Target IdentiÞcation by Diazirine Photo-Cross-Linking …contentscm.com/tevarepository/DownLoadFiles/A-F/Curr Protoc Chem... · Target IdentiÞcation by Diazirine Photo-Cross-Linking

Target IdentiÞcation by DiazirinePhoto-Cross-Linking and ClickChemistryAndrew L. MacKinnon1 and Jack Taunton1,2

1Program in Chemistry and Chemical Biology and Department of Cellular and MolecularPharmacology, University of California San Francisco, San Francisco, California2Howard Hughes Medical Institute, University of California San Francisco, San Francisco,California

ABSTRACT

Target identiÞcation is often the rate-determining step in deciphering the mechanism ofaction of biologically active small molecules. Photo-afÞnity labeling (PAL) representsa useful biochemical strategy for target identiÞcation in complex protein mixtures. Thisunit describes the use of alkyl diazirine-based photo-afÞnity probes and Cu(I)-catalyzedclick chemistry to covalently label and visualize the targets of biologically active smallmolecules. A general method for afÞnity puriÞcation of probe-modiÞed proteins, usefulfor identiÞcation of protein targets, is also described. Curr. Protoc. Chem. Biol. 1:55-73C© 2009 by John Wiley & Sons, Inc.

Keywords: photo-afÞnity labeling � diazirine � click chemistry � target identiÞcation �

afÞnity puriÞcation

INTRODUCTION

Target identiÞcation is often the rate-determining step in deciphering the mechanism ofaction of biologically active small molecules. Photo-afÞnity labeling (PAL) representsa useful biochemical strategy for target identiÞcation in complex protein mixtures. PALuses an analog of a biologically active small molecule, known as a photo-afÞnity probe,that bears photo-reactive and reporter functional groups, to identify macromolecularbinding partners. The photo-afÞnity probe is designed and synthesized based on SAR(structure-activity relationships) of a parent small molecule having known biologicalactivity. During PAL, the photo-afÞnity probe is incubated with a protein mixture andirradiated with UV light. Irradiation of the photo-reactive group generates a highlyreactive chemical species (e.g., carbene, nitrene, or radical) that covalently cross-linksthe photo-afÞnity probe to its macromolecular binding partner(s) based upon the closeproximity of the two constructs. Photo-cross-linked protein targets are then visualizedby means of the reporter group (e.g., ßuorophore, biotin, or radioactive label). Covalentbond formation between the probe and targets enables the subsequent puriÞcation andidentiÞcation of the targets using techniques such as SDS-PAGE, immunoprecipitation,biotin-streptavidin afÞnity puriÞcation, and mass spectrometry. AfÞnity puriÞcation ofprotein targets is often difÞcult with non�covalently bound small molecules, especiallythose with low to moderate binding afÞnity. The challenges are compounded with smallmolecules that target integral membrane proteins, which often show decreased functionafter solubilization with detergents, a prerequisite for afÞnity puriÞcation.

There are several photoreactive functional groups frequently used in PAL (e.g., ben-zophenone, trißuoromethyl phenyl diazirine, aryl azide). Like most useful photo-afÞnitygroups, the alkyl diazirine (Fig. 1A, I) is activated at a wavelength of light (∼355 nm)that is not damaging to protein(s). However, the alkyl diazirine holds unique advantages.

Current Protocols in Chemical Biology 1: 55-73, December 2009Published online December 2009 in Wiley Interscience (www.interscience.wiley.com).DOI: 10.1002/9780470559277.ch090167Copyright C© 2009 John Wiley & Sons, Inc.

Target ID byCrosslinking andClick Chemistry

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NH

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1. protein mixture

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RN2 R

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protein target protein target

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biotin/TAMRA

biotin/TAMRA-azide

CuSO4, TCEP, TBTA

Figure 1 (A) Generalized scheme for photo-afÞnity labeling and detection using a diazirine and alkyne-containing photo-afÞnity probe (I). UV irradiation of the diazirine generates a carbene intermediate (II) that covalently cross-links to theprotein target (III). The adduct is then detected by conjugation with an azide-containing reporter group under click chemistryconditions (IV). (B) Structures of the natural product HUN7293 ( 1), photo-afÞnity probe (2), and the photostable controlcompound (3).

First, it is compact in size, being nearly isosteric to a methyl group, and is accessedsynthetically via an alkyl ketone. This allows installation of the diazirine at positions ofa small molecule that would not tolerate larger, aryl-based photoreactive groups. Sec-ond, the carbene intermediate formed upon photoactivation of the diazirine (Fig. 1A,II) rapidly inserts into X-H bonds (X = N, S, O), as well as C-H bonds, to form stablecovalent insertion products (Brunner, 1993). When not poised for insertion into bondsof the macromolecular target, the alkyl carbene intermediate undergoes rapid quenchingby solvent or internal rearrangement to a stable oleÞn side product (Ford et al., 1998).The alkyl diazirine is stable toward acidic and basic conditions and toward ambientlight encountered during routine chemical synthesis. Several improved methods for thesynthesis of alkyl diazirines starting from alkyl ketone precursors have been recentlyreported (MacKinnon et al., 2007; Bond et al., 2009). Heterobifunctional amine-reactivealkyl diazirine cross-linkers, as well as alkyl diazirine-containing amino acid analogs,are commercially available (Pierce, Thermo ScientiÞc).

Cu(I)-catalyzed click chemistry is an important method for bioconjugation of probe-labeled proteins with reporter groups (Best, 2009). During the click reaction, Cu(I)catalyzes a highly selective 1,3 dipolar cycloaddition reaction between a terminal alkynegroup and an azide group to form a stable triazole product (Fig. 1A, III, IV). The terminalalkyne is typically present in the small-molecule probe, while the azide is present in aßuorescent or biotinylated reporter group. Alternatively, the azide can be incorporated


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into the probe and the alkyne incorporated into the reporter. However, this arrangementhas been shown to produce higher background labeling of proteins (Speers and Cravatt,2004). Following covalent labeling of protein targets via a (latent) chemically reactivemoiety within the probe, probe-modiÞed proteins are conjugated to the azide-bearingreporter under click chemistry conditions (Fig. 1A, IV). The reporter group is thusintroduced after covalent bond formation between the probe and target protein. Thisapproach thereby avoids directly introducing a bulky reporter into the small-moleculeprobe, which could perturb the interaction between probe and target. The terminal alkyne(or azide) is extremely compact and therefore minimally perturbs the structure of thesmall molecule, while providing the chemical functionality necessary for detection andafÞnity puriÞcation of targets. A variety of azide and alkyne reporters designed for use inbioconjugate click reactions have been described (Speers and Cravatt, 2004), and manyare commercially available (Invitrogen).

