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Review
. 2014 Feb;42(2):299-311.
doi: 10.1007/s10439-013-0878-3. Epub 2013 Aug 14.

Chemical tools for temporally and spatially resolved mass spectrometry-based proteomics

Kai P Yuet  1 , David A Tirrell
Affiliations
Review

Chemical tools for temporally and spatially resolved mass spectrometry-based proteomics

Kai P Yuet et al. Ann Biomed Eng. 2014 Feb.

Abstract

Accurate measurements of the abundances, synthesis rates and degradation rates of cellular proteins are critical for understanding how cells and organisms respond to changes in their environments. Over the past two decades, there has been increasing interest in the use of mass spectrometry for proteomic analysis. In many systems, however, protein diversity as well as cell and tissue heterogeneity limit the usefulness of mass spectrometry-based proteomics. As a result, researchers have had difficulty in systematically identifying proteins expressed within specified time intervals, or low abundance proteins expressed in specific tissues or in a few cells in complex microbial systems. In this review, we present recently-developed tools and strategies that probe these two subsets of the proteome: proteins synthesized during well-defined time intervals--temporally resolved proteomics--and proteins expressed in predetermined cell types, cells or cellular compartments--spatially resolved proteomics--with a focus on chemical and biological mass spectrometry-based methodologies.

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Figures

Figure 1
Figure 1
Cells can be metabolically labeled with a combination of Aha and/or stable isotopic variants of arginine and lysine in 5 workflows: standard SILAC (top left), pulse labeling with heavy amino acids (top middle), pSILAC (top right), BONCAT (bottom left) and QuaNCAT (bottom right).
Figure 2
Figure 2
Structures discussed in this review: amino acids for stable isotopic labeling (top row), methionine and analogs that are substrates for wild-type methionyl-tRNA synthetases (second row), methionine analogs that require the expression of mutant methionyl-tRNA synthetases for proteomic incorporation (third row), and uridine and uracil as well as their thio-substituted analogs 4-thiouridine and 4-thiouracil (last row).
Figure 3
Figure 3
(Top) Both puromycin and its alkyne-functionalized analog O-propargyl-puromycin incorporate into nascent polypeptide chains on translating ribosomes, resulting in premature termination of nascent polypeptide chains. (Bottom) Sectioning of mouse small intestine showed that OP-Puro labeling occurred primarily in cells in the crypts and the cells at the base of the villi. (Adapted with permission from Liu et al., Proc. Natl. Acad. Sci. U.S.A., 109, 413–418, 2012. Copyright 2012 Proceedings of the National Academy of Sciences USA.)
Figure 4
Figure 4
(Top) Cell-selective BONCAT performed in a mixture of cells. Restricting expression of a mutant synthetase to a certain cell restricts Anl labeling to that cell (highlighted in blue). Proteins synthesized in cells (highlighted in gray) that do not express the mutant synthetase are neither labeled nor detected following enrichment. (Bottom) Cell-selective labeling in mixtures of bacterial and mammalian cells. (a) In Anl-containing mixed cultures of E. coli and mouse alveolar macrophages, only E. coli cells constitutively expressing the mutant NLL-EcMetRS were labeled by TAMRA-alkyne. Macrophages were labeled with Mitotracker Deep Red and displayed low TAMRA-alkyne background emission. (b) In Aha-containing mixed cultures of E. coli and mouse alveolar macrophages, both wild-type E. coli cells and macrophages exhibited strong TAMRA-alkyne emission; incorporation of Aha occurs in both cell types. (c) Mixed cell lysate was subjected to conjugation with alkyne-functionalized biotin, and labeled proteins were enriched by NeutrAvidin affinity chromatography. Immunoblotting of unbound flow-through (FT), washes (W1, W3, W5) and eluent (E) reveals enrichment of the bacterial marker protein GFP. (Adapted with permission from Ngo et al., Nat. Chem. Biol., 5, 715–717, 2009. Copyright 2009 Nature Publishing Group.)
Figure 5
Figure 5
(Top) Selective labeling of the mitochondrial matrix proteome in living cells requires 1) genetically targeting APEX to the mitochondrial matrix (mito-APEX), 2) initiating biotinylation by adding biotin-phenol and H2O2 to the medium, and 3) stopping biotinylation by cell fixation or lysis. (Middle) In human embryonic kidney cells, only mitochondria that expressed mito-APEX and were exposed to both biotin-phenol and H2O2 contained biotinylated proteins (stained with NeutrAvidin-Alexa Fluor 647). Both confocal fluorescence imaging (Middle) and stochastic optical reconstruction microscopy (STORM) (Bottom) showed that biotinylated proteins (stained with Streptavidin-Cy3/Cy5 for STORM images) overlapped with mito-APEX only in the mitochondrial matrix. (Adapted with permission from Rhee et al., Science, 339, 1328–1331, 2013. Copyright 2013 The American Association for the Advancement of Science.)
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