1. Chemical Tags for Labeling Proteins Inside Living Cells

2. GFP ：green fluorescent protein
A breakthrough for live cell imaging came with the introduction of fluorescent proteins (FPs) in 1994 as selective, genetic protein tags. The original green fluorescent protein (GFP) from A. victoria is a 238 amino acid protein, which upon folding spontaneously forms a fluorescent chromo- phore by rearrangement and oxidation of Ser, Tyr, and Gly residues in the core of the 11-stranded β-barrel.

3. Advantages:
FPs have been optimized for spectral variation, increased brightness and other properties to provide a wealth of reagents for cell biologists.
FP tags are used routinely to observe the timing and location of protein expression in living cells, often providing significant mechanistic insight

4. Disadvantages:
As ∼30 kD proteins, FPs can disrupt the assembly, interaction,or function of the labeled protein.
FPs typically have broad absorption and emission spectra, making it technically demanding to monitor even just three different proteins simultaneously using multicolor imaging.
FPs can suffer from oligomerization and/or slow folding and chromophore maturation.
It is difficult to manipulate the fluorophore for specialized properties since it is inherent to the sequence of the FP.

5. Protein Labeling via Chemical Tags
Chemical tags have been developed to provide an alternative for labeling proteins with chemical probes directly in living cells.
Chemical tags retain the specificity of protein labeling achieved with FPs through genetic encoding but provide smaller, more robust tags and modular use of organic fluorophores with high photon-output and tailored functionalities.
The first report of a chemical surrogate to FPs for labeling proteins with organic fluorophores in living cells was FlAsH from Tsien and co-workers in 1998.

6. Fig. 1. Synthesis of FLASH (20) and proposed structure of its complex with an a-helical tetracysteine-containing peptide or protein domain. Although the structure is drawn with the i and i 1 4 thiols bridged by one arsenic and the
i + 1 and i + 5 thiols bridged by the other, we cannot rule out the isomeric complex in which one arsenic links the i and i + 1 thiols while the other links the i + 4 and i + 5 thiols.
Griffin, B. A.; Adams, S. R.; Tsien, R. Y. Specific Covalent Labeling of Recombinant Protein Molecules inside Live Cells. Science 1998, 281, 269–272.

7. Protein-based chemical tags
Issues:
The organic fluorophore ligand or substrate must be readily cell-permeable and not binding nonspecifically to endogenous proteins or other biomolecules or, partitioning to particular organelles within the cell.
The synthesis of the ligand or substrate derivatives should be straightforward and minimally disruptive to the receptor binding or enzyme function.
The protein receptor or enzyme should be small, monomeric, and well behaved for minimal perturbation of the biological pathway being studied.
The labelingreaction between the protein tag and the ligand/substrate-probe should be rapid and near quantitative.

9. TMP-tag
Fig. 2. Strategy for site-specific chemical labeling of proteins in vivo. Living cells are transfected with DNA encoding a protein of interest (POI) fused to a receptor domain, eDHFR (1). Upon expression of the receptor fusion, a cell-permeable small-molecule probe consisting of a ligand (TMP) coupled to a detectable tag is added to the cell growth medium (2). The fusion protein binds TMP and the complex can be analyzed in the cell(3).
Miller, L. W.; Cai, Y.; Sheetz, M. P.; Cornish, V. W. In Vivo Protein Labeling with Trimetho- prim Conjugates: A Flexible Chemical Tag. Nat. Methods 2005, 2,255–257.

10. Fig. 3. Heterodimeric conjugates of trimethoprim covalently linked to sensitized terbium chelates bind to Escherichia coli dihydrofolate reductase fusion proteins with nanomolar affinity. Terbium luminescence enables sensitive and time-resolved detection of labeled proteins in vitro and on the surface of living mammalian cells.
Rajapakse, H. E.; Reddy, D. R.; Mohandessi, S.; Butlin, N. G.; Miller, L. W. Luminescent Terbium Protein Labels for Time-Resolved Microscopy and Screening. Angew. Chem., Int. Ed. 2009, 48, 4990–4992.