The Basic Protocol presented below describes a method for photo-afÞnity labeling anddetection of photo-cross-linked proteins in complex protein mixtures. The method re-quires a photo-afÞnity probe that bears both an alkyl diazirine photoreactive group anda terminal alkyne group. The scope and limitations of the method, as well as essentialcontrols, parameters, and variables, are discussed. Key design strategies that lead to thesynthesis of photo-afÞnity probe 2 (labeled in Fig. 1B), as well as a summary of pros andcons of commonly used photoreactive groups, are also discussed. The Basic Protocoldescribes the method applied on an analytical scale, followed by a Support Protocol thatdescribes scale-up of the reactions, post�click chemistry work-up, and afÞnity puriÞca-tion of labeled proteins using monomeric avidin agarose or antibodies directed againstcarboxy-tetramethylrhodamine (TAMRA). The afÞnity-puriÞcation protocol is useful forpurifying and identifying unknown protein targets of biologically active small molecules.

STRATEGIC PLANNING

Design and synthesis of a photo-afÞnity probe can be one of themajor challenges of apply-ing PAL to small-molecule target identiÞcation. Structure-activity-relationships (SAR)of the parent molecule typically guide the choice and placement of the photoreactive orreporter groups within the parent scaffold. For example, in designing photo-afÞnity probe2 (Fig. 1B), a detailed SAR study of the HUN-7293 scaffold (Chen et al., 2002) revealedthat the N-methoxytryptophan side chain at position 5 (Fig. 1B) could be replaced witha smaller, phenylalanine side chain without signiÞcantly altering its biological activity.While this suggested that a phenyl azide at this position might also preserve biologicalactivity, photoactivation of the phenyl azide requires a wavelength of light (∼260 nm)that is damaging to protein structures. The SAR study also suggested that a larger aro-matic photoreactive group at this position, such as benzophenone, would signiÞcantlyreduce biological activity. To preserve biological activity, we therefore sought a compact,hydrophobic photoreactive group that could be placed into one of the many hydrophobicalkyl side chains of the molecule (Fig. 1B). The diazirine represented a suitable choice.Being nearly isosteric with a methyl group, the diazirine was intended to replace a termi-nal methyl group of a leucine residue in HUN-7293. To accomplish this, we synthesizeda diazirine-containing leucine analog, known as photo-leucine (Suchanek et al., 2005),starting from an alkyl ketone precursor, and used this precursor in the total synthesis ofphoto-afÞnity probe 2 (MacKinnon et al., 2007).

We also required a method a detect photo-cross-linked proteins. The SAR indicateda tolerance for smaller side chains at position 1. We therefore installed a propargylgroup at this position to enable detection of photo-cross-linked proteins using clickchemistry. In parallel, we also synthesized a photostable control compound (labeled 3 inFig. 1B) that was used in control experiments for distinguishing background from speciÞc


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Table 1 Comparison of Commonly Used Photoreactive Groups

Photoreactive group BeneÞts Downsides

Benzophenone Photoactivation at ∼350 nm is reversible,leading to high cross-linking yields withproteins. Selective for insertion into C-Hσ bonds over bulk solvent (Dorman andPrestwich, 1994). Chemically stable.

Large size. Reported to selectively react withmethionine residues in proteins leading toinaccurate determination of probe-binding sites(Wittelsberger et al., 2006).

Trißuoromethylphenyl diazirine

Generates a highly reactive carbeneintermediate upon photoactivation at∼350 nm. Photoinsertion of the carbeneinto proteins can proceed in high (>70%)yield (Brunner, 1993).

Relatively large size. Insertion products may bereversible under some conditions (Platz et al., 1991).Can undergo UV light-induced rearrangement toelectrophilic diazo isomer (Brunner, 1993), leadingto nonspeciÞc labeling. Challenging to synthesize.

Alkyl diazirine Small size. Generates highly reactivecarbene intermediate upon photoactivationat ∼350 nm. Good yield of insertion intoprotein targets (∼25%, MacKinnon et al.,2007). Synthesized directly from theketone precursor.

May undergo UV light-induced rearrangement toelectrophilic linear diazo isomer (Brunner, 1993),leading to nonspeciÞc labeling. Intramolecularrearrangement of the alkyl carbene intermediatemay compete with intermolecular insertion intoproteins (Ford et al., 1998).

Phenyl azide The singlet nitrene intermediate formed onphotoactivation is highly reactive.Photoactivation of nitro-substituted arylazides occurs at ∼340 nm and is thereforenot damaging to protein. Perßuoro phenylazides react primarily via the singletnitrene intermediate (Brunner, 1993).Easily synthesized.

Unsubstituted phenyl azides require activation atshort wavelengths (∼260 nm) that are damaging toprotein. In nonperßuorinated phenyl azides, thesinglet nitrene intermediate is prone toring-expansion to a long-lived electrophilic species(Brunner, 1993), resulting in nonspeciÞc labeling.Phenyl azide is chemically less stable than otherphotoreactive groups.

photo-cross-links to protein targets (discussed in Critical Parameters). Compounds 2 and3were found to maintain nanomolar potency in biological assays, indistinguishable fromthe natural product 1 (all labeled in bold in Fig. 1B).

Ideally, SAR-guided design of photo-afÞnity probes should involve replacing elementsfound in the parent molecule with photoreactive groups having similar chemical proper-ties. Several types of photoreactive groups that differ in size, hydrophobicity, and easeof chemical synthesis are commonly used (see Table 1). Due to intrinsic differences inchemical and photophysical properties between these groups, it is difÞcult to predict apriori which one will be best suited for a speciÞc PAL application. In some cases, itmay be possible to test different photoreactive groups in the same position of a probe,or the same photoreactive group at different positions within the probe. In all cases, it isimportant to evaluate the biological activity of photo-afÞnity compounds.

A brief comparison of beneÞts and downsides of commonly used photoreactive groupsis presented in Table 1. For more detailed descriptions of these photoreactive groups andtheir use in PAL, see Brunner (1993), Dorman and Prestwich (1994), Dorman (2000),and Sadakane and Hatanaka (2006).

Another important consideration in planning a PAL experiment is obtaining a pho-tostable competitor compound to be used in a critical control experiment to distin-guish background from speciÞc photo-cross-links to protein targets (discussed in CriticalParameters). The competitor is often the parent compound or other competitive an-tagonist. Considerable time and effort may be required to synthesize the photostablecompetitor.


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BASICPROTOCOL

DIAZIRINE PHOTOACTIVATION AND Cu(I)-CATALYZED CLICKCHEMISTRY FOR COVALENT LABELING AND DETECTION OF PROTEINTARGETS

This Basic Protocol describes the use of diazirine- and alkyne-containing photo-afÞnityprobes for detection of protein targets in complex protein mixtures. Following diazirinephotoactivation to covalently modify macromolecular binding partners, Cu(I)-catalyzedclick chemistry is used to install a ßuorescent or biotin reporter group for visualizingprobe-modiÞed proteins. While the method is described using photo-afÞnity probe 2(Fig. 1B) in a crude preparation of endoplasmic reticulum (ER) microsomes, it shouldserve as a general experimental guide for other PAL experiments. Critical experimentalvariables and essential controls are discussed.

