{{Short description|Analytical technique to study proteins}} [[File:Fp rhodamine.svg|thumb|200px|right|Fluorophosphonate-rhodamine (FP-Rhodamine) activity-based probe for profiling of the serine hydrolase superfamily. In this probe the fluorophosphonate is the reactive group (RG) as it binds irreversibly to the active-site serine nucleophile of serine hydrolases and the tag is rhodamine, a fluorophore for in-gel visualization.]] '''Activity-based proteomics''', or '''activity-based protein profiling''' ('''ABPP''') is a chemoproteomic strategy that employs modular probes to directly assess the functional state of enzymes within complex proteomes.<ref name="pmid15651898">{{cite journal | vauthors = Berger AB, Vitorino PM, Bogyo M | title = Activity-based protein profiling: applications to biomarker discovery, in vivo imaging and drug discovery | journal = American Journal of Pharmacogenomics | volume = 4 | issue = 6 | pages = 371–81 | date = 2004 | pmid = 15651898 | doi = 10.2165/00129785-200404060-00004 | s2cid = 18637390 }}</ref> Unlike expression-based proteomics, which measures protein abundance, ABPP provides information about catalytic activity, thereby enabling researchers to distinguish between active and inactive forms of enzymes. This functional perspective has proven valuable for identifying drug targets, mapping ligandable sites, and understanding enzyme regulation in physiological and pathological contexts.<ref name=":0">{{Cite journal|title=Activity-based protein profiling identifies alternating activation of enzymes involved in the bifidobacterium shunt pathway or mucin degradation in the gut microbiome response to soluble dietary fiber|journal=npj Biofilms and Microbiomes|date=2022-07-20|issn=2055-5008|pmc=9300575|pmid=35858888|page=60|volume=8|issue=1|doi=10.1038/s41522-022-00313-z|language=en|first1=Bryan J.|last1=Killinger|first2=Christopher|last2=Whidbey|first3=Natalie C.|last3=Sadler|first4=Adrian J.|last4=DeLeon|first5=Nathalie|last5=Munoz|first6=Young-Mo|last6=Kim|first7=Aaron T.|last7=Wright}}</ref><ref>{{Cite journal|title=Targeting the Undruggable Proteome: The Small Molecules of My Dreams|journal=Chemistry & Biology|date=2010-06-25|issn=1074-5521|pmc=2925121|pmid=20609404|pages=551–555|volume=17|issue=6|doi=10.1016/j.chembiol.2010.05.011|first=Craig M.|last=Crews}}</ref><ref>{{Cite journal|title=Advanced Activity-Based Protein Profiling Application Strategies for Drug Development|journal=Frontiers in Pharmacology|date=2018-04-09|issn=1663-9812|pmc=5900428|pmid=29686618|volume=9|doi=10.3389/fphar.2018.00353|language=en|first1=Shan|last1=Wang|first2=Yu|last2=Tian|first3=Min|last3=Wang|first4=Gui-bo|last4=Sun|first5=Xiao-bo|last5=Sun |article-number=353 |doi-access=free }}</ref><ref>{{Cite journal|title=Activity-based Protein Profiling Approaches for Transplantation|url=https://journals.lww.com/10.1097/TP.0000000000002752|journal=Transplantation|date=September 2019|issn=0041-1337|pages=1790–1798|volume=103|issue=9|doi=10.1097/TP.0000000000002752|language=en|first1=Mario|last1=Navarrete|first2=John A.|last2=Wilkins|first3=Ying|last3=Lao|first4=David N.|last4=Rush|first5=Peter W.|last5=Nickerson|first6=Julie|last6=Ho |pmid=30985576 |hdl=1993/35051 |hdl-access=free|url-access=subscription}}</ref> By covalently modifying active sites, '''activity-based probes''' ('''ABPs''') allow selective tagging, enrichment, and isolation of proteins, reducing the complexity of proteomic samples and facilitating downstream analysis.