Review

Activity-based probes as a tool for functional proteomic analysis of

Expert Rev. Proteomics 5(5), 721–730 (2008)

Marko Fonović† and Traditional proteomics methodology allows global ana­lysis of abundance but does not Matthew Bogyo provide information on the regulation of protein activity. Proteases, in particular, are known for †Author for correspondence their multilayered post-translational activity regulation that can lead to a significant difference Department of Biochemistry, between abundance levels and their activity. To address these issues, the field Molecular and Structural of activity-based proteomics has been established in order to characterize protein activity and Biology, Jožef Stefan Institute, monitor the functional regulation of in complex proteomes. In this review, we present Jamova Cesta 39, SI-1000 structural features of activity-based probes for proteases and discuss their applications in Ljubljana, Slovenia proteomic profiling of various catalytic classes of proteases. Tel.: +386 014 773 474 [email protected] Keywords: activity-based probe • protease • • proteomics

All organisms express a large variety of struct­ than activity do not always provide sufficient urally and catalytically diverse proteases. information to allow functional assignments of Genome-sequencing projects have revealed the specific proteases. presence of at least 569 human proteases that Traditional proteomics methodology, based on can be divided into several distinct catalytical 2D gel chromatography coupled with mass spec- classes: metallo­proteinases, serine, , trometry (MS), has recently been complemented threonine and aspartic proteases [1] . Interestingly, with non-gel-based methods, such as MudPIT the mouse and rat genomes show even higher and SELDI [3,4]. Chromatography separations protease variety, with 644 and 629 members, employed in these methods significantly increase respectively [2]. Proteases have the unique abil- resolving power and proteome coverage, but they ity to hydrolyze bonds and, therefore, still lack the ability to resolve active and inactive irreversibly modify the function of target pro- populations of enzymes. Activity-based proteo­ teins. They play crucial roles in diverse physio­ mics is a relatively new subdivision of proteomics logical processes, such as protein turnover, blood that has been developed to characterize protein coagulation, apoptosis, hormone processing and activity and directly monitor the functional regu- bone remodeling. Since proteolytic processing lation of enzymes in complex proteomes. This is an irreversible event, it must be tightly regu- method utilizes small molecule activity-based lated to avoid catastrophic consequences. The probes (ABPs) that covalently modify the enzyme most common way that proteases are regulated and enable detection and affinity puri- is by expression as an inactive proenzyme which fication of a target enzyme population (Fi g u r e 1). requires activation by proteolytic cleavage. The ABPs are highly selective and can be used for ana­ activation process can be regulated by cellular lysis of complex samples, such as cell lysates, intact signaling as well as by changes in the micro­ cells and even whole organisms. In this review, we environment. If this regulation fails, endo­ will outline advances in the development of ABPs genous extra­cellular and intracellular protease and highlight the numerous applications of these inhibitors (e.g., serpins and ) prevent reagents for the study of various protease classes any unwanted proteolysis. This multilayered and their roles in human disease. post-translational regulation can lead to signifi- cant differences between overall abundance lev- Structure of activity-based probes els and activity of proteases. Thus, methods that All ABPs share a similar basic design that rely on detection of protease abundance rather incorporates elements required for targeting, www.expert-reviews.com 10.1586/14789450.5.5.721 © 2008 Expert Reviews Ltd ISSN 1478-9450 721

Review Fonović & Bogyo y

Labeling with ABP Affinity capture Elution MS/MS Intensit

m/z Whole proteome Analysis Identification

Expert Rev. Proteomics © Future Science Group (2008)