Materials

Endoplasmic reticulum (ER) microsomes (∼1 mg/ml total protein) or other solubleor membrane protein lysate containing the unknown macromolecular target, inPBS (see recipe for PBS)

0.8 mM stock solution of photostable competitor compound (labeled 3 in Fig. 1)Dimethylsulfoxide (DMSO)20 μM stock solution of photo-afÞnity probe (labeled 2 in Fig. 1) in DMSO10% (w/v) sodium dodecyl sulfate (SDS) in H2O5 mM TAMRA-azide (labeled 4 in Fig. 2) or biotin-azide (labeled 5 in Fig. 2),synthesized by published methods (Speers and Cravatt, 2004; Weerapana et al.,2007); similar reagents are available commercially from Invitrogen, e.g., PEG4carboxamide-6-azidohexanyl biotin (Fig. 2)

1.7 mM TBTA in 80% t-butanol/20% DMSO (see recipe)50 mM CuSO4 in H2O50 mM Tris(2-carboxyethyl)phosphine (TCEP) in H2O, adjusted to pH ∼7 with1 M NaOH; prepare immediately before use

6× Laemmli sample buffer (see recipe)Fluorescent molecular weight markers (Pierce)

96-well plate or other open, shallow container1000 W Hg(Xe) lamp (Oriel Instruments, model 66923) with band-pass Þlter forirradiation at ∼355 nm (Oriel Instruments, cat. no. 59810) and a Þlter to absorbheat (Oriel Instruments, cat. no. 59044); http://www.oriel.com/

0.5-ml polypropylene microcentrifuge tubesTyphoon 9400 phosphor imager (Amersham)

Additional reagents and equipment for SDS-PAGE (e.g., Gallagher, 2006) andimmunoblotting (western blotting ; e.g., Gallagher et al., 2008)

Set up samples

1. In 0.5-ml polypropylene tubes, prepare Þve samples (labeledA to E), each containing19 μl of ER microsomes at a total protein concentration of ∼1 mg/ml in PBS.

Sample A is the experimental sampleSample B is the competition control,Sample C is the negative PAL probe controlSample D is the negative UV-irradiation controlSample E is the negative click chemistry control.

Other protein lysates (e.g., cytosolic proteins, subcellular fractions, crude plasma mem-brane fractions, or whole-cell lysates) containing the unknown protein target of the smallmolecule can also be tested. Prepare the lysate at a concentration of between 0.5 and10 mg/ml total protein in a buffer compatible with the click chemistry (see CriticalParameters).


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TAMRA-azide (4)

biotin-azide (5)

TEV recognition sequence

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PEG4 carboxamide-6-azidohexanyl biotin

Figure 2 Structures of TAMRA-azide (4) and biotin-azide (5) used in the protocols in this unit, and the structure of acommercially available biotin-azide reagent (Invitrogen, cat. no. B10184).

2. To sample B, add 0.5 μl of a 0.8 mM stock solution in DMSO of the photo-stablecompetitor compound (3) for a Þnal concentration of 20 μM. Add 0.5 μl of DMSOto samples A, C, D, and E and incubate all reactions for 15 min at 0◦C.Preincubation of sample B with a large excess of the photo-stable competitor (3) shouldpresaturate relevant protein targets. This controls for nonspeciÞc photo-cross-linkingduring irradiation and is used to distinguish speciÞcally photo-cross-linked proteins fromnonspeciÞc background. Typically a 10- to 100-fold molar excess of competitor is usedfor this control. Optimal preincubation times and temperatures may vary depending onthe kinetics of small-molecule binding to the protein target(s).

Introduce photo-afÞnity probe and perform cross-linking

3. To samples A, B, D, and E add 0.5 μl of a 20 μM stock solution in DMSO of thephoto-afÞnity probe (2) for a Þnal concentration of 500 nM. To sample C add 0.5 μlof DMSO. Incubate the reactions for an additional 15 min at 0◦C.The optimal concentration of the photo-afÞnity probe (2) and the optimal time and tem-perature of the incubation may depend on the particular system under study. The PALprobe is typically tested at between 0.1 and 10 μM. The concentration of DMSO in theÞnal reaction should be kept as low as possible.

4. Transfer each reaction to a well of a 96-well plate or other shallow container.

A shallow dish serves to maximize the surface area of the liquid for good exposure duringthe irradiation step.


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5. Position the reactions ∼6 cm from the source of a 1000-W Hg(Xe) lamp equippedwith a band-pass Þlter for irradiation at ∼350 nm. Irradiate samples A, B, C, and Efor 1 min. Keep sample D protected from irradiation with aluminum foil.

The samples can be irradiated simultaneously, provided they all lie within the boundariesof the incident light. Alternatively, samples can be irradiated sequentially.

Irradiation of the diazirine releases N2(g) and generates a carbene intermediate that cova-lently cross-links the photo-afÞnity probe to the protein target. The half-life of the diazirine(λmax ∼355 nm), and thus the optimal irradiation time, depends on the wavelength ofirradiation, the wattage of the UV light source, and the distance between the sample andthe source (i.e., the power per unit area). A 1000-W Hg(Xe) lamp provides an intensesource of radiation and requires short (≤ 1 min) irradiation times. A lower-intensity long-wavelength UV lamp that emits at∼365 nm is sufÞcient for diazirine photoactivation, butusually requires longer irradiation times (∼5 to 10 min for a 100-W lamp).

6. Transfer 19.5 μl each from samples A to E into new, labeled 0.5-ml polypropylenetubes for the click reaction.

Perform click chemistry

7. Add 2.5 μl of 10% SDS to each reaction and mix by vortexing.

Addition of SDS denatures proteins and exposes the terminal alkyne to the click reagents.

8. Add 0.5 μl of 5 mM TAMRA-azide (labeled 4 in Fig. 2) or 5 mM biotin-azide(labeled 5 in Fig. 2) to each reaction.

Other reporter-azide reagents are equally suitable and are commercially available (e.g.,PEG4 carboxamide-6-azidohexanyl biotin, Invitrogen; shown in Fig. 2).

Biotin-azide (5) has a TEV protease recognition sequence positioned between the bi-otin and azide groups (Fig. 2). This feature can be useful for proteolytically cleavingbiotinylated proteins or peptides from a streptavidin afÞnity matrix using TEV protease(Weerapana et al., 2007).

9. Prepare a master mix of the catalyst immediately before use by combining:

1.5 volumes 1.7 mM TBTA in 80% t-butanol/20% DMSO0.5 volumes 50 mM CuSO40.5 volumes 50 mM TCEPVortex to mix.

The catalyst master mix should have a faint blue color and be heterogeneous.