<ref name=":1">{{Cite journal|title=Activity-Based Protein Profiling: From Enzyme Chemistry to Proteomic Chemistry|url=https://www.annualreviews.org/content/journals/10.1146/annurev.biochem.75.101304.124125|journal=Annual Review of Biochemistry|date=2008-07-07|issn=0066-4154|pages=383–414|volume=77|doi=10.1146/annurev.biochem.75.101304.124125|language=en|first1=Benjamin F.|last1=Cravatt|first2=Aaron T.|last2=Wright|first3=John W.|last3=Kozarich |issue=1 |pmid=18366325 |bibcode=2008ARBio..77..383C |url-access=subscription}}</ref> ABPP has been applied across diverse biological systems, including cells, tissues, and whole organisms, and has contributed to advances in biomarker discovery, drug development, and in vivo imaging.<ref name=":0" /><ref>{{Cite journal|title=Profiling Cysteine Proteases Activities in Neuroinflammatory Cells|journal=ChemMedChem|date=2025|issn=1860-7187|pmc=11793851|pmid=39475209|article-number=e202400520|volume=20|issue=3|doi=10.1002/cmdc.202400520|language=en|first1=Laura|last1=Agost-Beltrán|first2=Ania|last2=Canseco-Rodríguez|first3=Tanja|last3=Schirmeister|first4=Santiago|last4=Rodríguez|first5=Ana María|last5=Sánchez-Pérez|first6=Florenci V.|last6=González}}</ref><ref>{{Cite journal|title=Chemoproteomics profiling of surfactin-producing nonribosomal peptide synthetases in living bacterial cells|url=https://www.cell.com/cell-chemical-biology/abstract/S2451-9456(21)00259-2|journal=Cell Chemical Biology|date=2022-01-20|issn=2451-9456|pmid=34133952|pages=145–156.e8|volume=29|issue=1|doi=10.1016/j.chembiol.2021.05.014|language=English|first1=Fumihiro|last1=Ishikawa|first2=Sho|last2=Konno|first3=Chiharu|last3=Uchida|first4=Takehiro|last4=Suzuki|first5=Katsuki|last5=Takashima|first6=Naoshi|last6=Dohmae|first7=Hideaki|last7=Kakeya|first8=Genzoh|last8=Tanabe|url-access=subscription}}</ref>
== Historical development == Early experiments resembling activity-based profiling were conducted in the 1970s, when small molecules were used to study the mechanism of action of the serine-modifying antibiotic penicillin.<ref>{{Cite journal|title=Interaction of penicillin with the bacterial cell: penicillin-binding proteins and penicillin-sensitive enzymes|journal=Bacteriological Reviews|date=September 1974|pmc=413858|pmid=4608953|pages=291–335|volume=38|issue=3|doi=10.1128/br.38.3.291-335.1974|first1=P M|last1=Blumberg|first2=J L|last2=Strominger}}</ref> The modern era of ABPP began in the 1990s with the development of ABPs compatible with proteomic workflows, and the first applications of ABPP were reported during this decade in studies of proteases.<ref name="pmid8305526">{{cite journal |vauthors=Kam CM, Abuelyaman AS, Li Z, Hudig D, Powers JC|title=Biotinylated isocoumarins, new inhibitors and reagents for detection, localization, and isolation of serine proteases|journal=Bioconjugate Chemistry|volume=4|issue=6|pages=560–7|date=1993|pmid=8305526|doi=10.1021/bc00024a021}}</ref><ref name="pmid7849068">{{cite journal |vauthors=Abuelyaman AS, Hudig D, Woodard SL, Powers JC|title=Fluorescent derivatives of diphenyl [1-(N-peptidylamino)alkyl]phosphonate esters: synthesis and use in the inhibition and cellular localization of serine proteases|journal=Bioconjugate Chemistry|volume=5|issue=5|pages=400–5|date=1994|pmid=7849068|doi=10.1021/bc00029a004}}</ref> In 1999, Liu and colleagues formally introduced the term "activity-based protein profiling," establishing a framework for systematic functional proteomics.