Figure 1. Workflow of activity-based proteomics. Target in the complex proteome are labeled with activity-based probes. Labeled proteins are enriched by affinity purification and identified by mass spectrometry. ABP: Activity-based probes; MS/MS: Tandem mass spectrometry. modification and detection of labeled proteins. Structurally, the linker can be as simple as an extended alkyl or polyethylene those elements can be divided into the reactive group, linker glycol spacer. Alternatively, the linker can be used to increase probe and tag (Fi g u r e 2A). specificity by the addition of structural elements (i.e., ) that enhance the specificity of the probe. For most protease probes, Reactive group the linker is a short peptide sequence that mimics a true protein The reactive functional group, also termed ‘the warhead’, provides . This peptide linker can be optimized to target distinct a covalent attachment of the probe to the catalytic residue of the subfamilies of proteases by changing its sequence [14] . protease active site. The majority of protease families have well- A more recent improvement in linker design has been the develop- established catalytic mechanisms that have been defined using ment of a region that can be enzymatically or chemically cleaved to abundant structural and kinetic data. These data have enabled the release probe-labeled proteins. This simplifies the elution process synthesis of a large variety of specific, irreversible small-­molecule and decreases background protein contamination during an affinity inhibitors that can be transformed into ABPs by the addition of purification. Various cleavable linker elements have recently been an appropriate reporter tag [5]. Probes for serine, cysteine and reported. Cravatt and coworkers developed a cleavable linker that threonine proteases utilize electrophile groups that form a cova- has a peptide recognition site for tobacco etch virus protease and lent bond with their active site nucleophile (serine, cysteine or applied it to the identification of proteins that had been labeled with threonine residue). The electrophile has to be strong enough a sulfonate ester probe [15,16] . One of the first chemically cleavable to react with the active site nucleophile but not with other free linkers described makes use of a bond that is readily cleaved nucleophiles in the proteome (Fi g u r e 2B). Various reactive groups in mild reductive conditions (Fi g u r e 3A) [17–19] . Disulfide linkers work have been successfully applied to protease probe designs (Tab l e 1). well in some applications, such as profiling of serine , but The phosphonate group is highly reactive towards the hydroxyl they are not compatible with reductive buffers that are needed for nucleophile of serine proteases and has therefore found use in a the profiling of cysteine proteases. are also quite labile number of probes for both trypsin and chymotrypsin fold serine and prone to disulfide exchange, which can also lead to a premature proteases [6]. The , on the other hand, has been tar- release and nonspecific labeling of proteins containing free thiol geted with probes containing a vinyl sulfone reactive group even groups [20]. These problems have been addressed with the develop- though this class of compounds was originally designed to react ment of a novel diazo cleavable linker that enables selective elution with cysteine proteases [7]. Interestingly, a,b-epoxy ketones are also under mild reductive conditions (Fi g u r e 3A) [21] . The diazo linker was known to form highly selective bonds with the N-terminal threo- successfully incorporated into ABPs that were used for labeling and nine of the proteasome [8]. Cysteine proteases are the family most proteomic identification of cysteine and serine proteases [22]. As an extensively studied using ABPs and probes containing a number alternative to cleavage by chemical reduction, acid cleavable linkers of different reactive functional groups, including epoxides, diazo­ have been developed and are also commercially available for isotope methyl ketones, acyloxymethyl ketones and vinyl sulfones, have coded affinity tag (ICAT) applications[23–25] . However, the strong been designed [9–11] . Unfortunately, the same electrophile-based acids required for cleavage, such as trifluoroacetic acid, also release approach does not work well for and aspartic nonspecifically bound proteins. Finally, the linker region can be used proteinases because they do not form covalent intermediates with for isotopic labeling in ICAT-like applications. Incorporation of light their substrates. In this case, probes are designed using reversible and heavy linkers have been demonstrated on the general inhibitor scaffolds coupled to a photoreactive group that provides family protease probe DCG‑04, which was used for quantitative covalent attachment upon irradiation with UV light [12,13]. profiling of cysteine proteases by direct MS-based methods [26].

Linker Tag The most basic function of the linker is to separate the reactive Incorporation of a tag group into an activity-based probe functional group from the tag. This improves probe accessi­bility enables the visualization and/or purification of labeled proteins. and reduces steric hindrance between both groups. For this purpose, Radioisotopes are commonly used in biological applications and

722 Expert Rev. Proteomics 5(5), (2008) Activity-based probes as a tool for functional proteomic analysis of proteases Review

A B

O O Epoxide Enzyme MTS-I-94 H OPh Enzyme N Cys N N P OPh O OS S H H SH H O O R OEt R OEt N N N N N HN H O H O OH O NH O O OH Tag Linker Warhead

O O O Phosphonate Enzyme H H DCG-04 O Ser N N OEt OPh O H2N N N OH OPh O H H O P P O O O R OPh R OSer Enzyme HN NH HO NH S O