11. SNAP-tag
Fig. 4. Design strategy for organelle-specific hydrogen peroxide reporters using the SNAP tag methodology.
Srikun, D.; Albers, A. E.; Nam, C. I.; Iavarone, A. T.; Chang, C. J. Organelle-Targetable Fluorescent Probes for Imaging Hydrogen Peroxide in Living Cells Via Snap-Tag Protein Labeling. J. Am. Chem. Soc. 2010, 132, 4455–4465.

12. Fig. 5. Scheme for Sumoylation Assay Based on SNAP Tag-Mediated Translation and RNA Polymerase-Based Amplification.
Yang Y.; Zhang C. Sensitive Detection of Intracellular Sumoylation via SNAP Tag-Mediated Translation and RNA Polymerase-Based Amplification. Anal. Chem. 2012, 84, 1229−1234.

13. HaloTag
Fig. 6. The protein tag (HaloTag) is a modified haloalkane dehalogenase designed to covalently bind to synthetic ligands (HaloTag ligands). The synthetic ligands comprise a chloroalkane linker attached to a variety of useful molecules, such as fluorescent dyes, affinity handles, or solid surfaces.
Los, G. V.; Encell, L. P.; McDougall, M. G.; Hartzell, D. D.; Karassina, N.; Zimprich, C.; Wood,M. G.; Learish, R.; Ohana, R. F.; Urh,M.; Simpson, D.;Mendez, J.; Zimmerman, K.; Otto, P.; Vidugiris, G.; Zhu, J.; Darzins, A.; Klaubert, D. H.; Bulleit, R. F.; Wood, K. V. Halotag: A Novel Protein Labeling Technology for Cell Imaging and Protein Analysis. ACS Chem. Biol. 2008, 3,373–382.

14. Coumarin ligase
Fig. 7. Natural and engineered ligation reactions catalyzed by lipoic acid ligase (LplA). The middle row shows two-step probe targeting via alkyl azide 7 ligation followed by [3 + 2] cycloaddition，and the bottom row shows direct fluorophore ligation by an LplA mutant. LAP = LplA Acceptor Peptide. The red circle represents any probe.
Uttamapinant, C.; White, K. A.; Baruah, H.; Thompson, S.; Fernandez-Suarez, M.; Puthenveetil, S.; Ting, A. Y. A Fluorophore Ligase for Site-Specific Protein Labeling inside Living Cells. Proc. Natl. Acad. Sci. U.S.A. 2010, 107, 10914–10919.

15. Peptide-Based Chemical Tags
Fig. 8. (a) Structures of biotin and ketone 1. (b) Structures of fluorescein hydrazide (FH) and benzophenone-biotin hydrazide (BP). (c) General method for labeling acceptor peptide (AP)-tagged recombinant cell surface proteins with biophysi- cal probes. Biotin ligase (BirA) catalyzes the ligation of ketone 1 to the AP (blue); a subsequ- ent bio-orthogonal ligation between ketone and hydrazide (or hydroxylamine) introduces the probe (green). c b a
Chen, I.; Howarth,M.; Lin,W.; Ting, A. Y. Site-Specific Labeling of Cell Surface Proteins with Biophysical Probes Using Biotin Ligase. Nat. Methods 2005, 2,99–104

16. Acyl-carrier protein (ACP)-based tag
Fig. 9. Sfp-catalyzed biotin labeling of PCP fusion proteins and direct spotting of the labeled protein on avidin glass slides.
Yin, J.; Liu, F.; Li, X.; Walsh, C. T. Labeling Proteins with Small Molecules by Site-Specific Posttranslational Modification. J. Am. Chem. Soc. 2004, 126, 7754–7755.

