(TOP-ABPP)—A General Method for Mapping Sites of Probe Modification in Proteomes

PROTOCOL Tandem orthogonal proteolysis-activity-based protein proﬁling (TOP-ABPP)—a general method for mapping sites of probe modiﬁcation in proteomes

Eranthie Weerapana, Anna E Speers & Benjamin F Cravatt

Departments of Cell Biology and Chemistry, The Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA. Correspondence should be addressed to B.F.C. ([email protected]).

Published online 31 May 2007; doi:10.1038/nprot.2007.194 s Activity-based protein profiling (ABPP) utilizes active site-directed chemical probes to monitor the functional state of enzymes directly in native biological systems. Identification of the specific sites of probe labeling on enzymes remains a major challenge in ABPP experiments. In this protocol, we describe an advanced ABPP platform that utilizes a tandem orthogonal proteolysis (TOP) strategy coupled with mass spectrometric analysis to simultaneously identify probe-labeled proteins together with their exact sites of natureprotocol / probe modification. Elucidation of probe modification sites reveals fundamental insights into the molecular basis of specific probe– m o

c protein interactions. The TOP-ABPP method can be applied to any type of proteomic sample, including those derived from in vitro or . e r in vivo labeling experiments, and is compatible with a variety of chemical probe structures. Completion of the entire protocol, u t

a including chemical synthesis of key reagents, requires approximately 8–10 days. n . w w w /

/ INTRODUCTION : p t Activity-based protein proﬁling (ABPP) torial or non-directed strategy has been introduced that relies on t h

The ﬁeld of proteomics aims to characterize and functionally the adaptation of mild carbon electrophiles to proﬁle enzymes from p

u annotate the large number of proteins encoded by eukaryotic and mechanistically diverse classes. Non-directed proﬁling efforts have o 14 r prokaryotic genomes. Utilizing a variety of analytical techniques, engendered useful classes of probes bearing sulfonate ester , G including two-dimensional gel electrophoresis and mass spectro- a-chloroacetamide15 and spiroepoxide reactive groups16, which, g n i metry (MS), proteomic researchers endeavor to identify and assign together, have greatly expanded the proteomic coverage of ABPP. h

s 1,2 i function to the myriad proteins expressed in biological systems . l b Owing to the vast size and diversity of the proteome, current Click chemistry u P

analytical methods struggle to characterize many of its constituents, The presence of a bulky biotin or ﬂuorophore reporter group e r impedes the cell permeability properties of ABPP probes. Hence,

u including those that are low abundance and display difﬁcult t a physicochemical properties (e.g., integral membrane proteins). initial protocols for ABPP typically involved homogenization of N

7 Additionally, conventional proteomic methods acquire informa- cells and tissues before treatment with probes, thereby removing 0

0 tion on protein expression level and, thus, offer only an indirect proteins from their native cellular environment. To circumvent this 2 estimate of the activity state of proteins. limitation, recent advances in the bio-orthogonal click chemistry © As many proteins are regulated by post-translational modifica- reaction were applied to the field of ABPP17,18. Click chemistry tions, protein–protein and/or protein–small molecule interactions, exploits a Cu(I)-catalyzed, stepwise analog of Huisgen’s concerted protein abundance is not always an accurate estimate of protein triazole synthesis to couple an alkyne to an azide19.ABPPprobes activity3. For these reasons, more advanced, ‘‘targeted proteomic’’ containing a sterically inert alkyne functionality can be adminis- methods have been introduced that enrich and profile specific tered to living cells/organisms allowing the protein labeling step to subsets of the proteome based on shared functional properties4 or occur in vivo. Click chemistry is then applied to couple the in vivo- post-translational modification state5–8. One of these emerging labeled proteins to azide-containing reporter molecules after cell targeted proteomic platforms is ABPP, which utilizes active site- lysis and homogenization. directed chemical probes to determine the functional state of many enzymes in parallel, directly in native biological samples4,9,10.ABPP A tandem orthogonal proteolysis (TOP) method for ABPP probes typically contain two core elements: (i) a reactive group for Initial analytical tools for ABPP relied on gel-based methods that binding/labeling and (ii) a reporter tag (biotin or fluorophore) for are problematic to automate and often fail to resolve highly related rapid and sensitive detection and isolation of labeled enzymes from protein species. With the development of increasingly sensitive and proteomes. Reactive groups often correspond to mild electrophiles accurate MS instruments and advanced chromatography tools, it that modify catalytic residues in enzyme active sites that demon- was desirable to shift the field of ABPP toward a ‘‘gel-free’’ platform. strate enhanced nucleophilicity. Several classes of ABPP probes With an MS-based platform, it would also be theoretically possible represent adaptations of known affinity agents that target specific to determine the exact site of probe modification, which would enzyme classes. Examples include fluorophosphonates that target offer mechanistic insights into probe–protein interactions and the serine hydrolase superfamily11, activated ketones and epoxides possibly facilitate the annotation of enzymes of unknown function. that target cysteine proteases12 and acyl phosphates that target With these goals in mind, we have developed an advanced LC-MS/ kinases13. For proteins lacking cognate affinity labels, a combina- MS method for ABPP that enables identification of probe-labeled

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proteins in conjunction with their sites of N 20 Alkyne probe TEV tag N modiﬁcation . This protocol unites click N N chemistry, biotin-streptavidin enrichment 3

techniques and a TOP strategy to selectively Proteome labeling Click chemistry capture probe-labeled proteins and serially release their unmodiﬁed and modiﬁed peptides for analysis in sequential LC-MS/MS runs (Fig. 1). In brief, proteomes are treated with an m/z LC/LC-MS/MS alkyne-containing ABPP probe and then N Streptavidin N (Protein ID) m/z N subjected to click chemistry to introduce a Trypsin digestion m/z ‘‘TEV-biotin tag’’ onto probe-labeled pro- Enrichment, LC/LC-MS/MS reduction and (Site of labeling) s teins. The tobacco etch virus (TEV)-biotin alkylation TEV digestion N N N tag consists of an azide moiety and a biotin N N group separated by a seven-amino-acid N TEV protease recognition sequence and a linker region (Fig. 2), and it can be prepared natureprotocol / using standard solid-phase peptide synth- m Figure 1 | The TOP-ABPP method. Proteomes are treated with ABPP probes to label active enzymes. Click o esis methods (Fig. 3)20.Taggedproteinsare c

. chemistry is then applied to append a TEV-biotin tag to probe-labeled proteins, which are enriched on e r then subjected to streptavidin enrichment, streptavidin beads and subjected to sequential trypsin and TEV-protease digestion. Sequential LC/LC-MS/ u t

a after which the proteins are treated with a MS (MudPIT) analyses of the trypsin digests and probe-labeled peptides provide identification of labeled n . reducing agent and iodoacetamide to proteins and their sites of modification, respectively (figure adapted from Speers and Cravatt20). w w reduce and alkylate all cysteine thiols in w / /