<ref name="pmid10611275">{{cite journal |vauthors=Liu Y, Patricelli MP, Cravatt BF|title=Activity-based protein profiling: the serine hydrolases|journal=Proceedings of the National Academy of Sciences of the United States of America|volume=96|issue=26|pages=14694–9|date=December 1999|pmid=10611275|pmc=24710|doi=10.1073/pnas.96.26.14694|bibcode=1999PNAS...9614694L|doi-access=free}}</ref> Subsequent work by Ben Cravatt at The Scripps Research Institute, [https://profiles.stanford.edu/matthew-bogyo Matthew Bogyo] at Stanford University, and [https://www.universiteitleiden.nl/en/staffmembers/hermen-overkleeft#tab-1 Herman S. Overkleeft] at Leiden University helped define the field through the design of probes targeting serine hydrolases,<ref name="pmid10611275" /> cysteine proteases,<ref name=":2">{{Cite journal|title=Chemical Approaches for Functionally Probing the Proteome *|url=https://www.mcponline.org/article/S1535-9476(20)34674-0/abstract|journal=Molecular & Cellular Proteomics|date=2002-01-01|issn=1535-9476|pmid=12096141|pages=60–68|volume=1|issue=1|doi=10.1074/mcp.T100003-MCP200|language=English|first1=Doron|last1=Greenbaum|first2=Amos|last2=Baruch|first3=Linda|last3=Hayrapetian|first4=Zsuzsanna|last4=Darula|first5=Alma|last5=Burlingame|first6=Katlin F.|last6=Medzihradszky|first7=Matthew|last7=Bogyo |doi-access=free }}</ref> oxidoreductases,<ref>{{Cite journal|title=Activity-Based Protein Profiling (ABPP) of Oxidoreductases|journal=ChemBioChem|date=2021|issn=1439-7633|pmc=7894341|pmid=32881211|pages=630–638|volume=22|issue=4|doi=10.1002/cbic.202000542|language=en|first1=Rita|last1=Fuerst|first2=Rolf|last2=Breinbauer}}</ref> human cytochrome P450s<ref>{{Cite journal|title=A Suite of Activity-Based Probes for Human Cytochrome P450 Enzymes|journal=Journal of the American Chemical Society|date=2009-08-05|issn=0002-7863|pmc=2737065|pmid=19583257|pages=10692–10700|volume=131|issue=30|doi=10.1021/ja9037609|first1=Aaron T.|last1=Wright|first2=Joongyu D.|last2=Song|first3=Benjamin F.|last3=Cravatt |bibcode=2009JAChS.13110692W }}</ref> and other enzyme families. Since its inception, ABPP has expanded rapidly, with bibliometric analyses documenting exponential growth in publications and widespread adoption across North America, Europe, and Asia.<ref>{{Cite journal|title=Mapping the Evolution of Activity-Based Protein Profiling: A Bibliometric Review|url=https://apb.tbzmed.ac.ir/Article/apb-39683|journal=Advanced Pharmaceutical Bulletin|date=2023-05-20|issn=2228-5881|pmc=10676541|pmid=38022804|pages=639–645|volume=13|issue=4|doi=10.34172/apb.2023.082|language=en|first=Exequiel Oscar Jesus|last=Porta}}</ref> Advances in mass spectrometry and protein separation technologies further accelerated the integration of ABPP into proteomic research, enabling the characterization of enzyme activity on a global scale and establishing ABPP as a cornerstone of functional proteomics.
==Probe design== The basic unit of ABPP is the activity-based probe (ABP), which are small-molecule probes engineered to profile enzymatic activities of a range of related enzymes within a complex proteome. Although specific architectures vary, most ABPs share three modular components: (1) a '''reactive group''' (RG, sometimes called a "warhead"), (2) a '''linker''' or '''binding element''', and (3) a '''tag''' used for detection or enrichment. This modularity allows probes to achieve broad yet mechanistically meaningful coverage of an enzyme class while minimizing off-target reactivity.<ref name=":1" />