Figure 2. Structure and binding of activity-based probes. (A) Structural features of two types of activity-based probes. Probe MTS-I-94 targets serine proteases. It is composed of a phosphonate warhead, a cleavable diazo linker and a biotin tag. DCG-04 is a probe with an epoxide warhead. (B) Probes that target serine and cysteine proteases irreversibly, and covalently bind to the active site nucleophile (serine or cysteine). were also the first to be used as tags for activity-based proteom- profiling[34,35] . Dipyrromethene boron difluoride (BODIPY) and 125 ics [27]. The γ-ray emitter, I, can be introduced by iodination cyanine dyes are photostable with high absorption coefficients, of any covalent inhibitor that has a phenol group. Labeled proteins high quantum yields and narrow absorption peaks. They are can then be analyzed by 1D or 2D electrophoresis and visualized also cell permeable, making them useful in a variety of biologi- by autoradiography. This approach was used in activity profiling cal applications. BODIPY-labeled epoxide probes have been used of cysteine proteases, such as [28], [29] and for labeling of cysteine proteases in lysates and intact cells [36]. 3 [30,31]. Protease probes that incorporate H as a tag probes containing the cyanine fluoro­phore have been have also been reported [32]. Tritium replaces hydrogen and this reported [37]. Owing to their superior chemical properties, cya- modification does not affect the probe structure. However, its nine dyes have a great potential for the use in imaging applica- use is limited by low, specific activity that requires long exposure tions. Unfortunately, they are also extremely expensive, limiting times to obtain labeling profiles. Although radiolabeled probes their use for large-scale synthesis. are easy to prepare, their storage time is limited by the half-life Fluorophores are perfect for rapid determination of labeling of the radiolabel. They also require special handling and labora- patterns after sodium dodecyl sulfate polyacrylamide gel electro- tory safety procedures and cannot be used to directly identify phoresis (SDS-PAGE) ana­lysis, since gels can be directly scanned target proteins. using a laser scanner and there is no need for time-consuming The application of fluorescent tags was a big step forward in blotting procedures required for biotinylated probes. However, the development of ABPs. Fluorophores are safe to use and are for the purpose of isolation and identification of proteins tar- commercially available in a variety of excitation and emission geted by ABPs, biotin still remains the affinity tag of choice. spectral ranges. The first fluorescent ABPs were demonstrated Strong, diffusion-limited interaction between biotin and immo- for cysteine proteases and serine hydrolases [33,34]. Various types bilized avidin provides efficient enrichment of even low abundant of dyes can be used as fluorophores, but their chemical prop- labeled targets. Avidin-based enrichment also reduces complexity erties can significantly influence their range of applications. of biological samples, which is extremely beneficial for proteomic Fluorescein and rodamine are inexpensive but are susceptible to applications. Unfortunately, the strong avidin–biotin interaction photobleaching, limiting their imaging applications. The fluo- requires harsh elution conditions, which are not directly compat- rophosphonate (FP) warhead containing a PEG linker labeled ible with MS ana­lysis. Eluted samples must first be analyzed by with rhoadamine tag has been demonstrated to be an effective SDS-PAGE and the proteins cut from a gel for identi­fication. probe for profiling of serine hydrolases in cell homogenates [33]. Alternatively, enriched proteins can be prepared for MS by ‘on Fluorescein and rodamine are not cell permeable and, therefore, bead’ digestion. In this approach, proteins are reduced, alky- cannot be used for the labeling of intracellular targets in vivo. lated and digested while they are still bound to immobilized Dansyl and nitrobenz-2-oxa-1,3-diazole (NBD) are inexpen- avidin. In both approaches, the final sample is contaminated sive fluorophores that have been used for in vitro activity-based with native biotinylated proteins and abundant, nonspecifically www.expert-reviews.com 723 Review Fonović & Bogyo

