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The emergence of proteome-wide technologies: systematic analysis of proteins comes of age

Nature Reviews Molecular Cell Biology volume 15, pages 453–464 (2014)Cite this article

Subjects

Key Points

  • Emerging technologies over the past decade have enabled efforts in systems biology to expand to whole-proteome analysis.

  • New methods for tracking proteins and their dynamic behaviour include the ease of creation of genetically encoded fluorescent fusion proteins, robust antibody production, emergence of whole-proteome mass spectrometry and robotic infrastructures.

  • The various large-scale technologies enable the evolution from the analysis of single proteins to whole proteomes, from population-level analysis to single cell studies and from static to dynamic measurements.

  • As proteomic data accumulate, they highlight that an enormous proportion of cellular regulation is post-translational. Hence, adding proteomic data to current cellular models provides a better description of the inherent complexity in biological systems.

  • By integrating molecular cues and buffering the constant fluctuations of the cell, protein-level changes are used to reduce the necessity of inducing a costly transcriptional response.

  • Future challenges should focus on: first, adapting technologies to be user-friendly, and cost- and time-effective, which will make proteomics the new transcriptomics; second, creating more single-cell technologies; third, pushing forward technologies that allow both mRNA and protein tracking in parallel; fourth, the development of analysis pipelines that enable the integration of proteome level data with additional systematic layers of cellular information (that is, glycomics, lipidomics and metabolomics).

Abstract

During the lifetime of a cell proteins can change their localization, alter their abundance and undergo modifications, all of which cannot be assayed by tracking mRNAs alone. Methods to study proteomes directly are coming of age, thereby opening new perspectives on the role of post-translational regulation in stabilizing the cellular milieu. Proteomics has undergone a revolution, and novel technologies for the systematic analysis of proteins have emerged. These methods can expand our ability to acquire information from single proteins to proteomes, from static to dynamic measures and from the population level to the level of single cells. Such approaches promise that proteomes will soon be studied at a similar level of dynamic resolution as has been the norm for transcriptomes.

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Figure 1: Modes of post-transcriptional regulation to control the functional protein pool of the cell.

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Acknowledgements

Work in the authors' laboratory is supported by a European Research Council (ERC) C Starting Grant (260395). M.S. is a European Molecular Biology Organization (EMBO) Young Investigator.

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Authors and Affiliations

  1. Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, 7610001, Israel

    Michal Breker & Maya Schuldiner

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  1. Michal Breker
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Correspondence to Maya Schuldiner.

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Glossary

Tandem affinity purification epitope

(TAP epitope). An epitope, against which specific and high-affinity antibodies exist, that can be used for purification and western blot analysis.

Shotgun proteomics

Techniques for the parallel identification of all proteins in a sample using high-performance liquid chromatography followed by mass spectrometry.

Fluorescence in situ hybridization

(FISH). A technique for detecting nucleic acids (DNA or mRNA) inside a single cell by use of a complementary oligonucleotide probe that is detected by fluorescence microscopy.

Ribosome profiling

A strategy for sequencing ribosome-protected mRNA fragments to provide a genome-wide map of translated proteins.

Protein microarray

A method to study protein modifications or physical interactions. Proteins are immobilized on glass slides and exposed to probe molecules (usually fluorescently labelled). A reaction between the probe and the arrayed protein emits a signal that is detected by a laser scanner.

Protein-fragment complementation assays

Assays for measuring protein–protein interactions based on the premise that when two proteins interact they also bring into proximity any protein fragments attached to them, thus enabling the fragments to complement each other and to fold into an active reporter protein.

Two-hybrid screens

Types of protein-fragment complementation assay in which each half of a split Gal4 transcription factor (the DNA-binding domain or the activation domain) is fused to one of two proteins of interest (bait and prey). Physical proximity of the two proteins enables the reconstitution of the Gal4 transcription factor, thus leading to transcriptional activation.

Split-ubiquitin assays

Types of protein-fragment complementation assay in which each half of a ubiquitin enzyme is fused to one of two proteins of interest (bait and prey). One half of the ubiquitin is also fused to a transcription factor. Physical proximity enables the reconstitution of the split protein into a ubiquitin moiety that is recognized by endogenous ubiquitin-directed proteases, which cleave between the ubiquitin and the transcription factor. The cleaved transcription factor can relocate to the nucleus and activate a reporter gene.

Split-DHFR assays

Types of protein-fragment complementation assay in which each half of a split dihydrofolate reductase (DHFR) enzyme is fused to one of two proteins of interest (bait and prey). Physical proximity enables the reconstitution of the split protein into a functional DHFR enzyme, which makes it possible for cells to grow in the presence of an inhibitor of the endogenous and essential DHFR enzyme (such as methotrexate).

Split-YFP approaches

Types of protein-fragment complementation assay in which each half of a split fluorescent protein (YFP, GFP or cyan fluorescent protein) is fused to one of two proteins of interest (bait and prey). Physical proximity enables the reconstitution of the split protein into a functionally fluorescent product.

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Breker, M., Schuldiner, M. The emergence of proteome-wide technologies: systematic analysis of proteins comes of age. Nat Rev Mol Cell Biol 15, 453–464 (2014). https://doi.org/10.1038/nrm3821

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