Mix again, then add 2.5 μl of catalyst mix to samples A, B, C, and D and mix byvortexing.

10. Prepare mock catalyst mix as described in step 9, but substitute deionized water forthe CuSO4. Add 2.5 μl of mock catalyst mix to sample E and mix by vortexing.

Without CuSO4 in the mock catalyst, the click reaction should not proceed. Sample Etherefore serves as a negative click chemistry control.

11. Incubate the reactions 30 min at 32◦C.Incubation for 1 hr at room temperature is also sufÞcient for labeling in the click reaction.

Following the incubation, reactions can be diluted∼10-fold in afÞnity puriÞcation buffer(see recipe) to reduce the concentration of SDS, and immunoprecipitated using speciÞcantibodies directed against candidate target proteins.


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Resolve proteins by electrophoresis

12. After the incubation, add 5 μl of 6× Laemmli sample buffer to each tube and mix.

It is not necessary to heat the samples after addition of sample buffer. Some proteins,including hydrophobic membrane proteins, irreversibly aggregate upon heating in thepresence of the click reagents.

13. Resolve 12.5 μl of samples A to E and 5 to 10 μl of ßuorescent or broad molecularweight markers by SDS-PAGE (Gallagher, 2006). Run the gel until the dye fronthas completely exited the gel.

Running the dye front (which also contains the click chemistry reagents) off the gelensures less carryover of free TAMRA-azide (labeled 4 in Fig. 2) into the imaging step(step 15).

Snap-freeze the remaining samples in liquid N2 and store at −80◦C. Storage of samplesat −20◦C leads to an increase in nonspeciÞc background labeling due to the clickchemistry reagents. The stored samples are stable for at least 1 week.

14. Wash the gel three times, each time for 10 min, with deionized water.

The gel is thoroughly washed with several changes of deionized water to help removeresidual traces of free TAMRA-azide from the gel.

Scan gels/perform immunoblotting

15a. If the click reaction was performed with TAMRA-azide (labeled 4 in Fig. 2): Scanthe gel using a Typhoon ßuorescent gel scanner (excitation wavelength 532 nm,emission wavelength 580 nm).

15b. If the click reaction was performed with biotin-azide (labeled 5 in Fig. 2) or otherbiotin-azide: Transfer proteins to nitrocellulose or PVDFmembrane with a westernblot transfer apparatus (Gallagher et al., 2008) and probe for biotinylated proteinsusing streptavidin-HRP.

SUPPORTPROTOCOL

AFFINITY PURIFICATION OF PROBE-MODIFIED PROTEINS

In this Support Protocol, probe-modiÞed proteins labeled with TAMRA-azide (labeled 4in Fig. 2) or biotin-azide (labeled 5 in Fig. 2) under click chemistry conditions are afÞnitypuriÞed. This is accomplished usingmonomeric avidin agarose (Pierce) for biotin-labeledproteins, or antibodies directed against TAMRA (Invitrogen) for TAMRA-labeled pro-teins. The use of monomeric avidin agarose or anti-TAMRA antibodies enables mildelution of labeled proteins. The protocol can be followed after optimizing the photo-labeling and click reaction steps described in the Basic Protocol. The Support Protocolis useful for ultimately identifying the protein target(s) using techniques such as Edmansequencing or mass spectrometry.

Additional Materials (also see Basic Protocol)

Protein mixture labeled with photo-afÞnity probe (Basic Protocol, steps 1 to 5)Liquid N2Acetone cooled to −20◦C1% SDS in PBS (see recipe for PBS)AfÞnity puriÞcation buffer (see recipe)Protein A�Sepharose beads (GE Healthcare)Anti-TAMRA antibody (Invitrogen, cat. no. A6397)Monomeric avidin�agarose beads (Pierce)Wash buffer (see recipe)Elution buffer (see recipe)


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Polyallomer 1.5-ml microcentrifuge tubes (Beckman-Coulter)Benchtop ultracentrifugeSonicating water bathRefrigerated microcentrifugeRotating tube mixer

Perform photo-activation and click chemistry

1. Complete steps 1 to 5 of the Basic Protocol on a 25× to 50× scale (e.g., 0.7 ml of1 mg/ml total protein).

The complete set of control experiments described in the Basic Protocol are not necessaryhere if they were previously performed in analytical-scale experiments.

During scale-up, a larger vessel, such as a well of a 12- or 24-well tissue culture dish,can be used during the irradiation step. For proper irradiation, it is important that theentire sample lie within the bounds of the incident light (a circle∼6 cm in diameter usingthe lamp setup described here). A 1-min irradiation of a 0.7-ml of sample in a well ofa 24-well dish, as used here, is sufÞcient for complete photoactivation of the diazirine.For sample volumes signiÞcantly larger than 0.7 ml, longer irradiation times, coincidentsample mixing, or irradiation in batches may be required. A small aliquot of the mixturecan be removed and analyzed by SDS-PAGE following click chemistry to determine theextent of cross-linking.

2. Following irradiation, transfer the mixture containing the ER microsomes to a 1.5-ml polyallomer microcentrifuge tube and sediment the microsomes in a benchtopultracentrifuge 10 min at 50,000 × g, 4◦C.Sedimentation concentrates the microsomes and allows the click reaction to be conductedon a smaller volume. For other types of protein mixtures that cannot be concentrated bysedimentation, other protein-precipitation methods can be tested in pilot experiments, orthe mixture can be directly subjected to the click chemistry.

3. Aspirate the supernatant and resuspend the microsomes in 97.5 μl of PBS.

4. Follow steps 7 to 11 of the Basic Protocol, scaling up the click reagent volumesaccording to the volume of the resuspended microsome pellet. For 97.5 μl of re-suspended microsomes, add 12.5 μl 10% SDS, 2.5 μl TAMRA-azide (labeled 4 inFig. 2) or biotin-azide (labeled 5 in Fig. 2), and 12.5 μl of the catalyst mix (seesteps 9 and 10 in the Basic Protocol).

Precipitate and redissolve proteins

5. Following the click reaction, remove a 5-μl aliquot, add 6× Laemmli sample buffer(see step 12 of Basic Protocol), snap-freeze in liquid nitrogen, and store the sampleat −80◦C.

The saved sample should be stable for at least one week at −80◦C.

This saved aliquot represents the �input� into the afÞnity puriÞcation. At this stage,detection of speciÞc photo-cross-linked proteins can be determined by SDS-PAGE (step13 of the Basic Protocol).

An aliquot of the sample can also be diluted ∼10-fold in afÞnity puriÞcation buffer(see recipe) to reduce the concentration of SDS, and immunoprecipitated using speciÞcantibodies directed against candidate target proteins.