=== Reactive Group === The reactive group, often referred to as the "warhead," covalently binds to conserved residues in enzyme active sites. It is the core determinant of enzyme selectivity. It mediates irreversible or photo-induced covalent attachment to residues located in the active site. Broadly, these fall into two functional classes: '''Electrophilic warheads''' and '''Photoreactive warheads.''' Electrophilic warheads react with conserved nucleophiles, for example, fluorophosphonates are widely used to target serine hydrolases,<ref name="pmid10611275" /> while epoxides and vinyl sulfones have been applied to cysteine proteases<ref name=":2" /> Photoreactive warheads are used when an enzyme class lacks a catalytic nucleophile, as in the case of metalloproteases<ref name="pmid15220480">{{cite journal |vauthors=Saghatelian A, Jessani N, Joseph A, Humphrey M, Cravatt BF|title=Activity-based probes for the proteomic profiling of metalloproteases|journal=Proceedings of the National Academy of Sciences of the United States of America|volume=101|issue=27|pages=10000–5|date=July 2004|pmid=15220480|pmc=454150|doi=10.1073/pnas.0402784101|bibcode=2004PNAS..10110000S|doi-access=free}}</ref> or histone deacetylases.<ref>{{Cite journal|title=Activity-based probes for proteomic profiling of histone deacetylase complexes|journal=Proceedings of the National Academy of Sciences|date=2007-01-23|pmc=1783107|pmid=17227860|pages=1171–1176|volume=104|issue=4|doi=10.1073/pnas.0608659104|first1=Cleo M.|last1=Salisbury|first2=Benjamin F.|last2=Cravatt |bibcode=2007PNAS..104.1171S |doi-access=free }}</ref> These probes incorporate benzophenone<ref>{{Cite journal|title=Optimization of Activity-Based Probes for Proteomic Profiling of Histone Deacetylase Complexes|journal=Journal of the American Chemical Society|date=2008-02-01|issn=0002-7863|pages=2184–2194|volume=130|issue=7|doi=10.1021/ja074138u|first1=Cleo M.|last1=Salisbury|first2=Benjamin F.|last2=Cravatt |pmid=18217751 |bibcode=2008JAChS.130.2184S }}</ref> or diazirine<ref name=ewsc>{{Cite journal|title=Developing Photoactive Affinity Probes for Proteomic Profiling: Hydroxamate-based Probes for Metalloproteases|journal=Journal of the American Chemical Society|date=2004-11-01|issn=0002-7863|pages=14435–14446|volume=126|issue=44|doi=10.1021/ja047044i|first1=Elaine W. S.|last1=Chan|first2=Souvik|last2=Chattopadhaya|first3=Resmi C.|last3=Panicker|first4=Xuan|last4=Huang|first5=Shao Q.|last5=Yao |pmid=15521763 |bibcode=2004JAChS.12614435C |url=http://scholarbank.nus.edu.sg/handle/10635/93568 }}</ref> moieties that, upon UV irradiation, produce radical intermediates that form covalent crosslinks with nearby residues in the enzyme active site.
With classes of enzymes such as the serine hydrolases<ref name="pmid10611275" /> and metalloproteases<ref name="pmid15220480" /> that often interact with endogenous inhibitors or that exist as inactive zymogens, ABPP offers a valuable advantage over traditional techniques that rely on abundance rather than activity. Because enzymatic activity is required for productive engagement with the reactive group, inactive precursors and inhibitor-bound enzyme forms typically remain unlabeled.<ref>{{Cite journal|title=Enzyme activity profiles of the secreted and membrane proteome that depict cancer cell invasiveness|journal=Proceedings of the National Academy of Sciences|date=2002-08-06|pmc=124915|pmid=12149457|pages=10335–10340|volume=99|issue=16|doi=10.1073/pnas.162187599|first1=Nadim|last1=Jessani|first2=Yongsheng|last2=Liu|first3=Mark|last3=Humphrey|first4=Benjamin F.|last4=Cravatt |bibcode=2002PNAS...9910335J |doi-access=free }}</ref> However, because photocrosslinking does not require catalytic turnover, inactive proteases can still be labeled, reducing the intrinsic activity-dependence of photoreactive probes. This limitation has motivated the incorporation of selectivity-enhancing functional groups directly into the probe design. In the case of metalloproteases, active enzymes coordinate a catalytic metal ion. By adding a metal-chelating moiety to the probe scaffold, photoreactive ABPs can be biased toward the catalytically competent, metal-bound form of the enzyme, thereby improving discrimination between active and inactive form.