: the proteome. The proteins are then digested on-bead with trypsin Proteins that appear in both data sets are considered to be specific p t t and the supernatant is isolated by filtration to provide the tryptic labeling events where probe modification occurred at a precise site. h

peptide sample for LC-MS/MS analysis. To obtain the TEV digest Proteins observed only in tryptic digests but not in the TEV digest p u for LC-MS/MS analysis, the probe-labeled peptides are eluted from may signify nonspeciﬁc labeling/binding events (e.g., nonspeciﬁc o r

G the beads by incubation with TEV protease. The TEV protease was binding of proteins to streptavidin beads and/or abundant proteins

g chosen due to its unique recognition domain, which renders it labeled on multiple sites). Conversely, proteins present in only the n i h orthogonal to almost all proteases typically used for protein TEV data sets may signify false predictions by the SEQUEST s i l digestion. Cleavage occurs between the Gln-Gly residues of the algorithm, which can make incorrect protein assignments based b 21 2 u sequence Glu-Asn-Leu-Tyr-Phe-Gln-Gly , leaving the bulky biotin on MS spectra of a single peptide. P

e group on the bead and releasing the labeled peptides into solution. Alternative methods for the identiﬁcation of probe-labeled r u

t The trypsin and TEV digests are analyzed in sequential tandem peptides involve the selective enrichment and elution of probe- a

N LC-MS/MS experiments, utilizing MudPIT (multidimensional labeled peptide fragments but discard the rest of the proteome

7 protein identiﬁcation technology)22, followed by analysis by the digest23,24. In contrast, analysis of the two complementary data sets 0 0

2 SEQUEST algorithm, to characterize probe-labeled proteins and obtained in TOP-ABPP circumvents potential ‘false positive’ hits

© sites of modification, respectively. The exact residue that is mod- resulting from nonspecific labeling/enrichment as well as mis- ified by the probe can be determined by specifying a differential annotations owing to the assignment of protein identities from a modification in the SEQUEST search corresponding to the single labeled peptide. The TOP-ABPP method also removes expected mass of the probe modification. We have found that the affinity tags appended to the probe-labeled peptide that can tryptic and TEV digests constitute complementary data sets. complicate analysis by reducing the sensitivity of the mass spectrometric detection, replacing them instead with an additional amine functionality H N O to further enhance ionization in MS instru- S N H ments. The acquisition of tryptic data Linker TEV protease recognition sequence Linker sets also strengthens the quantitative analysis of enzyme activities, allowing investi- HN O OH O gators to use emerging isotope-free H N NH2 2 methods such as spectral counting to OH OOO OO O OO O H H H H H H H estimate the relative abundance of probe- N N N N N N N N N N N N N N NH 2 labeled proteins in two or more pro- H OOOH H H OOOH H H O 25–27 TEV teomes . As the sample size is much O OH cleavage site greater in tryptic digests compared to TEV N 3 digests (owing to the greater number of accessible peptides in the former data Biotin Azide set), spectral counts in the tryptic phase Figure 2 | Structure of the TEV-biotin tag. A biotin group for affinity purification is separated from an provide a more reliable estimate of azide handle for click chemistry by a seven-amino-acid TEV protease recognition sequence. protein abundance.

NATURE PROTOCOLS | VOL.2 NO.6 | 2007 | 1415 PROTOCOL

O O FmocHN NaN3, Tf2O, FmocHN OH OH

ZnCl2 H2O/CH2Cl2 NH2 N3

Fmoc-Lys(N3)-OH

O O (1) 20% piperidine/DMF H (1) 20% piperidine/DMF FmocHN N FmocHN N FmocHN N TEV-biotin tag H (2) Fmoc-Gly-OH H (2) Fmoc-Lys(N3)-OH O PyBOP, DIPEA, DMF PyBOP, DIPEA, DMF N 3 N3

Figure 3 | Synthesis of the TEV-biotin tag. s

We have found the TOP-ABPP protocol to be quite versatile. It complete recovery of all the proteins in the sample. Additionally, can be applied to any proteome of interest and functions with a we have observed that some probe-labeled peptides are either too variety of alkyne-containing ABPP probes. Owing to the steric long or too short for accurate characterization by MS. To circum- natureprotocol / inertness of the alkyne moiety, the protein-labeling step can be vent this limitation, it is possible to perform the ﬁrst digestion with m o performed in living systems16–18, so as to minimize perturbations an alternative protease to trypsin so as to generate peptides of c . e

r to protein activity/post-translational modiﬁcation state potentially suitable length/properties for characterization. We have also u t

a induced by sample lysing/homogenization. The protocol detailed observed that, in a few instances, SEQUEST is unable to assign a n . herein provides extensive experimental procedures for the synthesis probe modification to a specific, single residue. Instead, several w w of the TEV-biotin tag, the click chemistry reaction to append the residues within a peptide are annotated as potential modified sites. w / /

: TEV-biotin tag to labeled proteins, streptavidin enrichment of In these cases, it is imperative to manually analyze the fragmenta- p t t tagged proteins, trypsin and TEV digestion steps, and subsequent tion data to assign the exact site of probe modiﬁcation. Finally, the h

MS analysis of the resulting peptide mixtures. structural complexity of the ABPP probe can complicate data p u analysis. Some probes may be either too large or labile, making o r

G Present limitations the assignment of labeling sites problematic. Emerging fragmenta-

g One limitation of the protocol is the incomplete precipitation of tion techniques such as electron capture dissociation and electron n i 28 h proteins after click chemistry, which we currently estimate to be transfer dissociation may preserve the structure of labile probe s i l approximately 50% of the total proteins in the sample. Currently, modiﬁcations during the fragmentation process, facilitating the b u work is underway to optimize this precipitation step to allow assignment of labeling sites for chemically complex probes. P e r u t a

