Merging molecular electron microscopy and mass spectrometry by carbon film-assisted endoproteinase digestion.
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.
Richter
Florian M
FM
Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.
Sander
Bjoern
B
Golas
Monika M
MM
Stark
Holger
H
Urlaub
Henning
H
D000595:Amino Acid Sequence; D002244:Carbon / Q000737:chemistry*; D018835:Chaperonin 10 / Q000737:chemistry / Q000648:ultrastructure; D018834:Chaperonin 60 / Q000737:chemistry / Q000648:ultrastructure; D002853:Chromatography, Liquid; D003432:Cross-Linking Reagents / Q000494:pharmacology; D010450:Endopeptidases / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D008854:Microscopy, Electron / Q000379:methods*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D015203:Reproducibility of Results; D017411:Ribonucleoproteins, Small Nuclear / Q000737:chemistry / Q000378:metabolism
D018835:Chaperonin 10; D018834:Chaperonin 60; D003432:Cross-Linking Reagents; D010455:Peptides; D017411:Ribonucleoproteins, Small Nuclear; D002244:Carbon; D010450:Endopeptidases