<ref name="pmid15220480" /><ref name=ewsc/> Another major design challenge is achieving sufficient reactivity to label the intended enzyme without indiscriminate modification of unrelated proteins. Strategies such as masked warheads, which become activated only in the presence of a target enzyme, have been developed to improve specificity.<ref>{{Cite journal|title=Diacylfuroxans Are Masked Nitrile Oxides That Inhibit GPX4 Covalently|journal=Journal of the American Chemical Society|date=2019-12-26|issn=0002-7863|pages=20407–20415|volume=141|issue=51|doi=10.1021/jacs.9b10769|first1=John K.|last1=Eaton|first2=Richard A.|last2=Ruberto|first3=Anneke|last3=Kramm|first4=Vasanthi S.|last4=Viswanathan|first5=Stuart L.|last5=Schreiber |pmid=31841309 |bibcode=2019JAChS.14120407E |url=https://figshare.com/articles/Diacylfuroxans_Are_Masked_Nitrile_Oxides_That_Inhibit_GPX4_Covalently/11371935 }}</ref>
=== Linker === Between the reactive group and the tag, ABPs often include a '''linker''' / '''spacer''' / '''biorecognition element''', which can tune probe solubility, steric accessibility, and substrate mimicry. Simple linkers may consist of alkyl chains or polyethylene glycol (PEG) spacers which adjust hydrophobicity and improve labeling across diverse proteomes. More complex designs incorporate biorecognition elements which can impart enzyme family or subfamily selectivity. For example, substrate-mimetic phosphonates have been used to target specific serine proteases<ref>{{Cite journal|title=Selective Chemical Functional Probes of Granzymes A and B Reveal Granzyme B Is a Major Effector of Natural Killer Cell-Mediated Lysis of Target Cells|url=https://www.cell.com/cell-chemical-biology/abstract/S1074-5521(05)00102-X|journal=Chemistry & Biology|date=2005-05-01|issn=1074-5521|pmid=15911377|pages=567–577|volume=12|issue=5|doi=10.1016/j.chembiol.2005.03.006|language=English|first1=Sami|last1=Mahrus|first2=Charles S.|last2=Craik}}</ref> and optimized peptide sequences can discriminate caspase isoforms.<ref>{{Cite journal|title=Identification of Early Intermediates of Caspase Activation Using Selective Inhibitors and Activity-Based Probes|url=https://www.cell.com/molecular-cell/abstract/S1097-2765(06)00435-7|journal=Molecular Cell|date=2006-08-18|issn=1097-2765|pmid=16916639|pages=509–521|volume=23|issue=4|doi=10.1016/j.molcel.2006.06.021|language=English|first1=Alicia B.|last1=Berger|first2=Martin D.|last2=Witte|first3=Jean-Bernard|last3=Denault|first4=Amir Masoud|last4=Sadaghiani|first5=Kelly M. B.|last5=Sexton|first6=Guy S.|last6=Salvesen|first7=Matthew|last7=Bogyo|hdl=11370/1556432a-189a-4c2e-860e-02c9af08c3ac|hdl-access=free}}</ref> Binding or targeting motifs within the linker can further enhance interactions with enzymes whose active sites impose structural constraints, thereby improving probe specificity.<ref>{{Cite journal|title=Activity-Based Profiling of Proteases|url=https://www.annualreviews.org/content/journals/10.1146/annurev-biochem-060713-035352|journal=Annual Review of Biochemistry|date=2014-06-02|issn=0066-4154|pages=249–273|volume=83|doi=10.1146/annurev-biochem-060713-035352|language=en|first1=Laura E.|last1=Sanman|first2=Matthew|last2=Bogyo |pmid=24905783 }}</ref> In addition, linker length and composition can modulate probe permeability and distribution in cellular or in vivo contexts. This component allows probe designers to balance breadth (profiling an entire enzyme class) versus specificity (targeting individual members).