N MATERIALS

7 REAGENTS .Pyridine (Sigma-Aldrich, cat. no. 360570) 0

0 .Trifluoromethanesulfonic anhydride (Sigma-Aldrich, cat. no. 91737) .Trifluoroacetic acid (TFA) (Sigma-Aldrich, cat. no. 302031) 2 .Sodium azide (Sigma-Aldrich, cat. no. S8032) .Triethylsilane (Sigma-Aldrich, cat. no. 90550) © .Dichloromethane (CH2Cl2; Fisher Scientific, cat. no. AC610050040) .Diethylether (Sigma-Aldrich, cat. no. 309958) .Sodium bicarbonate (Sigma-Aldrich, cat. no. S6014) .Dimethylsulfoxide (DMSO; Sigma-Aldrich, cat. no. D5879) .Fmoc-Lys-OH (EMD Biosciences, cat. no. 04-12-1042) .Dulbecco’s phosphate-buffered saline (PBS) (Invitrogen, cat. no. .Zinc chloride (Sigma-Aldrich, cat. no. 211273) 14040-133) .Triethylamine (Sigma-Aldrich, cat. no. T0886) .Tris 2-carboxyethyl phosphine (TCEP; Sigma-Aldrich, cat. no. 93284) .Methanol (Fisher Scientific, cat. no. A411-4) m CRITICAL TCEP should be stored at 4 1C under argon. .Silica gel 60 (Fisher Scientific, cat. no. M77363) .Tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (‘ligand’) (Sigma-Aldrich, .Silica gel 60 F254 precoated plates for thin-layer chromatography (Fisher cat. no. 678937) Scientific, cat. no. M57892) .t-Butanol (Sigma-Aldrich, cat. no. 360538) .Rink amide MBHA resin (EMD Biosciences, cat. no. 01-64-0037) .Copper (II) sulfate (Sigma-Aldrich, cat. no. 451657) .Piperidine (Sigma-Aldrich, cat. no. 104094) .Sodium dodecyl sulfate (SDS; Sigma-Aldrich, cat. no. L6026) .N,N-dimethylformamide (DMF; Sigma-Aldrich, cat. no. 319937) .Resuspension buffer: 1.2% (w/v) SDS in PBS .Fmoc-Gly-OH (EMD Biosciences, cat. no. 04-12-1001) .Immobilized streptavidin (Pierce, cat. no. 20349) .Fmoc-Gln(Trt)-OH (EMD Biosciences, cat. no. 04-12-1090) .Wash buffer: 0.2% (w/v) SDS in PBS .Fmoc-Phe-OH (EMD Biosciences, cat. no. 04-12-1030) .Urea (Sigma-Aldrich, cat. no. U5378) .Fmoc-Tyr(tBu)-OH (EMD Biosciences, cat. no. 04-12-1037) .Denaturing buffer: 6 M urea in PBS .Fmoc-Leu-OH (EMD Biosciences, cat. no. 04-12-1025) .Iodoacetamide (Sigma-Aldrich, cat. no. I1149) .Fmoc-Asn(Trt)-OH (EMD Biosciences, cat. no. 04-12-1089) .Sequencing-grade modified trypsin and trypsin resuspension buffer .Fmoc-Glu(OtBu)-OH (EMD Biosciences, cat. no. 04-12-1020) (Promega, cat. no. V5111) .Fmoc-Thr(tBu)-OH (EMD Biosciences, cat. no. 04-12-1000) .Calcium chloride (Sigma-Aldrich, cat. no. C1016) .Fmoc-6-aminohexanoic acid (EMD Biosciences, cat. no. 04-12-1111) .Formic acid (Sigma-Aldrich, cat. no. 94318) .Fmoc-Lys(biotin)-OH (EMD Biosciences, cat. no. 04-12-1237 ) .High-purity water (Burdick & Jackson, cat. no. 365-4) .Diisopropylethylamine (Sigma-Aldrich, cat. no. 387649) .Optima-grade acetonitrile (Fisher Scientific, cat. no. A955-1) .Benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate .Ammonium acetate (Sigma-Aldrich, cat. no. A7330) (EMD Biosciences, cat. no. 01-62-0016) .Ac-TEV protease, 20 TEV buffer and 0.1 M DTT (Invitrogen, cat. no. .Acetic anhydride (Sigma-Aldrich, cat. no. 320102) 12575-015) m CRITICAL To avoid subjecting the Ac-TEV protease to multiple

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freeze–thaw cycles, aliquot the Invitrogen stock into 10 ml fractions and store .Glass pipettes the aliquots at 80 1C until use (aliquots can be stored for 4–6 months .Glass wool at 80 1C). .Eppendorf tubes (1 ml) .MS buffer A: 95% high-purity water, 5% Optima-grade acetonitrile and .Low-adhesion screw-top microcentrifuge tubes (Sarstedt, cat. no. 0.1% formic acid 72.692) .MS buffer B: 20% high-purity water, 80% Optima-grade acetonitrile and .Conical tubes (15 ml) 0.1% formic acid .Labshake shaker/rotisserie (Barnstead Thermolyne) .MS buffer C: 95% high-purity water, 5% Optima-grade acetonitrile, 0.1% .Centrifuge 5810R (Eppendorf) formic acid and 500 mM ammonium acetate .Centrifuge 5415D (Eppendorf) .5 mm Aqua C18 reverse-phase resin (Phenomenex, cat. no. 04A-4299) .Branson sonifier 250 (Branson) .5 mm Partisphere strong cation exchange resin (Whatman, cat. no. 4621- .Heat block (65–90 1C) 1507) .Incubator (35 and 29 1C) EQUIPMENT .Micro Bio-Spin columns (Bio-Rad, cat. no. 732-6204) .Glass round-bottom flasks (50, 100 and 500 ml) .Low-adhesion microcentrifuge tubes (Life Science Products Inc., cat. no. .Magnetic stir-bars and stirring unit 8510-250LRT-2) s .Dewer for ice bath .Fused silica capillary tubing (365 mm outer diameter, 100 mm inner .Glass separation funnel (50 ml) diameter; Agilent, cat. no. 160-2255-10) .Erlenmeyer flasks (50 ml) .Model P-2000 CO2 laser puller (Sutter Instrument) .Glass syringes (1 and 25 ml) .Fused silica capillary tubing (365 mm outer diameter, 250 mm inner .Rotary evaporator diameter; Agilent, cat. no. 160-2635-10) .Glass chromatographic column (3.4 cm internal diameter (i.d.) 50 cm .Inline micro filter assembly (Upchurch, cat. no. M-520) natureprotocol / long with a 500 ml reservoir) .Filter end fitting 0.5 mm peek (Upchurch, cat. no. M-120X) m o .UV lamp with 254 nm bulb .Microtight green sleeve (Upchurch, cat. no. F-185) c . e .Disposable test tubes, glass (10 cm (length) 1.3 cm (outer diameter)) .Micro tee-assembly (Upchurch, cat. no. P888) r u . . t Fritted glass peptide-synthesis vessel (10 ml) (Chemglass) Agilent 1100 series HPLC coupled to an LTQ ion trap mass spectrometer a . n Nitrogen and vacuum lines (Thermo Electron) . w .Filter funnel and side-arm flask .SEQUEST software w .Glass vials (10 and 25 ml) .DTASelect software w / / : p t t h

