Label-free visual proteomics: Coupling MS- and EM-based approaches in structural biology

Abstract

Combining diverse experimental structural and interactomic methods allows for the construction of comprehensible molecular encyclopedias of biological systems. Typically, this involves merging several independent approaches that provide complementary structural and functional information from multiple perspectives and at different resolution ranges. A particularly potent combination lies in coupling structural information from cryoelectron microscopy or tomography (cryo-EM or cryo-ET) with interactomic and structural information from mass spectrometry (MS)-based structural proteomics. Cryo-EM/ET allows for sub-nanometer visualization of biological specimens in purified and near-native states, while MS provides bioanalytical information for proteins and protein complexes without introducing additional labels. Here we highlight recent achievements in protein structure and interactome determination using cryo-EM/ET that benefit from additional MS analysis. We also give our perspective on how combining cryo-EM/ET and MS will continue bridging gaps between molecular and cellular studies by capturing and describing 3D snapshots of proteomes and interactomes.

Keywords: crosslinking MS; cryo-EM; cryo-ET; integrative structural biology; label-free visual proteomics; protein-protein interactions; structural proteomics.

Figure 1.

Schematic overview of EM and MS approaches described in this review, and illustrated by two domains of AMPA protein in complex with auxiliary subunit γ5 (PDB ID 7RYY). The initial sample is an intact organism, tissue, or cells. SPA cryo-EM and nMS require purification of the protein or protein complex of interest, while ex-situ XL-MS and Shotgun Proteomics work with both purified proteins or protein complexes and highly heterogeneous samples like whole cell lysates. In-situ XL-MS and cryo-ET can be applied to intact samples, however, for samples thicker than ~500 nm thinning with FIB-milling must be performed prior to cryo-ET imaging. Each method provides complementary information about the biological system of interest, which are combined later to answer varieties of biological questions.

Figure 2.

Data workflows for generating molecular encyclopedias. (A) Ex-situ data generation workflow for samples prepared from whole cell lysates. First, samples are lysed and fractionated to reduce the sample complexity. Next, each fraction is subjected to XL-MS or Shotgun-EM in parallel. From the generated data, structural models of protein complexes are generated in two ways. In the case of integrative structural modelling, structural models of individual proteins are generated computationally and then models of protein complexes are assembled with input from XL-MS and Shotgun-EM. Alternatively, structural models of protein complexes are generated computationally, and then the best models are chosen based on XL-MS and Shotgun-EM data. (B) In-situ data generation workflow for creating molecular encyclopedias. Samples are first crosslinked with a membrane-permeable reagent and processed as in the proteome-wide XL-MS workflow. For acquiring in-situ cryo-ET data, samples are vitrified in their intact state either by plunge-freezing or HPF. In the case of thin-edge plunge-frozen cell experiments, cryo-ET data are collected from the thin cellular areas where FIB-milling is not required. For all other cryo-ET experiments, including HPF samples and thick cellular regions, an additional FIB-milling step is performed to obtain thin lamellae.

Figure 3.

Label-free visual proteomics with in-situ Shotgun Proteomics, XL-MS, and cryo-ET. (A) Cross-section of a cellular tomogram with ribosome densities highlighted in green. In this case, ribosome quantification performed with Shotgun Proteomics would identify the relative amount of ribosomes present in the sample as opposed to quantification by means of visual proteomics which would omit the ribosomes not present in the field of view (analogous to Albert et al., 2020). Identification of the most abundant proteins with Shotgun MS allows for correlation of the most often detected protein in the tomogram. (B) Cross-section of a cellular tomogram with ribosome highlighted in green and unidentified ribosome-associated densities highlighted in red. In this case, XL-MS would pinpoint the most probable ribosome interaction partner and help to identify those densities (analogous to O’Reilly et al., 2020). (C) Cross-section of a cellular tomogram highlighting the heterogeneous gingipains proteins in orange. Additional conformational stabilization of gingipains by XL-MS reagents would reduce flexibility and help in the high-resolution cryo-ET pipeline (analogous to Ke et al., 2020). (D) Cross-section of a cellular tomogram with membrane-bound chemosensory arrays and unidentified associated densities highlighted in yellow and purple, respectively. In this case, XL-MS alongside with quantitative Shotgun MS data generated in parallel would help to identify unassigned densities, protein complex orientation, and higher-order protein organization through protein self-links (analogous to Leung et al., 2021). The cross-sections through the cellular tomograms are taken from the publicly available Atlas of Bacterial and Archaeal Cell Structure (https://www.cellstructureatlas.org) with the protein densities assigned based on the information from the atlas. Figures 3A, B, D represent data from M. hungatei (10.22002/D1.1357) and Figure 3C are acquired on P. gingivalis (10.22002/D1.1578).

Figure 4.

The front view of a G3 Titan Krios column showing an available octagon port (A, B) and a back view of the same column showing an adaptor for a Residual Gas Analyzer (RGA) that is connected to the vacuum manifold (C, D). There is an extra octagon port available in the front of a G3 Titan Krios TEM column with ample space within the enclosure (not shown). (E) Image of the RGA itself which allows for measuring of low-molecular weight contaminants in the TEM column. Instrument parts containing Mass Analyzers are highlighted with red boxes. (F) Schematic representation of a combined EM and MS instrument for real-time localization of ROIs. The sample is ionized in the area close to the ROI with high-precision and then ions are guided towards the advanced mass analyzer which is schematically represented as an Orbitrap. After confirming the presence of the protein target in the vicinity, the ROI is either imaged by TEM or subjected to FIB-milling.