Activity assays are indispensable for studying biochemical properties of enzymes. The purposes of measuring activity are wide ranging from a simple detection of the presence of an enzyme to kinetic experiments evaluating the substrate specificity, reaction mechanisms, and susceptibility to inhibitors. Common activity assay methods include spectroscopy, electrochemical sensors, or liquid chromatography coupled with various detection techniques. This review focuses on the use of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) as a growing and modern alternative, which offers high speed of analysis, sensitivity, versatility, possibility of automation, and cost-effectiveness. It may reveal reaction intermediates, side products or measure more enzymes at once. The addition of an internal standard or calculating the ratios of the substrate and product peak intensities and areas overcome the inherent inhomogeneous distribution of analyte and matrix in the sample spot, which otherwise results in a poor reproducibility. Examples of the application of MALDI-TOF MS for assaying hydrolases (including peptidases and β-lactamases for antibiotic resistance tests) and other enzymes are provided. Concluding remarks summarize advantages and challenges coming from the present experience, and draw future perspectives such as a screening of large libraries of chemical compounds for their substrate or inhibitory properties towards enzymes.

Department of Biochemistry Faculty of Science and CATRIN Palacký University Olomouc Czech Republic

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Abraham EP, Chain E. An enzyme from bacteria able to destroy penicillin. Nature 1940;146:837. https://doi.org/10.1038/146837a0

Aebersold R, Mann M. Mass-spectrometric exploration of proteome structure and function. Nature 2016;537:347-355. https://doi.org/10.1038/nature19949

Armstrong LA, Lange SM, Dee Cesare V, Matthews SP, Nirujogi RS, Cole I, Hope A, Cunningham F, Toth R, Mukherjee R, Bojkova D, Gruber F, Gray D, Wyatt PG, Cinatl J, Dikic I, Davies P, Kulathu Y. Biochemical characterization of protease activity of Nsp3 from SARS-CoV-2 and its inhibition by nanobodies. PLoS ONE 2021;16:e0253364. https://doi.org/10.1371/journal.pone.0253364

Arnosti C. Microbial extracellular enzymes and the marine carbon cycle. Annu. Rev. Marine Sci. 2011;3:401-425. https://doi.org/10.1146/annurev-marine-120709-142731

Bartolini B, Tipton KF, Bianchi L, Stephenson D, Cunningham C, Della Corte L. Determination of monoamine oxidase activity by HPLC with fluorimetric detection. Neurobiology (Bp) 1999;7:109-121.

Bayramoglu G, Salih B, Akbulut A, Arica MY, Biodegradation of Cibacron Blue 3GA by insolubilized laccase and identification of enzymatic byproduct using MALDI-ToF-MS: toxicity assessment studies by Daphnia magna and Chlorella vulgaris, Ecotoxicol. Environ. Safe. 2019;170:453-460. https://doi.org/10.1016/j.ecoenv.2018.12.014

Beloqui A, Sanchez-Ruiz A, Martin-Lomas M, Reichardt NC. A surface-based mass spectrometry method for screening glycosidase specificity in environmental samples. Chem. Commun. 2012;48:1701-1703. https://doi.org/10.1039/c2cc16537f

Blake DA, McLean NV. A colorimetric assay for the measurement of D-glucose consumption by cultured cells. Anal. Biochem. 1989;177:156-160. https://doi.org/10.1016/0003-2697(89)90031-6

Börnsen KO. Influence of salts, buffers, detergents, solvents, and matrices on MALDI-MS protein analysis in complex mixtures. Methods Mol. Biol. 2000;146:387-404. https://doi.org/10.1385/1-59259-045-4:387

Brasseur C, Bauwens J, Tarayre C, Mattéotti C, Thonart P, Destain J, Francis F, Haubruge E, Portetelle D, Vandenbol M, Focant JF, De Pauw E. MALDI-TOF MS analysis of cellodextrins and xylo-oligosaccharides produced by hindgut homogenates of Reticulitermes santonensis. Molecules 2014;19:4578-4594. https://doi.org/10.3390/molecules19044578

Bungert D, Bastian S, Heckmann-Pohl DM, Giffhorn F, Heinzle E, Tholey A. Screening of sugar converting enzyme using quantitative MALDI-ToF mass spectrometry. Biotechnol. Lett. 2004;26:1025-1030. https://doi.org/10.1023/B:BILE.0000032965.18721.62