PROCEDURE p u Synthesis of Fmoc-Lys(N3)-OH TIMING 8h o r 1| Add sodium azide (1.0 g, 16 mmol, 8 equiv.) to a 50 ml round-bottom ﬂask equipped with a magnetic stir bar. Add 2.5 ml G

g of dichloromethane (CH2Cl2) to dissolve and cool to 0 1C in an ice bath. n i

h ! CAUTION Sodium azide is a highly toxic chemical. s i l b 2| Add triﬂuoromethanesulfonic anhydride (1.4 ml, 8 mmol, 4 equiv.) dropwise to the ﬂask with stirring at 0 1C. u P

e 3| Stir at 0 1C for 2 h and quench the reaction with saturated sodium bicarbonate solution (5 ml). r u t a 4| Transfer the mixture to a glass separation funnel and collect the lower layer (CH2Cl2)inanErlenmeyerﬂask(50ml). N

7 B 0 5| Wash the top aqueous layer with CH2Cl2 (2 5 ml) and combine all CH2Cl2 washes ( 10–12 ml) with the CH2Cl2 layer from 0

2 Step 4. This is the triflyl azide to be used in Step 8 below. © 6| In a separate 100 ml round-bottom flask equipped with a magnetic stir bar, dissolve Fmoc-Lys-OH (740 mg, 2 mmol, 1 equiv.) and zinc chloride (2.7 mg, 0.02 mmol, 1 mol%) in water (6.5 ml). 7| Add triethylamine (840 ml, 6 mmol, 3 equiv.) and methanol (22 ml) dropwise, using glass syringes and with constant stirring. 8| To this mixture, add the freshly prepared triflyl azide from Step 5 above and stir for 1 h at room temperature (25 1C). 9| Remove all the solvent by rotary evaporation at room temperature under reduced pressure to obtain a yellow residue.

10| Dissolve the residue in a minimal amount of CH2Cl2 and add to the top of a silica gel layer (B10 cm) in the glass chromatography column. Let the solution soak into the silica gel, add 100 ml of CH2Cl2 and allow the solvent to drain from the column. After the initial CH2Cl2 wash, fill the reservoir with a 2% solution of methanol/CH2Cl2 and elute the product from the column. Collect 5 ml fractions in disposable glass test tubes. Test each fraction with thin-layer chromatography using UV detection and pool all fractions containing the major product (Rf ¼ 0.5 in CH2Cl2:MeOH 4:1) in a 100 ml round-bottom flask. Remove all solvent using the rotary evaporator at room temperature, to obtain pure compound as a pale yellow solid (80–90% yield). ’ PAUSE POINT The purified Fmoc-Lys(N3)-OH (Fig. 3) can be stored for several weeks/months at 20 1C. Synthesis of the TEV-biotin tag TIMING 2–3 days 11| Weigh out 500 mg of Rink amide MBHA resin (500 mg, 0.3 mmol, 1 equiv.) and transfer to a 10 ml glass, fritted, peptide- synthesis vessel.

12| Swell the resin by adding 5 ml of CH2Cl2 to the resin and agitate by bubbling N2 gas through the inlet for 5 min. Remove the CH2Cl2 by attaching the outlet to a vacuum line and collecting the waste in a round-bottom ﬂask (500 ml).

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13| Deprotect the Fmoc group on the resin by adding a solution of 20% piperidine/DMF (5 ml) and agitate by bubbling N2 for 5 min. Remove the piperidine/DMF mixture by vacuum filtration, and add a fresh 5 ml of the same solution and repeat (3 5 min in total). After the complete deprotection cycle, wash the resin by adding 1 ml of DMF, agitate by bubbling N2 for 1 min, then remove the DMF by vacuum filtration. Repeat the DMF wash five times.

14| Weigh out the Fmoc-Lys(N3)-OH (260 mg, 0.7 mmol, 2 equiv.) from Step 10 and benzotriazole-1-yl-oxy-tris-pyrrolidino- phosphonium hexaﬂuorophosphate (340 mg, 0.7 mmol, 2 equiv.) into a glass vial. Dissolve in DMF (3 ml) and add this premixed solution to the resin. Using a syringe, add diisopropylethylamine (230 ml, 1.3 mmol, 4 equiv.) to the resin and agitate by bubbling N2 for 2 h. 15| Wash the resin (5 1 ml DMF), deprotect with 20% piperidine and wash again (5 1mlDMF)asinStep13. 16| Couple the remaining amino acids (0.7 mmol, 2 equiv.) using the standard protocol outlined in Steps 14 and 15 in the s following order: Fmoc-Gly-OH (200 mg), Fmoc-Gly-OH (200 mg), Fmoc-Gln(Trt)-OH (400 mg), Fmoc-Phe-OH (260 mg), Fmoc-Tyr(tBu)-OH (300 mg), Fmoc-Leu-OH (230 mg), Fmoc-Asn(Trt)-OH (390 mg), Fmoc-Glu(OtBu)-OH (280 mg), Fmoc-Thr(tBu)-OH (260 mg), Fmoc-Gly-OH (200 mg), Fmoc-Gly-OH (200 mg), Fmoc-6-aminohexanoic acid (230 mg) and Fmoc-Lys(biotin)-OH (390 mg) (Fig. 3). ’ PAUSE POINT The resin can be stored at 20 1C for several days in between the coupling steps. natureprotocol / m o

c 17| After the ﬁnal deprotection step following the coupling of Fmoc-Lys(biotin)-OH, the N terminus of the peptide chain is . e r capped with an acetyl group. To cap the peptide, add DMF (5 ml) to the resin, followed by acetic anhydride (310 ml, 3.3 mmol, u t a 10 equiv.) and pyridine (270 ml, 3.3 mmol, 10 equiv.) and agitate by bubbling N2 for 1 h. n . w w 18| Wash the resin with DMF (5 1ml)andCH2Cl2 (3 1 ml) by adding the indicated amount of solvent, agitating by w / /

: bubbling N2 for 1 min then removing the solvent by vacuum ﬁltration. Transfer the resin to a glass vial and add a premixed p t t solution of 90% TFA/5% CH2Cl2/2.5% water/2.5% triethylsilane (5 ml total volume). Incubate this cleavage reaction at room h

temperature for 2 h and agitate briefly by shaking every 30 min. p u ! CAUTION TFA is highly caustic; make sure to work in a ventilated fume hood and wear protective clothing. o r G 19| Transfer the cleavage mixture into a glass pipette with a plug of glass wool to filter off the resin. Collect the flow-through g n i in a glass vial and remove the TFA under a stream of nitrogen. h s i l 20| To the dry residue, add 5 ml of diethylether. The organic solvent will solubilize the impurities, whereas the TEV-biotin tag b u B