17. Formylglycine-generating enzyme-based tag
Fig. 10. Site-pecific protein modification using the genetically encoded aldehyde tag. (A) FGE oxidizes a critical cystein to FGly within a conserved 13-amino acid sequence. (B) The aldehyde tag can be transported into a heterologous protein for site-specific modification with chemical probes.
Wu, P.; Shui, W.; Carlson, B. L.; Hu, N.; Rabuka, D.; Lee, J.; Bertozzi, C. R. Site-Specific Chemical Modification of Recombinant Proteins Produced inMammalian Cells by Using the Genetically Encoded Aldehyde Tag. Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 3000–3005.

18. Tetraserine peptide tag
Fig. 11. (A) Scheme illustrating a likely mode of condensation between RhoBo and a compound containing four hydroxyl groups. (B) Apparent equilibrium dissociation constant (Kapp) of complexes between RhoBo and the peptides and monosacchar ides shown.
Halo, T. L.; Appelbaum, J.; Hobert, E.M.; Balkin, D.M.; Schepartz, A. Selective Recognition of Protein Tetraserine Motifs with a Cell-Permeable, Pro-Fluorescent Bis-Boronic Acid. J. Am. Chem. Soc. 2009, 131, 438–439.

19. A N-terminal Cys residue-based tag
Fig. 12. Condensation reactions between cysteine/N-terminal cysteine residues and CBT derivatives in the synthesis of d-luciferin and site-specific protein labeling, and structures of the CBT probes used for labeling.
Ren, H.; Xiao, F.; Zhan, K.; Kim, Y.; Xie, H.; Xia, Z.; Rao, J. A Biocompatible Condensation Reaction for the Labeling of Terminal Cysteine Residues on Proteins. Angew. Chem. 2009, 121, 9838 –9842.

20. Application of Chemical Tags
High-Resolution Imaging Enabled by Chemical Tags
Themes and Variations on Cell Imaging with Chemical Tags.
Chemical Tags Are Biotin/Streptavidin Surrogates for in Vitro Applications.

21. High-Resolution Imaging Enabled by Chemical Tags
Single-molecule imaging:
FIONA(fluorescence imaging with one nanometer accuracy) is a fluorescence technique that is able to localize the position of a single dye within ~1nm in the x-y plane. It is done simply by taking the point spread function of a single fluorophore excited with wide field illumination and locating the center of the fluorescent spot by a two-dimensional Gaussian fit.
High photon-output imaging
SR is a super-resolution imaging technologiesStochastic. SR imaging technologies including PALM (photoactivatable localization microscopy) and STORM (stochastic optical reconstruction microscopy) hinge on photoswitchable fluorophores with high photon-output that enable the locations of subsets of the total fluorophore population to be determined precisely over time.

22. FIONA
Fig. 13. Hand-over-hand model vs Inchworm model for myosinV. In the hand-over-hand model, the rear head passes by the front head, translating a total of 74 nm, while the front head stays stationary. Therefore, heads move alternating 74 and 0 nm steps. If the dye is on the lightchain, it moves 37-2x, followed by37+2x nm, where x is the distance between the dye and the center. In the inchworm model, the dye moves 37 nm regardless to where it is labeled.
Yildiz, A.; Forkey, J. N.;McKinney, S. A.; Ha, T.; Goldman, Y. E.; Selvin, P. R.Myosin VWalks Hand-over-Hand: Single Fluorophore Imaging with 1.5-Nm Localization. Science 2003, 300, 2061–2065.

23. Fig. 14. Preparation and analysis of fluorescently labeled spliceosome subcomplexes by CoSMoS. Integration of two orthogonal tags allows for simultaneous monitoring of two different subcomplexes by CoSMoS.
Hoskins,A.A.;Friedman,L.J.; Gallagher,S.S.;Crawford,D.J.;Anderson,E.G.; Wombacher, R.; Ramirez, N.; Cornish, V.W.; Gelles, J.;Moore,M. J. Ordered and Dynamic Assembly of Single Spliceosomes. Science 2011, 331, 1289–1295.