=== Tag === The tag may be either a direct reporter, such as a fluorophore, or an affinity label such as biotin, or it may consist of a latent handle like an alkyne or azide for use with Huisgen 1,3-dipolar cycloaddition (also known as click chemistry).<ref name="pmid12696868">{{cite journal |vauthors=Speers AE, Adam GC, Cravatt BF|title=Activity-based protein profiling in vivo using a copper(i)-catalyzed azide-alkyne [3 + 2] cycloaddition|journal=Journal of the American Chemical Society|volume=125|issue=16|pages=4686–7|date=April 2003|pmid=12696868|doi=10.1021/ja034490h |bibcode=2003JAChS.125.4686S }}</ref> Reporter tags facilitate detection and isolation of labeled proteins. Common examples include fluorophores used for visualization via in-gel fluorescence and high-throughput gel-based screens, biotin for streptavidin-based enrichment followed by mass spectrometry, and isotopic labels<ref name=":3">{{Cite journal|title=Quantitative reactivity profiling predicts functional cysteines in proteomes|journal=Nature|date=December 2010|issn=1476-4687|pmc=3058684|pmid=21085121|pages=790–795|volume=468|issue=7325|doi=10.1038/nature09472|language=en|first1=Eranthie|last1=Weerapana|first2=Chu|last2=Wang|first3=Gabriel M.|last3=Simon|first4=Florian|last4=Richter|first5=Sagar|last5=Khare|first6=Myles B. D.|last6=Dillon|first7=Daniel A.|last7=Bachovchin|first8=Kerri|last8=Mowen|first9=David|last9=Baker|first10=Benjamin F.|last10=Cravatt |bibcode=2010Natur.468..790W }}</ref><ref name=":4">{{Cite journal|title=Isotopically Labeled Desthiobiotin Azide (isoDTB) Tags Enable Global Profiling of the Bacterial Cysteinome|journal=Angewandte Chemie International Edition|date=2020|issn=1521-3773|pmc=7027453|pmid=31782878|pages=2829–2836|volume=59|issue=7|doi=10.1002/anie.201912075|language=en|first1=Patrick R. A.|last1=Zanon|first2=Lisa|last2=Lewald|first3=Stephan M.|last3=Hacker |bibcode=2020ACIE...59.2829Z }}</ref> for quantitative mass spectrometry. Alternatively, alkynes or azides can be incorporated as bio-orthogonal handles for post-labeling conjugation via click chemistry, enabling modular addition of fluorophores, affinity tags, or isotopic labels<ref name=":3" /><ref name=":4" /> after proteome labeling.<ref name="pmid12696868" /><ref>{{Cite journal|title=Profiling Enzyme Activities In Vivo Using Click Chemistry Methods|url=https://www.cell.com/cell-chemical-biology/abstract/S1074-5521(04)00096-1|journal=Chemistry & Biology|date=2004-04-01|issn=1074-5521|pmid=15123248|pages=535–546|volume=11|issue=4|doi=10.1016/j.chembiol.2004.03.012|language=English|first1=Anna E.|last1=Speers|first2=Benjamin F.|last2=Cravatt}}</ref> These "clickable" designs minimize steric hindrance at the active site and expand analytical flexibility, particularly for high-resolution liquid chromatography-mass spectrometry methods.
==Detection methods== [[Image:Gel-abpp eg.png|frame|right|In-gel ABPP using probes with different fluorophores in the same lane to simultaneously profile differences in enzyme activities]] ABPP can be analyzed using several complementary detection strategies, each suited to different experimental contexts. These methods generally visualize or enrich the enzyme-probe adduct, enabling qualitative assessment, quantitative comparison, or protein identification by mass spectrometry.
One of the earliest and most widely used ABPP workflows employs '''direct visualization on SDS-PAGE gels'''.<ref name="pmid10611275" /> Probes with fluorescent reporter tags (e.g. rhodamine) generate distinct bands corresponding to labeled enzymes, allowing rapid assessment of activity across samples or treatment conditions. This approach is commonly used for broad enzyme families such as serine hydrolases<ref name="pmid10611275" /> or cysteine proteases<ref name=":2" /> and is compatible with high-throughput screening for inhibitors. Because the readout is based on in-gel fluorescence rather than protein abundance, gel-based ABPP readily distinguishes active from inactive enzyme species.