P will reprecipitate as a white solid ( 600 mg). e r

u 21| Remove the ether solution by vacuum filtration using a filter funnel and side-arm flask, and dry the white solid under t a vacuum for at least 3–4 h. N

7 ’ PAUSE POINT The white solid can be dried under vacuum overnight. 0 0 2 22| Dissolve the TEV-biotin tag from Step 21 in DMSO (20 mg in 2.2 ml) to yield a 5 mM stock solution to be used in the click © chemistry reaction (Step 27). ’ PAUSE POINT The white solid and the DMSO stock solution of the TEV-biotin tag can both be stored at 20 1C for several months. Protein labeling and cycloaddition TIMING 5h 23| Dilute the desired proteome sample to a 2 mg ml1 solution in PBS. 24| Add 500 mlofdiluted2mgml1 proteome solution to each of the four Eppendorf tubes. 25| Add 1–20 mM of the desired alkyne probe from a concentrated DMSO stock: do not exceed 10 mlofDMSOineach500ml reaction. After addition of the probe, vortex and leave at room temperature for 1 h. For instance, in our laboratory, we added 2 ml of a 5 mM stock solution of a phenylsulfonate ester alkyne probe (synthesis described in ref. 18) in DMSO to each 500 ml proteome sample, to a ﬁnal probe concentration of 20 mM. 26| Prepare a fresh solution of 50 mM TCEP in water (14.4 mg ml1) and a 1.7 mM stock of ‘ligand’ (see REAGENTS) in DMSO: t-butanol 1:4 (0.9 mg ml1). m CRITICAL STEP The TCEP should be stored at 4 1C under argon and a stock solution in water prepared fresh on the day it is to be used. 27| To each Eppendorf tube, add 11.6 ml of the TEV-biotin tag (from the 5 mM stock in DMSO from Step 22) and vortex. 28| Add 11.6 ml of fresh TCEP solution (from Step 26) without vortexing.

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29| Add 35 ml of ‘ligand’ solution (from Step 26) and vortex. 30| Add 11.6 ml of copper (II) sulfate (50 mM stock in water (12.5 mg ml1)) and vortex. 31| Leave the tubes at room temperature for 1 h (vortex after 30 min). At this stage, the proteins will start to precipitate and the solution will turn cloudy. 32| Combine the tubes pairwise and centrifuge for 4 min, at 6,500g at 4 1C. A protein pellet will form. 33| Remove the supernatant and add 500 ml of cold methanol to each of the tubes, sonicate for 3–4 s and combine tubes pairwise. m CRITICAL STEP Ensure that the methanol used for washing the protein pellet is cooled on ice before use. The use of warm methanol results in loss of protein from the pellet. s 34| Rotate at 4 1C for 10 min and centrifuge for 4 min, at 6,500g at 4 1C.

35| Remove the supernatant and repeat the wash (Steps 33 and 34) with 500 mlofcoldmethanol. 36| Remove the supernatant, add 1 ml of 1.2% SDS/PBS, sonicate for 3–4 s and heat to 80–90 1C for 5 min. natureprotocol / m 37| Transfer the solution to a 15 ml conical tube and dilute to 0.2% SDS with 5 ml of PBS. o c . ’ 1 e PAUSE POINT Samples can be stored at 80 C for several days. r u t a Afﬁnity puriﬁcation TIMING 5h n

. w 38| Add 100 ml of streptavidin beads to a 15 ml conical tube and wash the beads with 5 ml of PBS, centrifuging at 1,400g for w

w 1

/ 3 min at 25 C to remove the solvent from the beads. Repeat the wash two more times. / : p t t

h 39| Add the labeled proteome sample (from Step 37) to the beads and incubate for 3 h at room temperature.

p u 40| Centrifuge at 1,400g for 3 min and remove the supernatant. o r G 41| Wash the beads by adding 5 ml of 0.2% SDS/PBS, place on a rotator for 10 min, then centrifuge at 1,400g for 3 min and g n i remove the supernatant. h s i l b 42| Add 5 ml PBS to the beads and place on a rotator for 1 min, centrifuge at 1,400g for 3 min and remove the supernatant. u P

Repeat the PBS wash three times. e r u t 43| Wash with 3 5 ml water as described in Step 42. a N

7 44| Transfer beads to a screw-top Eppendorf tube using 1 ml of water, centrifuge, let beads settle and pipette off the supernatant. 0 0 2 On-bead trypsin digestion TIMING 12–15 h © 45| Add 500 ml of a 6 M urea/PBS solution to the beads.

46| Add 25 ml of a 200 mM solution of TCEP in water (57 mg ml1). 47| Place the tube in a 65 1C heat-block for 15 min and cool to 35 1C.

48| Add 25 ml of a 400 mM solution of iodoacetamide in water (74 mg ml1) and leave for 30 min in a 35 1C incubator with agitation.

49| Dilute the reaction with 950 ml of PBS, centrifuge at 1,400g for 2 min and remove the supernatant. 50| Add a premixed solution of 200 ml of 2 M urea/PBS, 2 ml of 100 mM calcium chloride in water and 4 ml of trypsin (20 mg reconstituted in 40 mloftheTrypsinbuffer). m CRITICAL STEP Ensure that the trypsin digestion is performed in 2 M urea, as excess denaturing agent can affect trypsin activity. ’ PAUSE POINT Allow the reaction to proceed overnight in a 37 1C incubator with agitation.

51| Transfer the supernatant and beads to a Bio-Spin column and elute into a low adhesion tube by centrifugation (1,000g).

52| Wash the beads with 2 50 ml of water and combine the water washings with the eluent from Step 51.

53| Add 15 ml of formic acid to the sample and store tryptic samples at 20 1C until mass spectrometric analysis. ’ PAUSE POINT The samples can be stored for several months at 20 1C.

NATURE PROTOCOLS | VOL.2 NO.6 | 2007 | 1419 PROTOCOL

m CRITICAL STEP It is important to acidify the samples below pH 3.0 because the peptides are not fully charged at higher pH and therefore would not bind efﬁciently to the strong cation exchange resin used in the LC-MS/MS step. On-bead TEV digestion TIMING 12–15 h 54| Wash the beads in the Bio-Spin column (from Step 53) with 3 600 mlofPBSand3 600 ml of water. m CRITICAL STEP Ensure that all the urea from the trypsin digestion step is removed from the beads, as urea can signiﬁcantly affect Ac-TEV protease activity. 55| Transfer beads to a screw-top Eppendorf.