6. To the remaining sample (120 μl), add 0.5 ml of acetone, cooled to −20◦C, for aÞnal concentration of ∼80% (v/v). Vortex brießy and place at −20◦C for 30 min.A white precipitate should form, which contains precipitated proteins. Cold acetoneprecipitation removes the large molar excess of free TAMRA-azide (4) or biotin-azide (5)that would interfere with the binding to the TAMRA-antibody or monomeric avidin beads,


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respectively. The azide label is soluble in acetone while the proteins are not, permittingtheir separation.

Following addition of cold acetone, the sample can also be stored overnight at −20◦C

7. Sediment the precipitated protein in a by microcentrifuging 10 min at 20,000 × g,4◦C.A white pellet containing precipitated protein should be observed in the bottom of thetube.

8. Aspirate the supernatant and add 0.5 ml of cold acetone.

9. Use a sonicating water bath to break up and disperse the precipitated protein pelletin the acetone.

Avoid heating the sample during this process by sonicating only for brief periods.

10. Return the sample to −20◦C for 10 min.Longer incubation times at −20◦C may improve the recovery of precipitated protein.

11. Repeat steps 7 to 11 of this protocol two more times.

It is essential to remove all traces of free TAMRA-azide (4) or biotin-azide (5) formaximumyield of afÞnity-puriÞed proteins.

12. Aspirate the supernatant and air-dry the pellet brießy for∼10 min at room tempera-ture.

Air drying removes residual acetone from the protein pellet. Do not over-dry the pellet asit will become difÞcult to resolubilize.

13. Add 50 μl of 1% SDS in PBS to the pellet and gently dislodge the pellet from theside of the tube by vortexing and/or sonication.

Do not use a pipet tip to dislodge the pellet, as the pellet may stick to the tip.

The SDS aids in resolubilizing the protein pellet.

14. Once the pellet has completely dissolved, dilute the sample with 0.5 ml of afÞnitypuriÞcation buffer.

The SDS must be diluted to ≤0.1% for efÞcient puriÞcation in steps 16 to 19. The NP-40detergent helps �mask� the SDS in mixed micelles.

15. Remove a 20-μl aliquot of the mixture, add 4 μl of 6× Laemmli sample buffer for aÞnal concentration of 1×, snap-freeze in liquid nitrogen, and store at −80◦C.The sample should be stable for at least 1 week at −80◦C.

This sample can be used to evaluate the efÞciency of the precipitation and resolubilization(steps 6 to 14) by comparison to an aliquot of the �input� click reaction (saved in step 5).

Perform afÞnity chromatography

Follow steps 16a to 17a and 24a for TAMRA-labeled proteins; follow steps 16b to 17band 24b for biotin-labeled proteins.

For TAMRA-labeled proteins

16a. Equilibrate 30 μl of protein A�Sepharose in afÞnity puriÞcation buffer and preparea 50% slurry in the same buffer.

17a. Add 30 μl of the protein A�Sepharose slurry and 5 μl of anti-TAMRA antibody(directly as received from manufacturer) to the resolubilized protein pellet.


65


For biotin-labeled proteins

16b. Prepare the monomeric avidin beads according to the manufacturer�s direction,equilibrate with afÞnity puriÞcation buffer, and prepare a 50% slurry in afÞnitypuriÞcation buffer.

17b. Add 50μl of the monomeric avidin agarose slurry to the resolubilized protein pellet.

18. Incubate samples (from step 17a or 17b) on a rotating tube mixer for 3 hr at 4◦C.

Samples can also be incubated overnight at 4◦C.

19. Sediment the agarose beads for 1 min at 10,000× g, 4◦C, in a microcentrifuge andremove the supernatant.

20. Save a 20-μl aliquot of the supernatant, add 4 μl of 6× Laemmli sample buffer fora Þnal concentration of 1×, snap-freeze in liquid nitrogen, and store at −80◦C.The saved sample should be stable for at least 1 week at −80◦C.

The saved aliquot of the supernatant can be used to evaluate the efÞciency of the afÞnitypuriÞcation step by comparison to an equal aliquot of the resolubilized pellet (saved instep 15).

21. Add 1 ml of afÞnity puriÞcation buffer to the sedimented agarose beads and rotateon a rotating tube mixer for 10 min at 4◦C.Longer mixing times may be more effective at removing nonspeciÞcally bound proteinsfrom the agarose resin.

22. Repeat steps 19 to 21 (the supernatants from the washes can be discarded).

23. Repeat steps 19 to 21 two more times, but replace the afÞnity puriÞcation bufferwith wash buffer.

Elute and resolve proteins

24a. For TAMRA-labeled proteins: After the Þnal wash, elute bound proteins with 50 μlof 1× Laemmli sample buffer for 20 min at room temperature.

24b. For biotin-labeled proteins:After the Þnal wash, elute bound proteins with 50 μl ofelution buffer for 20 min at room temperature, or as described by the manufacturer.

The NP-40 detergent in the elution buffer is included to help maintain proteins in solutionafter elution. The detergent may not be required when eluting soluble proteins.

25. Resolve equivalent amounts of the �input� sample (saved in step 5, 4.5 μl), theresolubilized protein pellet (saved in step 15, 20 μl), the post-afÞnity puriÞedsupernatant (saved in step 20, 20 μl), and eluent (saved in step 24, 1.8 μl) bySDS-PAGE (Gallagher, 2006). Scan the gel for ßuorescence (step 15 of the BasicProtocol) or transfer proteins to a nitrocellulose or PVDFmembrane and probe withstreptavidin-HRP (Gallagher et al., 2008).

Comparison of the signal intensity of the �input� and resolubilized pellet samplesindicates the efÞciency of the acetone precipitation and resolubilization steps (steps 6to 15). Comparison of the signal intensity of the resolubilized pellet with post-afÞnitypuriÞed supernatant indicates the efÞciency of the pull down (steps 16 to 19). Comparisonof the signal intensity of the resolubilized pellet with the eluent indicates the recovery oflabeled proteins (steps 21 to 24).


66


REAGENTS AND SOLUTIONSUse deionized, distilled water in all recipes and protocol steps.

AfÞnity puriÞcation buffer

50 mM HEPES, pH 7.4100 mM NaCl1% (v/v) NP-40 or Triton X-100Store up to 1 month at 4◦C

Elution buffer

Phosphate-buffered saline (PBS; see recipe) containing:2 mM D-biotin1% (v/v) NP-40 or Triton X-100Store up to 1 month at 4◦C

Laemmli sample buffer, 6×12% (w/v) sodium dodecyl sulfate (SDS)60% (v/v) glycerol375 mM Tris·Cl, pH 8.0 (see recipe)0.015% (w/v) bromphenol blue30% (v/v) 2-mercaptoethanolStore up to 1 year at −20◦C

Phosphate-buffered saline (PBS)

137 mM NaCl10 mM Na2HPO42.7 mM KClStore up to 1 year at 4◦C

TBTA in 80% t-butanol/20% DMSO

Solid Tris(benzyltriazolylmethyl)amine (TBTA) is commercially available(Anaspec) or can be synthesized by published methods (Chan et al., 2004). Theworking stock is prepared by mixing one volume 8.5 mM TBTA stock in DMSOwith four volumes t-butanol. This solution is stable for months when stored at−20◦C.

