Cell-Type-Specific Proteomics: A Neuroscience Perspective

Abstract

Cell-type-specific analysis has become a major focus for many investigators in the field of neuroscience, particularly because of the large number of different cell populations found in brain tissue that play roles in a variety of developmental and behavioral disorders. However, isolation of these specific cell types can be challenging due to their nonuniformity and complex projections to different brain regions. Moreover, many analytical techniques used for protein detection and quantitation remain insensitive to the low amounts of protein extracted from specific cell populations. Despite these challenges, methods to improve proteomic yield and increase resolution continue to develop at a rapid rate. In this review, we highlight the importance of cell-type-specific proteomics in neuroscience and the technical difficulties associated. Furthermore, current progress and technological advancements in cell-type-specific proteomics research are discussed with an emphasis in neuroscience.

Keywords: affinity chromatography; biotinylation; cell type; mass spectrometry; neuron; neuroproteomics; neuroscience; proteomics; proximity labeling.

Figure 1

Methods for cell-type-specific isolation and proteome enrichment. (A) Two methods for specific cell isolation from a total cell population. Animal models can be generated that express fluorescent markers in a cell type of interest. Fluorescent cells can be detected and isolated using fluorescence-activated cell sorting (FACS) or laser capture microdissection (LCM). FACS requires homogenization of tissue prior to cell sorting, while LCM enables cells isolation from intact tissue slices. (B) Basic workflow of induced pluripotent stem cell (iPSC) differentiation. Skin or blood cells are collected from a biological organism of interest and used to generate induced pluripotent stem cells (iPSCs). Factors are then added to iPSCs for differentiation into neural progenitor cells (NPCs). (C) Cell-type-specific labeling methods enable stochastic incorporation of a non-canonical amino acid or puromycin into the target proteome. The cell-type-specific expression of a tRNA synthetase is accomplished either by genetic engineering of a Cre-dependent transgenic mouse (BONCAT/FUNCAT) or via viral transduction (SORT). The incorporated amino acid can be further biotinylated for enrichment prior to LC-MS/MS analysis (BONCAT/SORT) or modified with a fluorescent probe for visualization (FUNCAT). Puromycin labeling occurs through introduction of a cell-type-specific enzyme-tagged antibody (Ab-Tz) followed by an inactive puromycin analog. Activation of puromycin occurs after Tz reacts with the inactive puromycin analog. (D) Experimental workflow for BioID and APEX proximity labeling techniques. BioID or APEX fusion target proteins are expressed in a specific cell type. Reactive biotin is supplemented, and target interacting proteins are biotinylated via BioID or APEX. Biotinylated interactors can be enriched using affinity chromatography techniques with a stationary phase such as streptavidin prior to LC-MS/MS.

Figure 2

Overview of common mass spectrometry-based methods that are currently used for cell-type-specific analyses. Tree includes method type (triangles), name (hexagon) and a list of features associated with each method (rectangle).