56| Wash the beads with 1 TEV buffer (140 mlwater,7.5mlof20 TEV buffer, 1.5 mlof100mM DTT). 57| To the beads, add a premixed solution of TEV buffer as above, containing 5 mlofAc-TEVprotease. s ’ PAUSE POINT Leave the reaction in a 29 1C incubator overnight with mild agitation. m CRITICAL STEP Avoid multiple freeze–thaw cycles with the Ac-TEV protease. Aliquot the Invitrogen stock into 10 ml fractions and store the aliquots at 80 1C until use. The frozen aliquots are stable for 4–6 months. 58| Transfer the supernatant and beads to a Bio-Spin column and elute into a low adhesion Eppendorf tube. natureprotocol /

m 59| Wash the beads with 2 75 ml of water and combine the water washings with the eluent from Step 58. o c . e 60| Add 15 ml of formic acid to the sample. r u t ’ PAUSE POINT Samples are ready for MS analysis and can be stored at 20 1C for several days to months. a n . w w MS (general) TIMING 2–3 h w / /

: 61| Cut 45 cm of the 100 mm i.d. fused silica capillary tubing and, using a ﬂame, burn off 2–3 cm of the polyimide coating in p t t the middle. Wipe the burnt polyimide with a methanol-soaked Kimwipe to expose the underlying silica. Place the tubing in the h

laser puller with the exposed silica in line with the laser and generate two 5 mmtips. p u o r 62| Pack the tips from Step 61 with 10 cm of Aqua C18 reverse-phase resin and 4 cm of strong cation exchange resin (Fig. 4) G

g by making a slurry of the resins in methanol and pressure loading it onto the capillary. n i h s i 63| Using the 250 mm i.d. fused silica capillary tubing, make a desalting column by inserting 12 cm of the tubing into an inline l b microfilter assembly. Use the microtight green sleeve adapters to fit the tubing into the filter assembly. u P e r 64| Pack the desalting column from Step 63 with 4 cm of Aqua C18 reverse-phase resin (Fig. 4)usingtheslurryfromStep62. u t a N

65| Equilibrate the tip from Step 62 and the desalting column from Step 63 on an Agilent 1100 series HPLC using a gradient of 7

0 40% buffer A; 60% buffer B to 100% buffer A; 0% buffer B over 30 min, followed by a 10 min wash with 100% buffer A. Use a 0

2 ﬂow rate of 0.1 ml min1 with a tee splitter to reduce the ﬂow rate to 300–400 nl min1. © MS (tryptic samples) TIMING 11 h 66| Pressure-load the tryptic peptide samples from Step 53 onto the equilibrated desalting column from Steps 63 and 65.

67| Combine the loaded desalting column (Step 66) with the equilibrated tip (Steps 62 and 65) and attach to the LC-MS/MS system. The peptides are eluted off the column in ﬁve steps; Step 1: no salt; Step 2: 25% ammonium acetate; Step 3: 50% ammonium acetate; Step 4: 80% ammonium acetate; Step 5: 100% ammonium acetate. Use Tables 1–5 to set up the HPLC elution gradients for the ﬁve steps outlined above.

68| For all tryptic MudPIT samples, set the ﬂow rate through the column to 0.25 mlmin1 and the applied distal spray voltage to 2.5–2.7 kV. Perform MS2 data collection using one full scan (400–1,800 MW) followed by seven data-dependent MS2 scans of the most abundant ions with dynamic exclusion enabled (repeat count ¼ 1; exclusion list size ¼ 300, exclusion duration ¼ 60). Filter Desalting column MudPIT column MS (TEV samples) TIMING 11 h 250 µm i.d. 100 µm i.d. Tip 69| Pressure-load the TEV peptide samples from Step 60 onto 5 µm i.d. the equilibrated desalting column from Steps 63 and 65. 4 cm C18 4 cm SCX 10 cm C18

70| Combine the loaded desalting column (Step 69) with the Figure 4 | MudPIT column for LC-LC/MS-MS analysis of tryptic and TEV equilibrated tip (Steps 62 and 65) (Fig. 4) and attach to the peptides.

1420 | VOL.2 NO.6 | 2007 | NATURE PROTOCOLS PROTOCOL

LC-MS/MS system. The peptides are eluted off the column in ﬁve steps: Step 1: no salt; Step 2: 50% ammonium acetate; Step 3: 80% ammonium acetate; Step 4: 100% ammonium acetate; Step 5: 100% ammonium acetate. Use Tables 6–10 to set up the HPLC elution gradients for the ﬁve steps outlined above.

71| For all TEV MudPIT samples, set the ﬂow rate through the column to 0.25 mlmin1 and the applied distal spray voltage to 2.5–2.7 kV. Perform MS2 data collection using one full scan (400–1,800 MW) followed by 18 data-dependent MS2 scans of the most abundant ions with dynamic exclusion disabled.

TABLE 1 | Tryptic step 1 (0% ammonium acetate). Time (min) Flow rate (ml min1) % Buffer A % Buffer B % Buffer C s 0.00 0.1 100 0 0 5.00 0.1 100 0 0 60.00 0.1 55 45 0 70.00 0.1 0 100 0 80.00 0.1 0 100 0 natureprotocol / 90.00 0.1 0 100 0 m o c . e r u t a n . TABLE 2 | Tryptic step 2 (25% ammonium acetate). w w 1 w Time (min) Flow rate (ml min ) % Buffer A % Buffer B % Buffer C / / :

p 0.00 0.1 100 0 0 t t h 3.00 0.1 100 0 0

p 3.10 0.1 70 5 25 u o 5.00 0.1 70 5 25 r G

5.10 0.1 95 5 0 g

n 15.00 0.1 85 15 0 i h 60.00 0.1 75 25 0 s i l 112.00 0.1 45 55 0 b u P e r u t a N

7 TABLE 3 | Tryptic step 3 (50% ammonium acetate). 0 0

2 Time (min) Flow rate (ml min1) % Buffer A % Buffer B % Buffer C © 0.00 0.1 100 0 0 3.00 0.1 100 0 0 3.10 0.1 45 5 50 5.00 0.1 45 5 50 5.10 0.1 95 5 0 15.00 0.1 85 15 0 60.00 0.1 75 25 0 112.00 0.1 45 55 0

TABLE 4 | Tryptic step 4 (80% ammonium acetate). Time (min) Flow rate (ml min1) % Buffer A % Buffer B % Buffer C 0.00 0.1 100 0 0 3.00 0.1 100 0 0 3.10 0.1 15 5 80 5.00 0.1 15 5 80 5.10 0.1 95 5 0 15.00 0.1 85 15 0 60.00 0.1 75 25 0 112.00 0.1 45 55 0

