Differential Visual Proteomics: Enabling the Proteome-Wide Comparison of Protein Structures of Single-Cells

Proteins are involved in all tasks of life, and their characterization is essential to understand the underlying mechanisms of biological processes. We present a method called "differential visual proteomics" geared to study proteome-wide structural changes of proteins and protein-complexes between a disturbed and an undisturbed cell or between two cell populations. To implement this method, the cells are lysed and the lysate is prepared in a lossless manner for single-particle electron microscopy (EM). The samples are subsequently imaged in the EM. Individual particles are computationally extracted from the images and pooled together, while keeping track of which particle originated from which specimen. The extracted particles are then aligned and classified. A final quantitative analysis of the particle classes found identifies the particle structures that differ between positive and negative control samples. The algorithm and a graphical user interface developed to perform the analysis and to visualize the results were tested with simulated and experimental data. The results are presented, and the potential and limitations of the current implementation are discussed. We envisage the method as a tool for the untargeted profiling of the structural changes in the proteome of single-cells as a response to a disturbing force.

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Related concepts (8)

Cryogenic electron microscopy

Cryogenic electron microscopy (cryo-EM) is a cryomicroscopy technique applied on samples cooled to cryogenic temperatures. For biological specimens, the structure is preserved by embedding in an environment of vitreous ice. An aqueous sample solution is applied to a grid-mesh and plunge-frozen in liquid ethane or a mixture of liquid ethane and propane. While development of the technique began in the 1970s, recent advances in detector technology and software algorithms have allowed for the determination of biomolecular structures at near-atomic resolution.

Transmission electron microscopy

Transmission electron microscopy (TEM) is a microscopy technique in which a beam of electrons is transmitted through a specimen to form an image. The specimen is most often an ultrathin section less than 100 nm thick or a suspension on a grid. An image is formed from the interaction of the electrons with the sample as the beam is transmitted through the specimen. The image is then magnified and focused onto an imaging device, such as a fluorescent screen, a layer of photographic film, or a sensor such as a scintillator attached to a charge-coupled device.

Protein complex

A protein complex or multiprotein complex is a group of two or more associated polypeptide chains. Protein complexes are distinct from multidomain enzymes, in which multiple catalytic domains are found in a single polypeptide chain. Protein complexes are a form of quaternary structure. Proteins in a protein complex are linked by non-covalent protein–protein interactions. These complexes are a cornerstone of many (if not most) biological processes.