Subcellular Transcriptomics and Proteomics: A Comparative Methods Review

Abstract

The internal environment of cells is molecularly crowded, which requires spatial organization via subcellular compartmentalization. These compartments harbor specific conditions for molecules to perform their biological functions, such as coordination of the cell cycle, cell survival, and growth. This compartmentalization is also not static, with molecules trafficking between these subcellular neighborhoods to carry out their functions. For example, some biomolecules are multifunctional, requiring an environment with differing conditions or interacting partners, and others traffic to export such molecules. Aberrant localization of proteins or RNA species has been linked to many pathological conditions, such as neurological, cancer, and pulmonary diseases. Differential expression studies in transcriptomics and proteomics are relatively common, but the majority have overlooked the importance of subcellular information. In addition, subcellular transcriptomics and proteomics data do not always colocate because of the biochemical processes that occur during and after translation, highlighting the complementary nature of these fields. In this review, we discuss and directly compare the current methods in spatial proteomics and transcriptomics, which include sequencing- and imaging-based strategies, to give the reader an overview of the current tools available. We also discuss current limitations of these strategies as well as future developments in the field of spatial -omics.

Keywords: cellular fractionation; imaging; proximity labeling; spatial proteomics; spatial transcriptomics.

Graphical abstract

Fig. 1

Microscopy-based imaging approaches for subcellular proteomics or transcriptomics, focusing on the probing strategies.A, traditional antibody staining involves probing subcellular targets (such as the mitochondrial substructure) using monoclonal antibodies. These may be directly conjugated to a fluorescent label (direct immunofluorescence) or with a fluorescently labeled secondary antibody (indirect immunofluorescence). To determine subcellular location of proteins, an antibody against an organelle marker or a dye must be used alongside an antibody against the protein of interest. Then analysis can be performed to determine and quantify the colocalization of these antibodies/dyes. B, fluorescent protein reporters, such as GFP, can be genetically engineered to be fused and expressed with a target gene/protein of interest. Therefore, allowing confocal imaging of molecules that have no antibody or require live-cell imaging. In MS2 labeling systems for RNA, fluorescent reporter proteins can be genetically fused to MCP. C, RNA aptamers are an alternative to MS2 systems for labeling RNA, which allow for fusion of an RNA structure that binds and stabilizes an exogenous fluorescent molecule (e.g., DFHBI). RNA aptamers can be used either as affinity reagents or as reporters. D, in situ hybridization (ISH) employs a variety of antisense nucleic acid probes for the detection of RNA of interest in permeabilized and fixed cellular material. Recent ISH strategies have allowed for highly multiplexed experimental designs using molecular barcoding (e.g., seqFISH and MERFISH). DFHBI, 3,5-difluoro-4-hydroxybenzylideneimidazolidinone; MCP, bacteriophage MS2 coat protein; MERFISH, multiplexed error-robust FISH; seqFISH, sequential barcoding FISH.

Fig. 2

Alternative imaging for subcellular proteomics and/or transcriptomics, which couple technologies in MS, microfluidics, and/or microdissection.A, instrumentation coupling flow cytometry and microscopy allows for multiplexing of several protein–RNA targets using fluorescent labels, gaining both spatial and single-cell information. B, microlaser ablation and ionization of molecules, such as peptides, lipids, or metabolites, directly from tissue or cell culture sample enables label-free acquisition of mass spectra across each “pixel” of sample. Very rich datasets but still have poor resolution because of current technical limitations. C, similar to MSI, microlaser ablation allows for acquisition of spectra per “pixel” of a sample. Though, this method has improved subcellular resolution and uses labeling of antibodies conjugated to non-naturally occurring metal isotopes to quantify ∼40 target proteins/RNAs of interest. The metal isotope signals have less signal overlap than fluorescent methods allowing improved multiplexing than traditional antibody probing. MSI, MS imaging.

Fig. 3

Sequencing-based approaches to subcellular proteomics and transcriptomics. The approaches consist of biochemical organellar separation (A and B) or biotinylation of proximal molecules to a bait protein (C). A, quantifying proteins/RNAs in a targeted organelle-enrichment preparation (via centrifugation or detergents) against crude contaminant samples can infer resident proteins/RNAs of the organelle of interest. Quantification of enriched samples can be performed using MS or RNA-Seq. B, more extensive sequential centrifugation or detergent strategies can determine cell-wide residence of proteins/RNAs. The quantitative profiles of proteins/RNAs across the fractions aid identification of their localization by using organellar markers and machine learning techniques. C, a bait protein of interest (e.g., associated with a particular subcellular localization) is fused to an enzyme that catalyzes the biotinylation of proximal proteins/RNAs in the cell once the substrate (e.g., biotin) is added to the cells in vivo. The biotinylated molecules can be purified and analyzed using either MS or RNA-Seq.