Identification of Direct Protein Targets of Small Molecules

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Abstract

Small-molecule target identification is a vital and daunting task for the chemical biology community as well as for researchers interested in applying the power of chemical genetics to impact biology and medicine. To overcome this “target ID” bottleneck, new technologies are being developed that analyze protein–drug interactions, such as drug affinity responsive target stability (DARTS), which aims to discover the direct binding targets (and off targets) of small molecules on a proteome scale without requiring chemical modification of the compound. Here, we review the DARTS method, discuss why it works, and provide new perspectives for future development in this area.

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Most cited references 93

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Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics.

Shao-En Ong, Blagoy Blagoev, Irina Kratchmarova … (2002)

Quantitative proteomics has traditionally been performed by two-dimensional gel electrophoresis, but recently, mass spectrometric methods based on stable isotope quantitation have shown great promise for the simultaneous and automated identification and quantitation of complex protein mixtures. Here we describe a method, termed SILAC, for stable isotope labeling by amino acids in cell culture, for the in vivo incorporation of specific amino acids into all mammalian proteins. Mammalian cell lines are grown in media lacking a standard essential amino acid but supplemented with a non-radioactive, isotopically labeled form of that amino acid, in this case deuterated leucine (Leu-d3). We find that growth of cells maintained in these media is no different from growth in normal media as evidenced by cell morphology, doubling time, and ability to differentiate. Complete incorporation of Leu-d3 occurred after five doublings in the cell lines and proteins studied. Protein populations from experimental and control samples are mixed directly after harvesting, and mass spectrometric identification is straightforward as every leucine-containing peptide incorporates either all normal leucine or all Leu-d3. We have applied this technique to the relative quantitation of changes in protein expression during the process of muscle cell differentiation. Proteins that were found to be up-regulated during this process include glyceraldehyde-3-phosphate dehydrogenase, fibronectin, and pyruvate kinase M2. SILAC is a simple, inexpensive, and accurate procedure that can be used as a quantitative proteomic approach in any cell culture system.

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Large-scale analysis of the yeast proteome by multidimensional protein identification technology.

Michael Washburn, Dirk Wolters, John R R Yates (2001)

We describe a largely unbiased method for rapid and large-scale proteome analysis by multidimensional liquid chromatography, tandem mass spectrometry, and database searching by the SEQUEST algorithm, named multidimensional protein identification technology (MudPIT). MudPIT was applied to the proteome of the Saccharomyces cerevisiae strain BJ5460 grown to mid-log phase and yielded the largest proteome analysis to date. A total of 1,484 proteins were detected and identified. Categorization of these hits demonstrated the ability of this technology to detect and identify proteins rarely seen in proteome analysis, including low-abundance proteins like transcription factors and protein kinases. Furthermore, we identified 131 proteins with three or more predicted transmembrane domains, which allowed us to map the soluble domains of many of the integral membrane proteins. MudPIT is useful for proteome analysis and may be specifically applied to integral membrane proteins to obtain detailed biochemical information on this unwieldy class of proteins.

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Quantitative analysis of complex protein mixtures using isotope-coded affinity tags.

S P Gygi, B Rist, S Gerber … (1999)

We describe an approach for the accurate quantification and concurrent sequence identification of the individual proteins within complex mixtures. The method is based on a class of new chemical reagents termed isotope-coded affinity tags (ICATs) and tandem mass spectrometry. Using this strategy, we compared protein expression in the yeast Saccharomyces cerevisiae, using either ethanol or galactose as a carbon source. The measured differences in protein expression correlated with known yeast metabolic function under glucose-repressed conditions. The method is redundant if multiple cysteinyl residues are present, and the relative quantification is highly accurate because it is based on stable isotope dilution techniques. The ICAT approach should provide a widely applicable means to compare quantitatively global protein expression in cells and tissues.

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Author and article information

Journal

Journal ID (nlm-ta): ACS Chem Biol

Journal ID (publisher-id): cb

Journal ID (coden): acbcct

Title: ACS Chemical Biology

Publisher: American Chemical Society

ISSN (Print): 1554-8929

ISSN (Electronic): 1554-8937

Publication date (Electronic): 16 November 2010

Publication date (Print): 21 January 2011

Volume: 6

Issue: 1

Pages: 34-46

Affiliations

Department of Molecular and Medical Pharmacology, University of California, Los Angeles, California 90095, United States

Author notes

[* ] Corresponding author, jinghuang@ 123456mednet.ucla.edu .

Article

DOI: 10.1021/cb100294v

PMC ID: 3031183

PubMed ID: 21077692

SO-VID: bf11795b-8f0c-46f2-bf80-a487230ca141

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This is an open-access article distributed under the ACS AuthorChoice Terms & Conditions. Any use of this article, must conform to the terms of that license which are available at http://pubs.acs.org.

History

Date received : 23 September 2010

Date accepted : 15 November 2010

Date: 30 November 2010

Date: 21 January 2011

Date: 16 November 2010

Funding

National Institutes of Health, United States

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document-id-old-9 cb100294v

document-id-new-14 cb-2010-00294v

ccc-price

ScienceOpen disciplines: Biochemistry

Keywords: degradomics: the subfield of proteomics devoted to the study of proteases and their biological substrates.,silac: stable isotope labeling of amino acids in cell culture.,target identification: determination of which proteins a particular small molecule binds in the cell.

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ScienceOpen disciplines: Biochemistry

Keywords: degradomics: the subfield of proteomics devoted to the study of proteases and their biological substrates., silac: stable isotope labeling of amino acids in cell culture., target identification: determination of which proteins a particular small molecule binds in the cell.

Identification of Direct Protein Targets of Small Molecules

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Abstract

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Drug_transporters

Most cited references 93

Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics.

Large-scale analysis of the yeast proteome by multidimensional protein identification technology.

Quantitative analysis of complex protein mixtures using isotope-coded affinity tags.

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