NATURE PROTOCOLS | VOL.2 NO.6 | 2007 | 1421 © 2007 Nature Publishing Group http://www.nature.com/natureprotocols 5.0011000 0 0 0 0 100 100 0 0 0 65 30 15 7 0 0 0 0 100 35 70 85 93 0 0 100 100 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 150.00 140.00 90.00 23.00 15.10 15.00 2.10 2.00 0.00 0.0011000 0 0 0 0 0 0 100 100 0 0 0 0 100 100 30 15 7 0 0 0 0 0 0 0 0 0 100 80 100 0 80 0 0 0 70 85 93 55 0 0 55 25 100 15 5 0 100 5 0 0.1 5 0 0.1 0 0 0 0.1 0 50 0.1 50 0.1 45 0.1 0 45 0.1 75 0 0.1 85 0.1 95 55 0.1 15 55 0.1 15 25 15 100 5 200.00 0 100 5 195.00 0 5 194.00 0 184.00 0 0 100.00 0 0.1 0 25.00 0.1 45 0.1 20.10 45 0.1 20.00 0.1 75 4.10 0.1 85 4.00 95 0.1 100 0.00 45 0.1 100 45 0.1 45 0 100 0 100 120.00 115.00 0.1 63.00 0.1 18.00 0 0.1 8.10 0 0.1 8.00 0.1 55 5.10 0.1 100 5.00 0.1 100 0.00 0.1 0.1 80.00 75.00 0.1 35.00 0.1 15.00 0.1 8.10 0.1 8.00 0.1 6.10 6.00 0.00 100.00 70.00 60.00 5.00 0.00 ie(i)Fo ae(lmin (ml rate Flow (min) Time min (ml rate Flow (min) Time min (ml rate Flow (min) Time min (ml rate Flow (min) Time min (ml rate Flow (min) Time AL 9 TABLE 8 TABLE 7 TABLE 6 TABLE 5 TABLE 1422 PROTOCOL | O. NO.6 VOL.2 | | | | | E tp4(0%amnu acetate). ammonium (100% 4 step TEV acetate). ammonium (80% 3 step TEV acetate). ammonium (50% 2 step TEV acetate). ammonium (0% 1 step TEV acetate). ammonium (100% 5 step Tryptic | 2007 | AUEPROTOCOLS NATURE 1 1 1 1 1 ufrA%Bfe ufrC Buffer % B Buffer % A Buffer % ) ufrA%Bfe ufrC Buffer % B Buffer % C A Buffer Buffer % % B ) Buffer % C Buffer % A Buffer % B Buffer % ) A Buffer % ) ufrA%Bfe ufrC Buffer % B Buffer % A Buffer % ) PROTOCOL

TABLE 10 | TEV step 5 (100% ammonium acetate). Time (min) Flow rate (ml min1) % Buffer A % Buffer B % Buffer C 0.00 0.1 100 0 0 4.00 0.1 100 0 0 4.10 0.1 0 0 100 14.00 0.1 0 0 100 14.10 0.1 93 7 0 30.00 0.1 70 30 0 50.00 0.1 0 100 0 55.00 0.1 0 100 0 56.00 0.1 100 0 0 60.00 0.1 100 0 0 s

Data analysis TIMING 2–4 h 72| Search the data from the MudPIT analyses against a protein sequence database (e.g., for human and mouse proteomes, natureprotocol

/ versions 3.23 of the human and mouse IPI databases) using the SEQUEST algorithm for peptide sequence identiﬁcation. m o c

. 73| For both tryptic and TEV runs, specify a static modiﬁcation of +57 on cysteine to account for alkylation with iodoacetamide e r

u (see Step 48). t a n .

w 74| For TEV runs, specify the probe modification as a differential modification on the desired amino acid (for probe modifica- w

w tions on cysteine, specify a differential modiﬁcation of 57 AMU less than the calculated mass of the probe to account for the / / :

p previously speciﬁed static modiﬁcation). t t h

75| Filter the SEQUEST output ﬁles using DTASelect. For tryptic runs, use the default parameters in the DTASelect Manual v1.9 p u (min Xcorr ¼ 1.8 (+1), 2.5 (+2), 3.5 (+3), min DeltaCN ¼ 0.08). For TEV runs, lower the Xcorr parameters to 1.0 (+1), 2.0 (+2) o r 2.0 (+3) and the DeltaCN to 0.06. Also specify that all peptides contain the probe modiﬁcation as well as two tryptic ends. G

g ? TROUBLESHOOTING n i h s i

l ANTICIPATED RESULTS b u Fmoc-Lys(N3)-OH P Yield, 79%; 1H NMR (CDCl , 400 MHz) d 7.77 (d, J 7.6, 2H), 7.61 (m, 1H), 7.55 (m, 1H), 7.41 (t, J 7.3, 2H), 7.33 e 3 ¼ ¼ r u (t, J 7.3, 2H), 5.48 (d, J 8.2, 1H), 4.56 (m, 1H), 4.45 (d, J 7.0, 2H), 4.24 (m, 1H), 3.27 (t, J 6.7, 2H), 1.93 (m, 1H), t ¼ ¼ ¼ ¼ a +

N 1.74 (m, 1H), 1.61 (m, 2H), 1.48 (m, 2H). MALDI-FTMS m/z 417.1535 (C21H22N4O4 +Na requires 417.1533).

7 0

0 Tev-biotin tag 2

© HPLC-MS (negative mode) m/z 1,802.8 (C80H121N23O23S-H requires 1,802.86). MS: representative example Application of this protocol to a mouse heart soluble proteome with 20 mM phenylsulfonate ester alkyne probe (as indicated in Step 25) provides the labeled peptides illustrated in Table 11 with representative spectral counts for both tryptic and TEV digests. The data show several peptides that were unambiguously labeled at one residue on the peptide (e.g., Delta3,5-delta2,4-dienoyl- CoA isomerase, mitochondrial precursor and acyl-CoA dehydrogenase, long-chain-specific, mitochondrial precursor). However, there are also instances in which SEQUEST assigned the labeling event to multiple residues (e.g., NAD(P)H menadione oxidoreductase 2, dioxin inducible and histidine triad nucleotide-binding protein). In these cases, it is necessary to manually investigate the MS2 data to identify the correct site of labeling. Additionally, there are several proteins identified in the TEV data set that are not found in the tryptic data set (e.g., heat-shock protein 1A and splice isoform of Q61738 integrin a-7 precursor). These are likely to be false-positives resulting from SEQUEST assigning protein identities based on a single peptide sequence. TIMING Synthesis of Fmoc-Lys(N3)-OH (Steps 1–10): 8 h Synthesis of the TEV-biotin tag (Steps 11–22): 2–3 days Protein labeling and cycloaddition (Steps 23–37): 5 h Affinity purification (Steps 38–44): 5 h On-bead trypsin digestion (Steps 45–53): 12–15 h On-bead TEV digestion (Steps 54–60): 12–15 h MS (Steps 61–71): 20–22 h Data analysis (Steps 72–75): 2–4 h