based on the structure of their active site. The two most abundant A Reduction and intensively studied clans are the CA and CD clans. Clan R S SR R SH HS R 1 2 Dithiothreitol 1 2 CA includes papain and families of cysteine proteases, as well as various families of the -processing peptidases. Reduction E-64 is a well-known natural inhibitor of this family of R1 NNR2 R1 NH2 H2NR2 Sodium hydrosulfite cysteine proteases, and its structure and reactive epoxide were a logical choice for the use in the design of cysteine protease B ABPs [41] . The general epoxide probe DCG-04 has a wide speci- N R1 R1 Cu(I) N N N ficity towards and cysteine cathepsins, and was used N N to determine their roles in various physiological and pathologi- R cal processes. DCG‑04 was used to identify m-calpain and the R2 2 related Lp82 as the main active cysteine proteases present in the eye lens that mediate γ-crystalin cleavage during cataract forma- Figure 3. Cleavable and ‘click’ chemistry linkers. tion [42]. Activity-based labeling and affinity purification with (A) Reduction cleavage of disulfide and diazo cleavable linkers. (B) Mechanism of ‘click’ chemistry. Cycloaddition of an azide DCG-04 was used for identification of the papain-like protease and alkyne functional group is used for the tag attachment after L as the prohormone-processing enzyme responsible protein labeling. for cleaving proenkephalin [43]. This probe was also successfully used to identify cysteine proteases involved in host cell inva- bound proteins. This contamination can be minimized by the use sion by the parasite Plasmodium falciparum and in the proteomic of probes with cleavable linkers that enable specific elution[22] . The identification of papain-like proteases in plants[44,45] . Papain-like largest disadvantage of the biotin tag is its overall poor cell perme- cathepsins were found to be involved in various stages of ability that limits its use for in situ and in vivo applications. Since progression. Fluorescent versions of DCG-04 were used to profile protease activity is often regulated by factors within its intracellular upregulation of cathepsin activity during tumorigenesis in several microenvironment, labeling of cell lysates does not often provide mouse cancer models [46,47]. an accurate picture of protease activity profiles. To resolve this Mammalian deubiquitinating enzymes (DUBs) are another problem, a tandem labeling strategy that utilizes ‘click chemistry’ important group of clan CA cysteine proteases. Many of these was developed. Click chemistry is based on a cycloaddition of an enzymes act as oncogenes, tumor suppressors or have some other azide and alkyne functional group in the presence of a copper connection to cancer and, therefore, represent an important target catalyst (Fi g u r e 3B) [2,3]. This method was originally developed by for drug development. The first DUB-specific ABPs were designed Sharpless and coworkers, but, in the modified form, was applied based on a full-length ubiquitin (Ub) derivatized at the C-terminus to activity-based proteomics [38,39]. In this approach, intact cells with a reactive vinyl sulfone. Fluorogenic substrates for DUBs, such are labeled with a cell-permeable probe, which carries an alkyne or as Ub-7-amido-4-methyl coumarin (UbAMC), as well as DUB azido group instead of a biotin tag. After labeling, cells are homog- inhibitors Ub-aldehyde and Ub-nitrile, were also developed [48–51] . enized and the biotin tag, which carries a complementary alkyne or Development of the irreversible DUB inhibitor, ubiquitin vinyl azido functional group, is added by cycloaddition. Tagged proteins sulfone (UbVS), represented an important advancement in the can then be enriched and identified by MS [40]. study of DUB function. UbVS binding is SDS resistant and also enables radioiodination with subsequent autoradiographic visual- Application of probes for the profiling of proteases ization of labeled enzymes [52]. However, this probe still lacks the Cysteine, serine and threonine proteases all have an active site ability to enrich and isolate targets for the purpose of proteomic nucleophile that can be covalently modified by electrophiles, and it is identification. To achieve this goal, an intein-based chemical of no surprise that the majority of reported ABPs have been designed ligation method was introduced. Here, an N-terminally epitope- against these protease classes. ABPs for numerous cysteine and serine tagged ubiquitin derivative, with a C-terminal electrophile, was proteases are available but, so far, the proteasome is the only threo- prepared by combination of recombinant protein expression and nine protease to be profiled by ABPs. Probe designs for proteases synthetic chemistry [53]. This probe allowed activity-based profil- that do not form a covalent intermed­iate with its substrate have ing and proteomic identification of previously uncharacterized proven to be much more challenging. Probes that target metallo­ DUBs in complex biological samples [54]. In a more complex study, proteses were reported just a few years ago, but ABPs that target DUB activity was monitored in tumor cell lines and was found aspartic proteases still remain elusive. In this part of the review, to be increased as a direct consequence of cell transformation [55]. we will outline the development and application of activity-based ABPs have also been used for proteomic identification of DUBs proteomics for each of the various protease classes. in various pathogenic organisms, such as the herpes simplex virus, human cytomegalovirus, Chlamydia, Plasmodium falciparum and Cysteine proteases Escherichia coli [56–59]. The main catalytical feature of cysteine proteases is the use of a Clan CD is the second most abundant clan of cysteine proteases. Cys residue that is activated for nucleophilic attack by a nearby Its members include caspases, gingipains, legumains, clostripains His residue. Cysteine proteases are further divided into six clans and . Caspases are of particular interest due to their