Tris·Cl [tris(hydroxymethyl)aminomethane], 1 MDissolve 121 g Tris base in 800 ml H2OAdjust to desired pH with concentrated HClMix and add H2O to 1 liter

Approximately 70 ml of HCl is needed to achieve a pH 7.4 solution, and approximately42 ml for a solution that is pH 8.0.

IMPORTANT NOTE: The pH of Tris buffers changes signiÞcantly with temperature, de-creasing approximately 0.028 pH units per 1◦C. Tris-buffered solutions should be adjustedto the desired pH at the temperature at which they will be used. Because the pKaof Tris is8.08, Tris should not be used as a buffer below pH ∼7.2 or above pH ∼9.0.

Wash buffer

50 mM HEPES, pH 7.4500 mM NaCl1% (v/v) NP-40 or Triton X-100Store up to 1 month at 4◦C


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COMMENTARY

Background InformationIdentifying the target of a biologically ac-

tive small molecule is a major step towardunderstanding its underlying mechanism ofaction. A traditional biochemical method forsmall-molecule target identiÞcation employsafÞnity chromatography of the target fol-lowed by identiÞcation by mass spectrometryor Edman degradation (Harding et al., 1989;Taunton et al., 1996; Ding et al., 2004). In thismethod, a complex protein mixture is passedover a resin matrix that has been covalentlymodiÞed with the small molecule of inter-est. The afÞnity matrix is stringently washed,and speciÞcally bound proteins are eluted, re-solved by SDS-PAGE, and identiÞed. The suc-cess of this approach requires that the targetand small molecule have a sufÞciently strongbinding afÞnity (typically in the nM range) tosurvive the extensive washing steps requiredto reduce nonspeciÞc binding of proteins tothe afÞnity matrix. In a recent variation ofthis technique, less stringent washing condi-tions coupled with highly sensitive quantita-tive mass spectrometry were used to identifyspeciÞc protein targets of inhibitors with mi-cromolar afÞnity (Ong et al., 2009). The ap-proach works best with soluble protein targets,since integral membrane proteins require de-tergent solubilization prior to chromatography,which often prevents binding to the immobi-lized small-molecule.PAL has several features that distinguish it

from the traditional afÞnity chromatographyapproach. First, since photoactivation is per-formed under native conditions, PAL providesthe opportunity for detection and identiÞcationof integral membrane protein targets (Colcaet al., 2004; Saghatelian et al., 2004), an im-portant class of proteins targeted by a largenumber of small-molecule drugs. PAL can alsobe used to characterize and map the ligandbinding sites of known integral membrane pro-teins or other targets that lack high-resolutionstructural information (Al-Mawsawi et al.,2006; Xi et al., 2006). Furthermore, sincePAL establishes a stable, covalent bond be-tween the small-molecule probe and the target,the targets of even moderately potent small-molecules can, in principle, be identiÞed.Several different click reaction conditions

have been described in the literature. In theversion described here, Cu(II)SO4 serves asthe precursor to the Cu(I) species that cat-alyzes triazole formation between the ter-minal alkyne and azide. Tris-carboxyethyl

phosphine (TCEP) presumably reduces Cu(II)to Cu(I) in situ during preparation ofthe catalyst mix (Basic Protocol step 9).Tris(benzyltriazolylmethyl) amine (TBTA) isa polytriazole ligand that stabilizes the Cu(I)ion and enhances its catalytic activity in solu-tion (Chan et al., 2004). Highly pure Cu(I)Br(99.999%) (Dieterich et al., 2007) or Cu(I) tri-ßate (Strable et al., 2008) have also been usedto effect the click reaction. However, we pre-fer in situ generation of Cu(I), since Cu(I)Br issparingly soluble and aqueous Cu(I) solutionsare prone to oxidation by dissolved oxygen.Tagging of probe-modiÞed proteins with

reporter groups by click chemistry requiresthe presence of only a small alkyne or azidegroup in the probe. This avoids introducing abulky reporter directly into the small-moleculescaffold. While there are examples of success-ful target identiÞcation using chemically reac-tive probes that have been directly modiÞedwith a biotin reporter (Sin et al., 1997; Kwoket al., 2001), such bulky groups can perturbthe interaction between the probe and proteintargets. This point is exempliÞed in a studyof compounds 6, 7, and 8 (labeled in bold inFig. 3), where the rhodamine reporter is con-jugated directly to the natural product scaffoldvia a triazole linkage and variable-length alkylspacer arm. Photo-cross-linking to the target,Sec61α, in ER microsomes, was only∼5% to10% as efÞcient using 6 compared to photo-cross-linking followed by click chemistry us-ing 2. Compound 7 cross-linked even less ef-Þciently than 6, and speciÞc photo-cross-linksto Sec61αwere undetectable using compound8 (data not shown). The reduced photo-cross-linking yield presumably reßects a reduction inbinding afÞnity after conjugating the moleculewith a bulky rhodamine reporter. Introductionof the alkyne group preserved the nanomolarpotency of the compound, while providing thechemical functionality needed to detect photo-cross-linked proteins in a second, click chem-istry step.Introduction of a radiolabel into the small-

molecule probe is a widely used approach todetect probe-modiÞed proteins. While radio-labels are small in size, extremely sensitive,and offer a high ratio of signal to noise, radio-labeled probes can be costly to synthesize andradioactive materials require special handlingand dedicated equipment. Click chemistry pro-vides a nonradioactive alternativewhich is alsohighly sensitive, and has the added advantageof coincidently installing a chemical handle


68


NH

N

O

O

ONH

O

N

O

HN

N

O

O

O

NN

O

O

N

N

N

HN

O

O

O

OH N

N

6

NH

N

O

O

ONH

O

N

O

HN

N

O

O

O

NN

N

N

N

HN

O

O

O

OH N

N

O

O

7

NH

N

O

O

ONH

O

N

O

HN

N

O

O

O

NN

N

N

N

HN

O

O

O

OH N

N

8

Figure 3 Structures of compounds 6, 7, and 8, which have a ßuorescent reporter group (TAMRA) directly incorporatedinto the natural product scaffold.