24. SR
Fig. 15. Microscope. (a) Excitation pulses are followed by stimulated emission depletion pulses for fluorescence inhibition. After passing dichroic mirrors and emission filters, fluorescence is detected through a confocal pinhole by a counting photodiode. (b)Measured excitation PSF. (c)Measured STED-beam-PSF featuring local minimum at the center and intense maxima above and
below the focal plane. Z denotes optic axis. The measurements of b and c are carried out with the pinhole removed.
Klar, T. A.; Jakobs, S.; Dyba, M.; Egner, A.; Hell, S. W. Fluorescence Microscopy with
Diffraction Resolution Barrier Broken by Stimulated Emission. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 8206–8210.

25. PALM
Fig. 16. It is a method for optically imaging intracellular proteins at nanometer spatial resolution. Numerous sparse subsets of photoactivatable fluorescent protein molecules were activated, localized (to ~2 to 25 nanometers), and then bleached. The aggregate position information from all subsets was then assembled into a superresolution image.
Betzig, E.; Patterson, G. H.; Sougrat, R.; Lindwasser, O. W.; Olenych, S.; Bonifacino, J. S.; Davidson, M. W.; Lippincott-Schwartz, J.; Hess, H. F. Imaging Intracellular Fluorescent
Proteins at Nanometer Resolution. Science 2006, 313,1642–1645.

26. STORM
Fig. 17. It is a high-resolution fluorescence microscopy method which based on high-accuracy localization of photoswitchable fluorophores. In each imaging cycle, only a fraction of the fluorophores were turned on, allowing their positions to be determined with nanometer accuracy. The fluorophore positions obtained from a series of imaging cycles were used to reconstruct the overall image. We demonstrated an imaging resolution of 20 nm. This technique can, in principle, reach molecular-scale resolution.
Rust, M. J.; Bates, M.; Zhuang, X. Sub-Diffraction-Limit Imaging by Stochastic
Optical Reconstruction Microscopy (Storm).
Nat. Methods 2006, 3, 793–795.

27. dSTORM
Fig. 18. dSTORM of core histones using the TMP tag. The spatiotemporal resolution of subdiffraction fluorescence imaging has been limited by the difficulty of labeling proteins in cells with suitable fluorophores.
Wombacher, R.; Heidbreder, M.; van de Linde, S.; Sheetz, M. P.; Heilemann, M.; Cornish,V. W.; Sauer, M. Live-Cell Super-Resolution Imaging with Trimethoprim Conjugates. Nat. Methods 2010, 7, 717–719.

28. Themes and Variations on Cell Imaging with Chemical Tags
Unlike FPs, chemical tags are not limited to traditional fluorescent imaging.
Connect to different apparatuses.
Enzyme-mediated peptide tags have been reinvented as unique reporters of endocytosis.
Many creative extensions of chemical tags have been exploited.

29. Connect to electron microscopy
Fig. 19. Pulse-chase labeling of Cx43 with FlAsH and ReAsH and correlative fluorescence and EM images. Oligomers of Cx43 form gap junctions on plasma membranes through which metabolites and signalingmolecules are exchanged between cells. Tetracysteine tags have enabled pulse-chase experiments to observe the dynamic assembly and turnover of junctional plaque with minimal disturbance on Cx43 structure and function.
Gaietta, G.; Deerinck, T. J.; Adams, S. R.; Bouwer, J.; Tour, O.; Laird, D.W.; Sosinsky, G. E.; Tsien, R. Y.; Ellisman, M. H. Multicolor and Electron Microscopic Imaging of Connexin
Trafficking. Science 2002, 296,503–507.

30. Endocytosis reporter
Fig. 20. Cartoon and live cell imaging of the BirA-based peptide tag used to visualize internalization of low density lipoprotein (LDL) receptors. (A) LDL receptors are fused to biotin acceptor peptide (AP) and are expressed both inside and on the surface of the cell. Only cell surface receptors can be labeled with Alexa568 viamonomerized streptavidin (mSA). After endocytosis, cell surface receptors are quenched with QSY quencher. (B) Fluorescence images of cells immediately before and after surface fluorescence quenching (+QSY). (C)With 5min incubation at 37 ℃, some LDL receptors are internalized and thus protected from QSY quenching.
Zou, P.; Ting, A. Y. Imaging Ldl Receptor Oligomerization During Endocytosis Using a Co-Internalization Assay. ACS Chem. Biol. 2011, 6, 308–313.