724 Expert Rev. Proteomics 5(5), (2008) Activity-based probes as a tool for functional proteomic analysis of proteases Review crucial role in apoptosis, and it is not surprising that they were cell culture and after tumor formation in mouse mammary fat targets of the first generation of ABPs for the CD clan. Caspase pads. These studies showed that cancer cells exhibited distinct probes utilizing acyloxymethyl ketone (AOMK) and aldehyde activity profiles when grown in vitro (in culture) and in vivo reactive groups coupled to a specific peptide sequence and a biotin (xenograft tumors). More than seven types of enzyme activities tag have been designed. In fact, the first caspase (,β -IL- with unique activity profiles were identified. These findings -sug converting enzyme) was identified using a biotinylated AOMK gest that studies using human cancer cell lines grown in culture activity-based probe [60]. Recently, a small-molecule positional may not be predictive of the behavior of these same cells in vivo. scanning library was used for the development of highly specific It was also noted that many dramatic alterations in enzyme activ- AOMK-based probes for caspases 3, 7, 8 and 9. These probes ities occurred as a result of post-transcriptional events, again could then be used for monitoring caspase activation kinetics confirming the value of activity-based profiling methods. upon stimulation of apoptosis in cell-free extracts and intact This cancer profiling methodology was soon enhanced by cells [61] . ABPs were also designed for separases and legumain. the incorporation of non-gel methods for proteomic ana­lysis of Acyloxymethyl- and chloromethyl ketone-based probes were used labeled samples. A two-phase functional proteomic platform was to study the role of in [62]. Legumain is reported, where, in the first phase, serine hydrolases were labeled thought to play a role in lysosomal protein degradation, although by fluorogenic FP-probe and an activity profile was determined by its exact role still remains elusive. Legumain can participate in the 1D SDS-PAGE ana­lysis. This stage required a minimal amount processing of antigenic peptides and in the generation of double- of sample and could be applied to a wide range of primary human chain forms of the papain-like cathepsins [63,64]. A highly specific specimens. In the second phase, labeled targets were identified by set of AOMK-based probes for legumain was recently developed multidimensional liquid chromatography-MS/MS (MudPIT). that could provide key insights into its physiological role [65]. Thus, the sensitivity and resolution of MudPIT was coupled to the high-content functional information obtained by activity- Serine proteases based profiling. Using this approach, more than 50 enzyme acti­ Serine proteases are part of the larger serine family, vities were identified in breast tumor samples [69]. Comparison which represents approximately 1% of all in the human of activity-based proteomics data with cDNA microarray ana­ genome. Besides proteases, the serine hydrolase family also includes lysis showed that for some enzymes, activity and mRNA levels numerous lipases, esterases, amidases and transacylases. All of were largely uncorrelated. This finding additionally emphasizes them share the same catalytical mechanism, involving a serine the importance of activity-based proteomics that can detect low nucleophile activated by a proton relay associated with an acidic abundance, disease-associated enzymes that might evade other residue (aspartate or glutamate) and a basic residue (usually histi- molecular profiling methods. As an alternative to the FP probe, dine). The majority of serine hydrolases are irreversibly inhibited which shows broad reactivity with serine hydrolyases, the diphe- by the FP reactive group. Thus, probes containing PEG or ali- nylphosphonate warhead has been used to design a phatic linkers and various tags attached to this warhead have been specific probe. P1 basic amino acid probes were found to target developed as broad-specificity serine hydrolyase ABPs [66]. These only trypsin family serine proteases and proved valuable for the probes have been used in a range of profiling experiments, most profiling of serine protease activity in mast cells [70]. notably in studies of serine hydrolase biomarkers in cancer. Cravatt and coworkers used fluorescent FP-Rhodamine probes Metalloproteases to profile serine hydrolase activities in a series of cancer cell Perhaps the most critical difference between metalloproteases lines [67]. Samples were labeled, analyzed by SDS-PAGE and and the serine and cysteine classes of proteases is that they do the labeled protein profile was visualized by laser scanning. In not use an amino acid side-chain as a nucleophile for direct a parallel experiment, a biotinylated version of the probe was covalent attack on the substrate. Instead, they utilize a zinc used to directly isolate targets by affin- ity chromatography for identification by Table 1. Types of reactive groups used for targeting various MS. The identified serine hydrolase pro- protease classes. files were used to classify cancer cell lines into functional subtypes based on tissue Reactive group Targeted protease class Ref. of origin and state of invasiveness, thus Phosphonate Serine proteases [6,67–70] demonstrating the diagnostic power of the Sulfonate Serine proteases [68] probes for disease classification. A similar approach was also used to pro- Vinyl sulphone Cysteine proteases threonine [7,11,30,79] file hydrolases found in different stages of proteases (proteasome) breast cancer [68]. Serine hydrolase-specific Epoxide Cysteine proteases [10,41–47] probes (FP and sulfonate ester reactive Diazomethyl ketone Cysteine proteases [5] groups) were used to study the differ- Acyloxymethyl ketone Cysteine proteases [9,89] ences in activity profiles of MDA-MB-231 human breast cancer cells when grown in Hydroxamate Metalloproteases [13,72–77] www.expert-reviews.com 725 Review Fonović & Bogyo ion in their active site to deprotonate a water molecule, which proteins and fulfills several vital regulatory functions. The pro- then mediates hydrolysis of the [71] . Since metal- teasome has six proteolytic sites in the central core particle that loproteases do not form acyl-enzyme intermediates, they cannot possess three distinct substrate