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Selective cross-linking of coinciding protein assemblies by in-gel cross-linking mass-spectrometry

ABSTRACT: Cross-linking mass spectrometry has developed into an important method to study protein structures and interactions. The in-solution cross-linking workflows involve time and sample consuming steps and do not provide sensible solutions for differentiating cross-links obtained from co-occurring protein oligomers, complexes, or conformers. Here we developed a cross-linking workflow combining blue native PAGE with in-gel cross-linking mass spectrometry (IGX-MS). This workflow circumvents steps, such as buffer exchange and cross-linker concentration optimization. Additionally, IGX-MS enables the parallel analysis of co-occurring protein complexes using only small amounts of sample. Another benefit of IGX-MS, demonstrated by experiments on GroEL and purified bovine heart mitochondria, is the substantial reduction of undesired over-length cross-links compared to in-solution cross-linking. We next used IGX-MS to investigate the complement components C5, C6, and their hetero-dimeric C5b6 complex. The obtained cross-links were used to generate a refined structural model of the complement component C6, resembling C6 in its inactivated state. This finding shows that IGX-MS can provide new insights into the initial stages of the terminal complement pathway.

SUBMITTER: Mr. Johannes, F Hevler

PROVIDER: S-SCDT-EMBOJ-2020-106174 | biostudies-other |

REPOSITORIES: biostudies-other

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Project description:With the goal of expanding the very limited toolkit of cross-linking agents available for nucleic acids and their protein complexes, we evaluated the merits of a wide range of bifunctional agents that may be capable of reacting with the functional groups characteristic of these types of biopolymers. The survey specifically focused on the ability of test reagents to produce desirable inter-molecular conjugates, which could reveal the identity of interacting components and the position of mutual contacts, while also considering a series of practical criteria for their utilization as viable nucleic acid probes. The survey employed models consisting of DNA, RNA, and corresponding protein complexes to mimic as close as possible typical applications. Denaturing polyacrylamide gel electrophoresis (PAGE) and mass spectrometric (MS) analyses were implemented in concert to monitor the formation of the desired conjugates. In particular, the former was used as a rapid and inexpensive tool for the efficient evaluation of cross-linker activity under a broad range of experimental conditions. The latter was applied after preliminary rounds of reaction optimization to enable full-fledged product characterization and, more significantly, differentiation between mono-functional and intra- versus inter-molecular conjugates. This information provided the feedback necessary to further optimize reaction conditions and explain possible outcomes. Among the reagents tested in the study, platinum complexes and nitrogen mustards manifested the most favorable characteristics for practical cross-linking applications, whereas other compounds provided inferior yields, or produced rather unstable conjugates that did not survive the selected analytical conditions. The observed outcomes will help guide the selection of the most appropriate cross-linking reagent for a specific task, whereas the experimental conditions described here will provide an excellent starting point for approaching these types of applications. As a whole, the results of the survey clearly emphasize that finding a universal reagent, which may afford excellent performance with all types of nucleic acid substrates, will require extending the exploration beyond the traditional chemistries employed to modify the constitutive functional groups of these vital biopolymers.

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Selective cross-linking of coinciding protein assemblies by in-gel cross-linking mass-spectrometry

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