However, a significant limitation of gel-based detection is lack of resolving ability, preventing the resolution and identification of low-abundance proteins. In recent years ABPP has been combined with tandem mass spectrometry enabling the identification of hundreds of active enzymes from a single sample. This technique, known as '''''ABPP-MudPIT''''' (multidimensional protein identification technology) is especially useful for profiling inhibitor selectivity as the potency of an inhibitor can be tested against hundreds of targets simultaneously.<ref>{{Cite book|title=Activity-Based Protein Profiling (ABPP) and Click Chemistry (CC)–ABPP by MudPIT Mass Spectrometry|journal=Current Protocols in Chemical Biology|date=2009|issn=2160-4762|pmc=3119539|pmid=21701697|pages=29–41|volume=1|issue=1|doi=10.1002/9780470559277.ch090138|language=en|first1=Anna E.|last1=Speers|first2=Benjamin F.|last2=Cravatt}}</ref> For proteome-wide analysis, ABPs incorporating affinity tags (e.g. biotin) or clickable handles (azides or alkynes) enable enrichment of labeled enzymes prior to identification by liquid chromatography-tandem mass spectrometry (LC-MS/MS). After covalent labeling in cells or lysates, enzyme-probe adducts are captured using streptavidin or other affinity matrices, washed to remove unlabeled proteins, and digested for MS-based peptide identification.<ref>{{Cite journal|title=Activity-based protein profiling reveals off-target proteins of the FAAH inhibitor BIA 10-2474|journal=Science|date=2017-06-09|pmc=5641481|pmid=28596366|pages=1084–1087|volume=356|issue=6342|doi=10.1126/science.aaf7497|first1=Annelot C. M.|last1=van Esbroeck|first2=Antonius P. A.|last2=Janssen|first3=Armand B.|last3=Cognetta|first4=Daisuke|last4=Ogasawara|first5=Guy|last5=Shpak|first6=Mark|last6=van der Kroeg|first7=Vasudev|last7=Kantae|first8=Marc P.|last8=Baggelaar|first9=Femke M. S.|last9=de Vrij|first10=Hui|last10=Deng|first11=Marco|last11=Allarà|first12=Filomena|last12=Fezza|first13=Zhanmin|last13=Lin|first14=Tom|last14=van der Wel|first15=Marjolein|last15=Soethoudt |bibcode=2017Sci...356.1084V }}</ref> This strategy provides high sensitivity and depth of coverage, which facilitates the discovery of novel enzyme targets, detection of low-abundance enzymes and analysis of complex enzyme families.
To compare enzyme activity across biological conditions, ABPP can be integrated with '''quantitative proteomic technologies''' to measure relative enzyme activity across samples.<ref>{{Cite journal|title=Target identification with quantitative activity based protein profiling (ABPP)|url=https://onlinelibrary.wiley.com/doi/abs/10.1002/pmic.201600212|journal=Proteomics|date=2017|issn=1615-9861|article-number=1600212|volume=17|issue=3–4|doi=10.1002/pmic.201600212|language=en|first1=Xiao|last1=Chen|first2=Yin Kwan|last2=Wong|first3=Jigang|last3=Wang|first4=Jianbin|last4=Zhang|first5=Yew-Mun|last5=Lee|first6=Han-Ming|last6=Shen|first7=Qingsong|last7=Lin|first8=Zi-Chun|last8=Hua |pmid=27723264 |url-access=subscription}}</ref> Techniques such as '''SILAC''' ('''Stable Isotope Labeling by Amino acids in Cell culture''') incorporate heavy or light isotope-encoded amino acids into cellular proteins, facilitating direct MS-based comparison of probe-labeled peptides from different samples.<ref>{{Cite journal|title=A road map to evaluate the proteome-wide selectivity of covalent kinase inhibitors|journal=Nature Chemical Biology|date=September 2014|issn=1552-4469|pmc=4138289|pmid=25038787|pages=760–767|volume=10|issue=9|doi=10.1038/nchembio.1582|language=en|first1=Bryan R.|last1=Lanning|first2=Landon R.|last2=Whitby|first3=Melissa M.|last3=Dix|first4=John|last4=Douhan|first5=Adam M.|last5=Gilbert|first6=Erik C.|last6=Hett|first7=Theodore O.|last7=Johnson|first8=Chris|last8=Joslyn|first9=John C.|last9=Kath|first10=Sherry|last10=Niessen|first11=Lee R.|last11=Roberts|first12=Mark E.|last12=Schnute|first13=Chu|last13=Wang|first14=Jonathan J.|last14=Hulce|first15=Baoxian|last15=Wei}}</ref> '''TMT''' ('''Tandem mass tagging''') which uses isobaric mass tags allows labeled peptides from multiple samples to be multiplexed and quantified simultaneously.<ref>{{Cite journal|title=Functionalizing tandem mass tags for streamlining click-based quantitative chemoproteomics|journal=Communications Chemistry|date=2024-04-10|issn=2399-3669|pmc=11006884|pmid=38600184|page=80|volume=7|issue=1|doi=10.1038/s42004-024-01162-x|language=en|first1=Nikolas R.|last1=Burton|first2=Keriann M.|last2=Backus |bibcode=2024CmChe...7...80B }}</ref> These strategies have been widely applied to assess selectivity of covalent inhibitors and genome-wide changes in enzymatic activity. isoTOP-ABPP (Isotope Tagging of Optimized Probes for Activity-Based Protein Profiling)<ref name=":3" /> is a specialized workflow that was developed to address some of the limitations of conventional ABPP, including poor specificity, limited sensitivity, and the absence of selective chemical ligands for many human enzymes. This method uses stable isotopes into probe-derived tags to improve the sensitivity and selectivity of ABPP, while also enabling accurate quantification of protein interactions in complex biological samples.