69


2

3 ( M)

h

5 10 20

Coomassie

20011910886

53

27

*

TAMRA fluorescence

clic

k re

act

ion (1%

)re

-solu

bili

zed p

elle

t (1

%)

post

-avi

din

supern

ata

nt (1

%)

elu

ent (1

2%

)

75

50

37

25

20

100150

15

10

Strep-HRP

A B

*

Figure 4 (A) Photo-cross-linking in ER microsomes with 2 (see Fig. 1) followed by click chemistry with TAMRA-azide(4; see Fig. 2) as described in the Basic Protocol. Sec61α is marked with an asterisk (Þgure adapted with permissionfrom MacKinnon et al., 2007). (B) Photo-cross-linking in ER microsomes with 2 followed by click chemistry with biotin-azide (5) and afÞnity puriÞcation using monomeric avidin as described in the Support Protocol. Samples representing theclick reaction (lane 1), the resolubilized protein pellet following acetone precipitation (lane 2), the post-monomeric avidinsupernatant (lane 3), and the eluent (lane 4) were resolved by SDS-PAGE, transferred to nitrocellulose, and probed forbiotinylated proteins with streptavidin-HRP (Strep-HRP). Percentages indicate the fraction of the total sample that wasloaded in each lane. The position of Sec61α is marked with an asterisk. The biotinylated protein at∼21 kDa is a backgroundband.

(biotin or TAMRA) that can be used to afÞnitypurify and identify probe-modiÞed proteins. Insome cases, this method can target the precisesite of probe modiÞcation at the amino acidlevel (Adam et al., 2004; Speers and Cravatt,2005; Weerapana et al., 2007).AfÞnity puriÞcation of probe-modiÞed tar-

gets is an essential step in target identiÞca-tion. PuriÞcation of biotinylated moleculeswith matrix-immobilized tetrameric strepta-vidin is a widely used technique that takes ad-vantage of the extremely tight interaction be-tween biotin and streptavidin (Kd ∼10−15 M).While this tight interaction permits stringentwashing conditions resulting in low back-ground, elution of speciÞcally bound mate-rial requires harsh conditions, typically boilingin SDS-PAGE sample buffer. Such conditionsmay not be suitable for some proteins, andSDS present in sample buffer is not com-patible with many downstream applicationsincluding liquid chromatography/mass spec-trometry (LC/MS). Several novel cleavable bi-otin reagents have been described (Verhelstet al., 2007;Weerapana et al., 2007) and othersare commercially available (e.g., Pierce, cat.

nos. 21331 and 21442). These reagents allowelution of streptavidin-bound material chemi-cally or enzymatically without disrupting thestrong biotin-streptavidin interaction. How-ever, many of these cleavable reagents stillsuffer from low elution efÞciencies. For ex-ample, biotin-azide (labeled 5 in Fig. 2), usedin the present protocol, has a TEV proteaserecognition sequence positioned between thebiotin and azide groups (Fig. 2). This feature isdesigned to permit elution of biotinylated pro-teins by incubation with TEV protease. How-ever, in our case, we were unable to efÞcientlyelute bound Sec61α by incubation with TEVprotease, possibly due to steric occlusion ofthe protease recognition sequence.To circumvent problems associated with

tetrameric streptavidin and cleavable biotin-azide reagents, the Support Protocol utilizesmonomeric avidin (Pierce) for afÞnity pu-riÞcation of biotinylated targets (Fig. 4B).Monomeric avidin has a lower afÞnity forbiotin (Kd ∼10−8 M), permitting elution ofbound material with 2 mM biotin in PBS,a condition more suitable for diverse down-stream applications. This unit also presents a


70


mild capture and elution method for TAMRA-labeled proteins utilizing anti-TAMRA anti-bodies (Invitrogen) and protein A�Sepharose.AfÞnity puriÞcation with anti-TAMRA anti-bodies has the advantage that, following afÞn-ity puriÞcation, puriÞed protein targets canbe proteolytically digested and probe-labeledpeptide fragments visualized by ßuorescencedetection (Adam et al., 2004; Okerberg et al.,2005). AfÞnity puriÞcation with monomericavidin or anti-TAMRA antibodies both pro-vide ∼25% yield of labeled proteins.

Critical ParametersNonspeciÞc photo-cross-linking to highly

abundant or �sticky� proteins in a complexprotein mixture can be problematic in PAL.High background can obscure detection ofspeciÞc cross-links to less abundant proteins.Detection of speciÞc cross-links therefore de-pends on the relative abundance of the proteintarget (i.e., the amount of target protein pertotal protein). Subcellular or biochemical frac-tionation that enriches the sample for a puta-tive target can be employed to improve the ra-tio of speciÞc signal-to-background noise. Forexample, while we could not detect speciÞcphoto-cross-links to Sec61α in crude mam-malian cell extract (data not shown), robustcross-linking was observed in puriÞed ER mi-crosomes (Fig. 4). Even in the presence ofhigh background, valuable information on spe-ciÞc photo-cross-links to targets can be deter-mined by immunoprecipitation against candi-date proteins (Kukar et al., 2008). Extremelylow-abundance targets may be difÞcult or im-possible to detect, even in fractionated lysates.In such cases, it may be possible to detectthese targets following an afÞnity puriÞcationstep after the click reaction. Ultimately, detec-tion of targets in a crude mixture of proteinsdepends on a favorable conßuence of vari-ables, including the relative abundance of thetarget, the photo-cross-linking speciÞcity andyield, and the click chemistry yield (discussedbelow).Several controls are essential for distin-

guishing background from speciÞc photo-cross-linking. First, it is important to demon-strate that labeling of a putative target dependson the presence of both the photo-afÞnityprobe and on UV irradiation. When labelingis observed in the absence of the probe or UVlight, it may indicate background labeling dueto the click reaction (discussed below). Sec-ondly, it is important to conduct a competi-tion experiment to control for the speciÞcity

of photo-cross-linking. This is done by incu-bating the protein sample with a large excessof a photostable competitor compound prior toUV irradiation. Cross-links to speciÞcally la-beled proteins are dose-dependently competedby the photostable compound, whereas back-ground cross-links are weakly competed or notcompeted at all (Fig. 4A).Background labeling that is independent of

the photo-afÞnity probe or UV irradiation isdue to nonspeciÞc labeling during the click re-action. The level of background appears to bestrongly dependent on the total protein con-centration, with lower concentrations of totalprotein yielding less background. The opti-mal concentration of total protein that providesthe best ratio of speciÞc signal to backgroundnoise should be determined empirically. Re-ducing the concentration of the TAMRA-azide(4) or biotin-azide (5) in the reaction (wehave gone as low as ∼25 μM) can also helpreduce nonspeciÞc background labeling. Wehave found that background increases whenclick reactions are stored at −20◦C, even af-ter addition of Laemmli sample buffer. Quick-freezing samples in liquid N2 and storing at−80◦C prevents this. A low concentration(0.1% to 1%) of SDS in the click reaction alsoreduces nonspeciÞc background labeling.TAMRA-azide (4) or other commercially

available ßuorescent-azides (e.g., Invitrogen,cat. no. A10270 and T10182) used duringthe click reaction can trail though the gellanes when reactions are resolved by SDS-PAGE. Trailing ßuorophore contributes tobackground ßuorescence in the gel and re-duces the sensitivity of in-gel ßuorescent scan-ning. To mitigate this problem, the dye frontcontaining the ßuorophore should be run com-pletely off the bottom of the gel during elec-trophoresis. Removal of excess free TAMRA-azide by gel Þltration, dialysis, or protein pre-cipitation (e.g., acetone precipitation as used inthis protocol) prior to SDS-PAGE can also sig-niÞcantly reduce the background due to trail-ing ßuorophore. Gels should be thoroughlywashed with several changes of deionized wa-ter prior to in-gel ßuorescence scanning, toremove traces of residual ßuorophore.The click reaction tolerates a fairly broad

range of salt, buffer, and detergent concentra-tions, as well as a broad range of pH and tem-peratures. Metal chelators such as EDTA orEGTA should be avoided during preparation ofthe protein lysate, as these sequester the Cu(II)ions required for the reaction. We have alsofound (unpub. observ.) that 2-mercaptoethanol


