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 subnanometer 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.

Cryo-EM samples of gas-phase purified protein assemblies using native electrospray ion-beam deposition

“…Complementary information, in particular from structurally sensitive mass spectrometry (MS) based techniques, including native MS, hydrogen deuterium exchange (HDX), ion mobility spectrometry (IMS), crosslinking mass spectrometry (XL-MS), and different forms of ion activation, can help to nd optimal sample conditions, interpret and rene 3D structures, reveal native interactions, and provide information on small ligands and exible protein regions that may have lower resolution in cryo-EM density maps, due to conformational or chemical heterogeneity. 6,[8][9][10][11][12][13][14][15] These studies indicate the potential of establishing a direct link between MS and cryo-EM, to allow for improved correlation between complementary chemical information and high-resolution structures. Electrospray ion-beam deposition (ES-IBD), also known as so landing or preparative mass spectrometry, allows ultra-clean and molecularly pure samples to be prepared on solid surfaces for further analysis.…”

“…Complementary information, in particular from structurally sensitive mass spectrometry (MS) based techniques, including native MS, hydrogen deuterium exchange (HDX), ion mobility spectrometry (IMS), crosslinking mass spectrometry (XL-MS), and different forms of ion activation, can help to find optimal sample conditions, interpret and refine 3D structures, reveal native interactions, and provide information on small ligands and flexible protein regions that may have lower resolution in cryo-EM density maps, due to conformational or chemical heterogeneity. 6,8–15…”

Real-time library search increases cross-link identification depth across all levels of sample complexity

“…Cross-linking mass spectrometry (XL-MS) is a versatile tool that allows mapping of stable and transient protein-protein interactions (PPIs) in complex biological systems 1 . Moreover, it offers structural information on single proteins and protein complexes, thereby complementing structural biological techniques of higher resolution such as X-Ray crystallography and cryo-electron microscopy 2 . In XL-MS, the structural information is derived as distance restraints given by the length of the cross-linker and of the specific amino acid side chains it targets.…”

Unbiased spatial proteomics with single-cell resolution in tissues

“…Beyond genomics and MS-based shotgun proteomics, more modalities with a spatial dimension could be addressed. This even extends to the determination of the structures of protein complexes in situ and their intracellular localization (Klykov et al, 2022).…”