In addition, fluorescent and photoaffinity probes can be applied in situ or in vivo to visualize enzyme activity within cells, tissues, or whole organisms. These approaches provide spatial information about enzyme function and have been used to study processes such as protease activity in cancer and infection models. Emerging designs aim to enable real-time, non-invasive imaging of enzyme activity in living systems.<ref>{{Cite journal|title=Noninvasive optical imaging of cysteine protease activity using fluorescently quenched activity-based probes|url=https://www.nature.com/articles/nchembio.2007.26|journal=Nature Chemical Biology|date=October 2007|issn=1552-4469|pages=668–677|volume=3|issue=10|doi=10.1038/nchembio.2007.26|language=en|first1=Galia|last1=Blum|first2=Georges|last2=von Degenfeld|first3=Milton J.|last3=Merchant|first4=Helen M.|last4=Blau|first5=Matthew|last5=Bogyo |pmid=17828252 |url-access=subscription}}</ref>
== Advantages and limitations == A major advantage of ABPP is the ability to monitor the availability of the enzyme active site directly, rather than being limited to protein or mRNA abundance. Ideal ABPs would target a large, but manageable, number of enzymes (tens to hundreds) to provide researchers with a global view of the functional state of the proteome.<ref name=":1" /> This degree of target promiscuity must be balanced by minimal cross-reactivity with unrelated proteins. Most ABPs achieve this combination of intraclass coverage and limited extraclass reactivity by coupling appropriate reactive groups with binding elements that recognize conserved mechanistic or structural features within enzyme active sites. Furthermore, ABPP could be used to target specific proteins which were previously viewed as undruggable targets.<ref>{{cite journal |doi=10.1016/j.ejmech.2020.112151|title=Activity-based protein profiling: Recent advances in medicinal chemistry|date=2020|last1=Deng|first1=Hui|last2=Lei|first2=Qian|last3=Wu|first3=Yangping|last4=He|first4=Yang|last5=Li|first5=Weimin|journal=European Journal of Medicinal Chemistry|volume=191|article-number=112151 |pmid=32109778}}</ref>
A disadvantage of ABPs is that their design is somewhat restricted by the need to attach an electrophile or photocrosslinker, which can limit chemical diversity. Another potential drawback is that covalent modification of the active site irreversibly inhibits the target enzyme. However, in many cases it is possible to use concentrations of ABP such that only a small fraction of the active enzyme pool is labeled, minimizing perturbation of overall enzymatic activity.<ref>{{Cite journal|title=Functional imaging of proteases: recent advances in the design and application of substrate-based and activity-based probes|journal=Current Opinion in Chemical Biology|date=2011-12-01|issn=1367-5931|pmc=3237724|pmid=22098719|pages=798–805|volume=15|series=Molecular Imaging|issue=6|doi=10.1016/j.cbpa.2011.10.012|first1=Laura E|last1=Edgington|first2=Martijn|last2=Verdoes|first3=Matthew|last3=Bogyo}}</ref><ref>{{Cite journal|title=Caspase-1 activity is required to bypass macrophage apoptosis upon Salmonella infection|journal=Nature Chemical Biology|date=September 2012|issn=1552-4469|pmc=3461347|pmid=22797665|pages=745–747|volume=8|issue=9|doi=10.1038/nchembio.1023|language=en|first1=Aaron W.|last1=Puri|first2=Petr|last2=Broz|first3=Aimee|last3=Shen|first4=Denise M.|last4=Monack|first5=Matthew|last5=Bogyo}}</ref>
==See also== * Mass spectrometry * Proteomics * Related inhibitors MAFP and DIFP * Chemoproteomics * Thermal proteome profiling
==References== {{Reflist|30em}}
{{genomics-footer}}
{{DEFAULTSORT:Activity-Based Proteomics}} Category:Proteomics Category:Genomics Category:Protein methods Category:Mass spectrometry