Merging Molecular Electron Microscopy and Mass Spectrometry by Carbon Film-assisted Endoproteinase Digestion

Abstract

Many fundamental processes in the cell are performed by complex macromolecular assemblies that comprise a large number of proteins. Numerous macromolecular assemblies are structurally rather fragile and may suffer during purification, resulting in the partial dissociation of the complexes. These limitations can be overcome by chemical fixation of the assemblies, and recently introduced protocols such as gradient fixation during ultracentrifugation (GraFix) offer advantages for the analysis of fragile macromolecular assemblies. The irreversible fixation, however, is thought to render macromolecular samples useless for studying their protein composition. We therefore developed a novel approach that possesses the advantages of fixation for structure determination by single particle electron microscopy while still allowing a correlative compositional analysis by mass spectrometry. In this method, which we call "electron microscopy carbon film-assisted digestion", macromolecular assemblies are chemically fixed and then adsorbed onto electron microscopical carbon films. Parallel, identically prepared specimens are then subjected to structural investigation by electron microscopy and proteomics analysis by mass spectrometry of the digested sample. As identical sample preparation protocols are used for electron microscopy and mass spectrometry, the results of both methods can directly be correlated. In addition, we demonstrate improved sensitivity and reproducibility of electron microscopy carbon film-assisted digestion as compared with standard protocols. We show that sample amounts of as low as 50 fmol are sufficient to obtain a comprehensive protein composition of two model complexes. We suggest our approach to be an optimization technique for the compositional analysis of macromolecules by mass spectrometry in general. Molecular & Cellular Proteomics 9:1729 -1741, 2010.An increasing number of proteins are nowadays recognized to fulfill their cellular tasks as part of macromolecular machines (1). Recent advances in the isolation procedures allow purification of many of these assemblies for further biochemical and structural analysis (2-4). Thereby, MS of peptides derived by digestion of the proteins of the assemblies (5, 6) or MS of intact assemblies (7, 8) is used to determine the protein composition, whereas electron microscopy (EM) 1 can be used to provide information about the structure of the assembly using computational averaging and reconstruction techniques (9). Available protocols to combine information derived by MS and EM therefore either analyze the endoproteolytic digestion products (10 -13) or the intact assembly (14). However, the sample preparation protocols typically differ between EM and MS. Although for MS the sample is either directly digested in solution (6), subsequent to gel electrophoretic separation of the sample (15-17) or used in an intact form (7,8), EM typically requires the adsorption of the sample to carbon film (13, 18 -20). In the case of sample heterogeneity, the utilization of thes...

A Pipeline for Determining Protein–Protein Interactions and Proximities in the Cellular Milieu

“…This low molar ratio is optimized for stabilization of native interactions, efficient affinity isolation, and minimal interference with MS readout. Although glutaraldehyde has been widely used for stabilization purposes in electron and optical microscopy (5,30,32,60,61), it has been seldom used with MS in the cellular context, and in these cases, it has been used largely for visualization of low molecular mass metabolites (62). Indeed, in the MS context, glutaraldehyde has often been considered detrimental (63).…”

“…Recently, glutaraldehyde has been applied in the "GraFix" protocol in which purified protein complexes are subjected to centrifugation through a density gradient that also contains a gradient of glutaraldehyde (30,31), allowing for optimal stabilization of authentic complexes and minimization of nonspecific associations and aggregation. GraFix has also been combined with mass spectrometry on purified complexes bound to EM grids to obtain a compositional analysis of the complexes (32), thereby raising the possibility that glutaraldehyde can be successfully utilized in conjunction with AC in complex cellular milieux directly.…”

A reversibly photoswitchable GFP-like protein with fluorescence excitation decoupled from switching

“…Tryptic peptides were separated by nano liquid chromatography (LC), mixed with matrix, spotted onto MALDI target and analyzed by MALDI MS on a 4800 Proteome Analyser (ABSciex), as described previously 42 . MSMS spectra were annotated manually.…”

Structure of glutaraldehyde cross-linked ryanodine receptor

“…However, within in the last few years several groups have used chemical fixatives (mainly glutaraldehyde) to stabilize large macromolecular complexes and determine their structures by single particle cryo-EM (Carlo et al, 2010; Lee et al, 2003; Richter et al, 2010; Southworth and Agar, 2011). In particular the “GraFix” (Kastner et al, 2008) procedure has proved useful for preparing various cytoplasmic complexes for cryo-EM including the ribosome, spliceosomes and anaphase promoting complex.…”

“…Weakly binding RyR ligands generally do not form sufficiently stable complexes with RyR in vitro for study by single particle cryo-EM. Glutaraldehyde has been used to stabilize large fragile cytoplasmic complexes for single particle cryo-EM (Carlo et al, 2010; Lee et al, 2003; Richter et al, 2010; Southworth and Agar, 2011; Yu et al, 2008), and could similarly be used to stabilize RyR or components of ECC. However this approach has not been tried with RyR before.…”