NATURE PROTOCOLS | VOL.2 NO.6 | 2007 | 1423 PROTOCOL

TABLE 11 | Representative data for the application of the TOP strategy to mouse soluble heart proteome with a phenylsulfonate ester alkyne probe (data adapted from ref. 20). Spectral counts Spectral counts Protein Peptide (TEV) (tryptic) Delta3,5-delta2,4-dienoyl-CoA isomerase, mitochondrial K.EVDMGLAAD*VGTLQR.L 1061 69 precursor Acyl-CoA dehydrogenase, long-chain-speciﬁc, K.GFYYLMQELPQE*R.L 86 62 mitochondrial precursor NAD(P)H menadione oxidoreductase 2, dioxin-inducible K.VLAPQISFGLD*VSSE*EER.K 85 29 Acyl-CoA dehydrogenase, very-long-chain-speciﬁc, R.IFE*GANDILR.L 5 36 mitochondrial precursor Histidine triad nucleotide-binding protein R.ISQAEE*DD*QQLLGHLLLVAK.K 21 31 s Methylmalonate-semialdehyde dehydrogenase R.C*MAILSTAILVGEAK.K 17 48 Hemoglobin alpha, adult chain 1 K.IGGHGAE*Y*GAE*ALE*R.M 19 64 Enolase 3, beta muscle K.VNQIGSVTESIQAC*K.L 16 43 Heat-shock protein 1A K.QTQTFTTYSDNQPGVLIQVY*E*GER.A 50 0 Splice isoform of Q61738 integrin alpha-7 precursor R.QQFKEE*KTGTIQR.S 5 0 natureprotocol

/ Acyl-CoA dehydrogenase, medium-chain-speciﬁc, K.IYQI*E*GTAQIQR.L 6 42 m mitochondrial precursor o c . Riken cDNA231000O14 gene (uncharacterized) R.AVLAGIY*NTE*LVMMQDSSPDFEDTWR.F 13 13 e r u Citrate synthase K.GLVY*E*TSVLDPDE*GIR.F 15 46 t a

n Isocitrate dehydrogenase K.SEGGFIWAC*K.N 5 15 . w The * refers to the speciﬁc residue containing the probe modiﬁcation and the period refers to the sites of tryptic cleavage. w w / / : p t t ? TROUBLESHOOTING h

Troubleshooting advice can be found in Table 12. p u o r |

G TABLE 12 Troubleshooting table. g

n Step Problem Possible reason Solution i h

s 75 Reduced peptide identiﬁcations in Incomplete trypsin digestion Trypsin activity is optimal at 2 M urea but can be i l

b the tryptic data set reduced by the high urea concentrations (42M) u

P left in the sample after the denaturation step. Wash the e

r beads with PBS, resuspend in 2 M urea/PBS, and u t resubject to trypsin digestion a N

0 75 Reduced peptide identiﬁcations in Incomplete TEV digestion Ac-TEV protease is very sensitive to traces of urea in 0

2 the TEV data set the buffer. Wash thoroughly with 3 PBS, 3 water

COMPETING INTERESTS STATEMENT The authors declare no competing financial 8. Vocadlo, D.J., Hang, H.C., Kim, E.J., Hanover, J.A. & Bertozzi, C.R. A chemical interests. approach for identifying O-GlcNAc-modified proteins in cells. Proc. Natl. Acad. Sci. USA 100, 9116–9121 (2003). Published online at http://www.natureprotocols.com 9. Adam, G.C., Sorensen, E.J. & Cravatt, B.F. Chemical strategies for functional Rights and permissions information is available online at http://npg.nature.com/ proteomics. Mol. Cell. Proteomics 1, 781–790 (2002). reprintsandpermissions 10. Speers, A.E. & Cravatt, B.F. Chemical strategies for activity-based proteomics. Chembiochem 5, 41–47 (2004). 1. Anderson, N.L. & Anderson, N.G. Proteome and proteomics: new 11. Liu, Y., Patricelli, M.P. & Cravatt, B.F. Activity-based protein profiling: the serine technologies, new concepts, and new words. Electrophoresis 19, 1853–1861 hydrolases. Proc. Natl. Acad. Sci. USA 96, 14694–14699 (1999). (1998). 12. Kato, D. et al. Activity-based probes that target diverse cysteine protease 2. Mann, M., Hendrickson, R.C. & Pandey, A. Analysis of proteins and proteomes by families. Nat. Chem. Biol. 1, 33–38 (2005). mass spectrometry. Annu. Rev. Biochem. 70, 437–473 (2001). 13. Patricelli, M.P. et al. Functional interrogation of the kinome using nucleotide acyl 3. Kobe, B. & Kemp, B.E. Active site-directed protein regulation. Nature 402,373– phosphates. Biochemistry 46, 350–358 (2007). 376 (1999). 14. Adam, G.C., Sorensen, E.J. & Cravatt, B.F. Proteomic profiling of mechanistically 4. Evans, M.J. & Cravatt, B.F. Mechanism-based profiling of enzyme families. Chem. distinct enzyme classes using a common chemotype. Nat. Biotechnol. 20,805– Rev. 106, 3279–3301 (2006). 809 (2002). 5. Oda, Y., Nagasu, T. & Chait, B.T. Enrichment analysis of phosphorylated 15. Barglow, K.T. & Cravatt, B.F. Discovering disease-associated enzymes by proteome proteins as a tool for probing the phosphoproteome. Nat. Biotechnol. 19, reactivity profiling. Chem. Biol. 11, 1523–31 (2004). 379–382 (2001). 16. Evans, M.J., Saghatelian, A., Sorensen, E.J. & Cravatt, B.F. Target discovery in 6. Zhou, H., Watts, J.D. & Aebersold, R. A systematic approach to the analysis of small-molecule cell-based screens by in situ proteome reactivity profiling. Nat. protein phosphorylation. Nat. Biotechnol. 19, 375–378 (2001). Biotechnol. 23, 1303–1307 (2005). 7. Tai, H.C., Khidekel, N., Ficarro, S.B., Peters, E.C. & Hsieh-Wilson, L.C. Parallel 17. Speers, A.E., Adam, G.C. & Cravatt, B.F. Activity-based protein profiling in vivo identification of O-GlcNAc-modified proteins from cell lysates. J. Am. Chem. Soc. using a copper(i)-catalyzed azide-alkyne [3 + 2] cycloaddition. J. Am. Chem. Soc. 126, 10500–10501 (2004). 125, 4686–4687 (2003).

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p u o r G g n i h s i l b u P e r u t a N

7 0 0 2 ©

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