31. Chromophore assisted laser inactivation (CALI)
Fig. 21. Selected time-lapse sequential DIC images of control (A) and Y27632-treated (B) cells spreading on fibronectin-coated coverslips at early times. White arrows show cytoplasm rounding and shrinkage. Scar bar:20μm.
Cai, Y.; Rossier, O.; Gauthier, N. C.; Biais, N.; Fardin,M. A.; Zhang, X.;Miller, L.W.; Ladoux, B.; Cornish, V. W.; Sheetz, M. P. Cytoskeletal Coherence Requires Myosin-Iia Contractility. J. Cell Sci. 2010, 123,413–423.

32. Detect protein-protein interactions
Fig. 22. Mechanism of S-CROSS: Cross-linking of CLIP-tag and/or SNAP-tag fusion proteins with bifunctional molecules in which the substrates of the two tags are connected via a fluorophore (either Cy5 or Cy3). This permits the irreversible trapping of homo- and heterotypic protein complexes.
Gautier, A.; Nakata, E.; Lukinavicius, G.; Tan, K. T.; Johnsson, K. Selective Cross-Linking of Interacting Proteins Using Self-Labeling Tags. J. Am. Chem. Soc. 2009, 131, 17954–17962.

33. Read out protein folding and association
Determination of Apparent Dissociation Constant (KDapp):
Where, Fobs is the observed fluorescence, Fmin and Fmax are the minimum and the maximum fluorescence value. The Fmax value was obtained by fitting the data to a single exponential expression.
Krishnan, B.; Gierasch, L. M. Cross-Strand Split Tetra-Cys Motifs as Structure Sensors in a Beta-Sheet Protein. Chem. Biol. 2008, 15, 1104–1115.

34. Chemical inducers of dimerization (CIDs)
Fig. 24. The membrane-associated Loc and soluble Cat domains are separated and fused to small molecule binding proteins. In the absence of the CID, the Cat domain has no mechanism for Golgi retention and is secreted from the cell. In the presence of the CID, the Cat domain associates with the Loc domain and is therefore retained in the Golgi compartment where it can act on substrates. In this depiction, the glycosyltransferase is fucosyltransferase 7, which adds fucose to a glycan substrate forming sialyl Lewis x.
Czlapinski, J. L.; Schelle, M. W.; Miller, L. W.; Laughlin, S. T.; Kohler, J. J.; Cornish, V. W.; Bertozzi, C. R. Conditional Glycosylation in Eukaryotic Cells Using a Biocompatible Chemical Inducer of Dimerization. J. Am. Chem. Soc. 2008, 130, 13186–13187.

35. Chemical Tags Are Biotin/Streptavidin Surrogates for in Vitro Application
Fig. 25. Labeling kinetics (TMR ligand) compared to streptavidin-biotin and DhaA.H272F. Reactions between 10 nM protein and 2.5 nM ligand at 25 ℃ were monitored over time by fluorescence polarization. Apparent second-order rate constants shown for GST-HaloTag and streptavidin were calculated using this data, whereas the rate constant for GST-DhaA.H272F was calculated from a reaction between 15 nM protein and 15 nM ligand (data not shown). The calculated binding rate for streptavidin-biotin is consistent with previously reported values
Same as Fig. 6

36. Future Development of the Chemical Tagging Technology
There is an immediate need formultiple orthogonal chemical tags for multicolor imaging in living cells and high photon-output fluorophore tags that work inside the cell.
The realization of the potential of chemical tags to dramatically decrease tag size.
The potential of these modular tags for introducing probes beyond fluorophores is surprisingly largely unexplored.