71


Table 2 Troubleshooting Guide for Target IdentiÞcation by Diazirine Photo-Cross-Linking and Click Chemistry

Problem Cause Solution

No signal observed ongel/blot following theclick reaction

Incorrect wavelength for diazirinephotoactivation; insufÞcient timefor irradiation

Check the wavelength settings on the lamp; performan irradiation time course

Concentration of the photo-afÞnityprobe is too low

Titrate the photo-afÞnity probe into a Þxed amount ofprotein lysate and conduct PAL and click reactions

The photo-afÞnity probe does notbind the target of the parent molecule

ConÞrm that the photo-afÞnity probe is biologicallyactive

The target�s relative abundance istoo low

Enrich the sample for the target by biochemical orsubcellular fractionation; afÞnity purify following theclick reaction

The click reaction failed Prepare new reagent stocks; use freshly preparedTCEP solution

High backgroundobserved on gel/blotfollowing the clickreaction

Non-speciÞc photo-cross-linking Reduce the concentration of the photo-afÞnity probeor the concentration of total protein; enrich the samplefor putative targets prior to PAL

NonspeciÞc background due toclick reaction

Reduce the total protein concentration; reduce theconcentration of the azide used during the clickreaction; store reactions at −80◦C

NonspeciÞc and speciÞc bandsoverlap on SDS-PAGE

Change SDS-PAGE conditions; for example, testdifferent acrylamide concentrations, different buffersystems (e.g., Tris-Tricine), or 2-D gel electrophoresis

Labeled proteins are notdepleted during afÞnitypuriÞcation

Contaminating free TAMRA-azide(4) or biotin-azide (5)

Repeat the acetone-precipitation steps to removecontaminating TAMRA-azide or biotin-azide andrepeat the afÞnity puriÞcation

(2-ME) and dithiothreitol (DTT) inhibit thereaction at fairly low concentrations (∼100μM). Labeling of some probe-modiÞed pro-teins requires or is improved by the presenceof a low concentration of SDS (0.1% to 1%)or other detergent including deoxy-BigChaps,TX-100, NP-40, or sodium cholate.

TroubleshootingA troubleshooting guide is presented in

Table 2.

Anticipated ResultsWe recently utilized alkyl diazirine photo-

activation and click chemistry methods toidentify the molecular target of �cotransins,�a class of cyclic heptadepsipeptides derivedfrom the fungal natural product HUN-7293(labeled 1 in Fig. 1B). Cotransins inhibit co-translational translocation of nascent proteinsacross the endoplasmic reticulum (ER) mem-brane in a signal-sequence-dependent manner(Garrison et al., 2005). Inhibition occurs at thelevel of insertion of the nascent protein into an

ER membrane�embedded multiprotein com-plex, termed the translocon, which recognizessignal sequences and forms a pore throughwhich substrate proteins traverse (Osborneet al., 2005). Utilizing an alkyl diazirine�basedphoto-afÞnity probe that bears an alkyne han-dle (labeled 2 in Fig. 1B), we identiÞed an inte-gral membrane protein subunit of the translo-con complex, Sec61α, as the molecular targetof cotransins (MacKinnon et al., 2007).Figure 4A shows a gel (adapted from

MacKinnon et al., 2007) resulting from fol-lowing the Basic Protocol in crude ER micro-somes using photo-afÞnity probe 2 and clickchemistry with TAMRA-azide (labeled 4 inFig. 2). Three proteinswere labeled in the pres-ence of the PAL probe (lane 1), including onemajor band at∼45 kDa (marked with an aster-isk). Labeling of the major band was depen-dent on UV light (lane 5) and the PAL probe2 (Lane 6), and was dose-dependently com-peted by preincubation with the photostablecompetitor 3 (Lanes 2 to 4), indicating speciÞcphoto-cross-linking to this protein. Labeling of


72


two other proteins at ∼60 kDa and ∼40 kDaalso depended on the PAL probe and UV light,but was not competed by excess 3, indicatingnonspeciÞc (i.e., nonsaturable) photo-cross-linking to these proteins. Coomassie stainingindicated equal loading of protein across allsamples. The ∼45 kDa protein, previouslyidentiÞed as Sec61α (MacKinnon et al., 2007),is present at about 1% of total ER proteinsand represented the major labeled protein.However, Sec61α did not represent a ma-jor Coomassie-stainable band, indicating thespeciÞcity of the reaction and the ability to de-tect a protein target of moderate abundance ina complex mixture of ER proteins. Labelingthat was independent of 2 and UV light wasbackground due to the click reaction (Lanes 5and 6).Figure 4B shows afÞnity puriÞcation of bi-

otinylated proteins using monomeric avidinfollowing photo-cross-linking with 2 and clickchemistry with biotin-azide (labeled 5 inFig. 2), as described in the Support Proto-col. The position of Sec61α is marked withan asterisk. Comparison of equivalent aliquotsof the starting click reaction (1% of total re-action, lane 1) with the resolubilized proteinpellet (1% of total sample, lane 2), showed∼50% protein recovery following the acetone-precipitation protocol. Comparison of equiva-lent aliquots of the resolubilized protein pellet(lane 2) with the post-monomeric avidin su-pernatant (1% of total sample, lane 3), showedquantitative depletion of biotinylated proteinsfrom the sample using monomeric avidin. Re-covery of biotinylated proteins by mild elutionwith 2 mM biotin proceeded in ∼25% yield,as determined by comparison of lanes 2 and 4.

Time ConsiderationsA signiÞcant investment of time and re-

sources is required for the design and synthe-sis of a photo-afÞnity probe that retains potentbiological activity. Additional time may be re-quired to synthesize a photostable competitor.Once a suitable probe is in hand, it can berapidly tested in the photo-cross-linking andclick reactions in <4 hr when using TAMRA-azide (4) in the click reaction. The time re-quired for optimization of the photo-cross-linking and click chemistry will vary, but maybe completed in<1week. AfÞnity puriÞcationand analysis of samples takes 1 to 2 days.

AcknowledgementsThis work was supported by the NIH

(GM81644) and the Howard Hughes MedicalInstitute.

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