprehensive and comparative structural interactomes by cross-linking mass spectrometry.

Progress toward Proteome-Wide Photo-Cross-Linking to Enable Residue-Level Visualization of Protein Structures and Networks In Vivo

“…Finally, studies that employ affinity purification or organelle isolation would likely be preferrable to undifferentiated lysates. 95 Although the ability to search a proteome-wide database with photo-cross-linkers is an important milestone demonstrated here, undifferentiated lysates typically overrepresent high-abundance proteins (such as the ribosome) and are likely not the best path forward to delve deeper into the proteome's dynamic range. 95 To summarize, we present new molecular tools, coupled with an experimental and computational workflow, that enable reliable proteome-wide identification of photo-cross-linked peptides.…”

“…95 Although the ability to search a proteome-wide database with photo-cross-linkers is an important milestone demonstrated here, undifferentiated lysates typically overrepresent high-abundance proteins (such as the ribosome) and are likely not the best path forward to delve deeper into the proteome's dynamic range. 95 To summarize, we present new molecular tools, coupled with an experimental and computational workflow, that enable reliable proteome-wide identification of photo-cross-linked peptides. This study has piloted these tools to produce a small, partial PPI network of E. coli but nevertheless one that is resolved down to the residue-level.…”

“…Future experiments will need to be more exacting in this choice of parameters. Finally, studies that employ affinity purification or organelle isolation would likely be preferrable to undifferentiated lysates . Although the ability to search a proteome-wide database with photo-cross-linkers is an important milestone demonstrated here, undifferentiated lysates typically overrepresent high-abundance proteins (such as the ribosome) and are likely not the best path forward to delve deeper into the proteome’s dynamic range …”

“…First, given the complexity of photo-cross-linked proteomes, it is likely that even greater fractionation than we employed here would be necessary to mitigate co-isolation problems. 95 A likely candidate is 2D fractionation combining SEC (as done here) with strong-cation exchange, which has proven successful for other high-complexity cross-linking experiments. 96 Second, our experiments would have likely benefitted from tighter instrument calibration and higher resolution on MS 2 scans.…”

A special issue ofEssays in Biochemistryon structural mass spectrometry

“…The spacer arm of the reagent constitutes a distance restraint between these cross-linked residues that can be deployed to model structures, validate models, or compare with other data [ 19 , 20 ]. XL-MS technology has found broad application, including for the study of proteins/assemblies in vitro and in cell, but faces several analytical challenges as outlined here by Lee and O’Reilly [ 21 ]. Similarly, new advances are occurring to study protein–nucleic acid assemblies using XL-MS methodologies as described by Steinmetz et al [ 22 ].…”