specificities: chymotrypsin-like, be labeled using simple electrophiles fused to a peptide that trypsin-like and peptidyl-glutamyl peptide-­hydro­lyzing [78]. The mimics a protein substrate. To overcome this problem, probes proteasome is a threonine protease since it uses the N-terminal containing a hydroxamate scaffold linked to a benzophenone threonine of the catalytic β-subunits as a nucleophile for the crosslinking group have been developed [72]. The hydroxam- attack on the peptide bond. Although initially designed to target ate is a strong zinc chelating agent that tightly binds in the cysteine proteases, C-terminal vinylsulfones have proven effective protease active site. While this is a high-affinity interaction, as irreversible inhibitors of the active site N-terminal threonine it is not irreversible, and the benzophenone photocrosslinking and are now widely used as warheads for the design of ABPs [30]. group is required for the attachment of the probe to the pro- The first probes developed for the characterization of catalytical tease active site. The main disadvantage of this type of ABP is proteasome β-subunits made use of the vinylsulfone warheads that they are not suitable for the use in living cells and whole coupled to a peptide that could be labeled with a radioisotope animals. Hydroxamate probes for metallo­proteases were suc- or biotin tag [79]. In addition, cell-permeable proteasome probes cessfully applied in the search for diagnostic markers in an have also been prepared by the incorporation of an azido linker invasive melanoma cell line [72]. In this study, a trifunctional (click chemistry) or a hapten tag [80,81] . Finally, cell-permeable hydroxamate probe (with a fluorescent and biotin tag) was used fluorescent probes were recently developed for visualization of for detection, affinity isolation and identification of neprilysin. labeled proteasomes in living cells and animals [82,83]. The natu- This metalloprotease was also found to be highly upregulated ral product, epoxomicin, was also found to be a highly selective, in melanoma cell lines. Neprilysin is a membrane-associated irreversible covalent inhibitor of the proteasome. This natural metalloprotease, which is considered to be a negative regulator product contains a reactive a,b-epoxyketone that forms a stable of tumorigenesis since it is known to degrade several mitogenic six-member ring with the N-terminal threonine. A labeled ver- peptides [73]. The results reported with the metalloprotease sion of this natural product was used as an ABP to identify the ABPs suggest that, in some tumor types, it may also promote target as the proteasome [84]. Thus, this class of compounds is cancer progression. likely to be used as a highly specific ABP of the proteasome. Activity-based probes usually only target a limited number of members within a specific enzyme class. This is problematic when Expert commentary & five-year view trying to globally profile large and structurally diverse protease The authors believe that the next 5 years will see a continued families, such as metalloproteases. To overcome this problem, growth in the development and application of ABPs for not only Cravatt and coworkers developed a profiling strategy using small proteases, but also for additional classes of important regulatory libraries of structurally diverse photoreactive hydroxamate probes. enzymes. The past 5 years has already seen a dramatic expan- By using libraries that were designed to have complementary sion in the diversity of enzyme families that can be studied using metalloprotease selectivity, it was possible to increase the overall activity-based proteomic methods. Creative work by chemists has coverage of the metalloproteases. The probes in the library were led to the development of probes for kinases, histone deacety- designed to be cell-permeable by incorporating the alkyne group lases, phosphatases and glycanases, to name just a few [85–88]. The so that a tag of choice (fluorophore or biotin) could be added development of ABPs that target diverse enzyme families has led after labeling using ‘click’ chemistry. Initial SDS-PAGE ana­lysis to a range of applications for activity-based proteomics. Probes enabled the selection of a ‘cocktail’ of optimal probes that pro- can be applied to a large collection of complex biological samples vided the greatest coverage of the family. This mixture of probes and enable global profiling of enzyme activities. Comparison of was used for more extensive ABP-MudPIT ana­lysis of cancer cell enzyme activity profiles from healthy and disease samples can lines [74]. With this approach, the authors were able to identify lead towards identification of novel biomarkers and drug tar- over 20 metalloproteases in various human and mouse samples. gets. An important advantage of ABPs is also in profiling of Among the identified metalloproteases were members of all the enzyme activities in vivo. Last year, Blum and coworkers devel- main branches of this enzyme family. Other examples of hydrox- oped protease probes that enable monitoring of enzyme activities amate probes for matrix metalloproteases using ‘click’ chemistry by whole-body imaging. Furthermore, those probes also allow have also been reported [75]. Metalloprotease probe libraries have subsequent ex vivo identification and biochemical characteriza- been created as well and used for the rapid determination of met- tion of labeled targets [89]. Those results show that ABPs have alloprotease inhibitor specificity fingerprints using a microarray a potential to become important imaging tools used for disease approach [76,77]. diagnosis and for preclinical and clinical testing of therapeutic agents in vivo. ABP-based in vivo assays significantly improve Threonine proteases high-throughput inhibitor screens needed for discovery of novel So far, the only threonine protease extensively studied using therapeutic agents. They enable screening of more than one target activity-based proteomics is the proteasome. This multisubunit in a single experiment, and because protein targets are screened complex is the central protease activity involved in ubiquitin- in their native environment, there is a better chance of detecting mediated protein degradation. It degrades old and damaged any unanticipated off-target inhibition or activation [90,91].

726 Expert Rev. Proteomics 5(5), (2008) Activity-based probes as a tool for functional proteomic analysis of proteases Review

We can expect that the number and types of applications for As the quality of instrumentation and the ability to analyze ABPs will continue to expand. This rapidly growing field is likely more complex, biologically relevant samples improve, we antici- to have a significant impact on a number of research disciplines, pate that ABPs will help to siphon off the most relevant and proteomics being perhaps the most directly affected. important information from these large data sets. This will Besides profiling of protease activities and target identification, require a widespread integration of the probes and continued chemical labeling has also become a powerful tool for proteomic efforts from chemists to develop probes with enhanced func- identification of protease substrates. In this approach, complex tionality. We are confident that the early developments in this proteomes are directly treated with specific proteases and newly field have created the necessary positive momentum to see these formed N-terminal ends of cleaved substrates are chemically modi- goals realized. fied by biotinylated reagents. After affinity purification, substrates and their exact cleavage sites are identified by MS [92]. Cleavage Financial & competing interests disclosure sites can also be quantitatively evaluated by the incorporation of The authors have no relevant affiliations or financial involvement with any quantitative proteomic techniques, such as isobaric tag for relative organization or entity with a financial interest in or financial conflict with and absolute quantitation (iTRAQ) [93,94]. Determination of the the subject matter or materials discussed in the manuscript. This includes whole complement of cellular protease substrates (degradome) employment, consultancies, honoraria, stock ownership or options, expert remains one of the main challenges that currently face the field testimony, grants or patents received or pending, or royalties. of proteolysis. No writing assistance was utilized in the production of this manuscript.

Key issues • The main advantage of activity-based proteomics is that it detects enzyme activity instead of abundance and, therefore, provides more accurate information on the biological roles of these enzymes in a given biological process. • Activity-based proteomics makes use of small-molecule probes that are designed to target specific enzyme classes. • Activity-based probes are composed of a functional group (warhead), linker and tag that enables specific labeling and enrichment of target proteins. • Activity-based probes for all the main protease classes of proteases (serine, cysteine, threonine and metalloproteases) have been successfully developed and are in use for global functional studies of proteases.

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