Bungert D, Heinzle E, Tholey A. Quantitative matrix-assisted laser desorption/ionization mass spectrometry for the determination of enzyme activities. Anal. Biochem. 2004;326:167-175. https://doi.org/10.1016/j.ab.2003.11.013

Burckhardt I, Zimmermann S. Using matrix-assisted laser desorption ionization-time of flight mass spectrometry to detect carbapenem resistance within 1 to 2.5 h. J. Clin. Microbiol. 2011;49:3321-3324. https://doi.org/10.1128/JCM.00287-11

Bush K. Past and present pespectives on β-lactamases. Antimicrob. Agents Chemother. 2018;62:e01076-18. https://doi.org/10.1128/AAC.01076-18

Carlin G, Djursäter R, Smedegård G, Gerdin B. 1985. Effect of anti-inflammatory drugs on xanthine oxidase and xanthine oxidase induced depolymerization of hyaluronic acid. Agents Actions 1985;16:377-384. https://doi.org/10.1007/BF01982876

Chang HL, Su KY, Goodman SD, Yen RS, Cheng WC, Yang YC, Lin LI, Chang SY, Fang W. Measurement of uracil-DNA glycosylase activity by matrix assisted laser desorption/ionization time-of-flight mass spectrometry technique. DNA Repair 2021;97:103028. https://doi.org/10.1016/j.dnarep.2020.103028

Chong PM, McCorrister SJ, Unger MS, Boyd DA, Mulvey MR, Westmacott GR. MALDI-TOF MS detection of carbapenemase activity in clinical isolates of Enterobacteriaceae spp., Pseudomonas aeruginosa, and Acinetobacter baumannii compared against the Carba-NP assay. J. Microbiol. Methods 2015;111:21-23. https://doi.org/10.1016/j.mimet.2015.01.024

Clark Jr. LC, Wolf R, Granger D, Taylor Z. Continuous recording of blood oxygen tensions by polarography. J. Appl. Physiol. 1953;6:189-193. https://doi.org/10.1152/jappl.1953.6.3.189

Cooper RDG. The carbacephems: a new beta-lactam antibiotic class. Am. J. Med. 1992;92:S2-S6. https://doi.org/10.1016/0002-9343(92)90600-g

Correa-Martínez CL, Idelevich EA, Sparbier K, Kostrzewa M, Becker K. Rapid detection of extended-spectrum β-lactamases (ESBL) and AmpC β-lactamases in Enterobacterales: development of a screening panel using the MALDI-TOF MS-based direct-on-target microdroplet growth assay. Front. Microbiol. 2019;10:13. https://doi.org/10.3389/fmicb.2019.00013

Correa-Martínez CL, Idelevich EA, Sparbier K, Kuczius T, Kostrzewa M, Becker K. Development of a MALDI-TOF MS-based screening panel for accelerated differential detection of carbapenemases in Enterobacterales using the direct-on-target microdroplet growth assay. Sci. Rep. 2020;10:4988. https://doi.org/10.1038/s41598-020-61890-7

De Cesare V, Johnson C, Barlow V, Hastie J, Knebel A, Trost M. The MALDI-TOF E2/E3 ligase assay as universal tool for drug discovery in the ubiquitin pathway. Cell Chem. Biol. 2018;25:1117-1127. https://doi.org/10.1016/j.chembiol.2018.06.004

Del Río LA, Ortega MG, López AL, Gorgé JL. A more sensitive modification of the catalase assay with the Clark oxygen electrode. Application to the kinetic study of the pea leaf enzyme. Anal. Biochem. 1977;80:409-415. https://doi.org/10.1016/0003-2697(77)90662-5

Deng Z, Ye M, Bian Y, Liu Z, Liu F, Wang C, Qin H, Zou H. Multiplex isotope dimethyl labeling of substrate peptides for high throughput kinase activity assay via quantitative MALDI MS. Chem. Commun. 2014;50:13960-13962. https://doi.org/10.1039/c4cc04906c

Elased KM, Cool DR, Morris M. Novel mass spectrometric methods for evaluation of plasma angiotensin converting enzyme 1 and renin activity. Hypertension 2005;46:953-959. https://doi.org/10.1161/01.HYP.0000174601.30793.b1

Elased KM, Cunha TS, Gurley SB, Coffman TM, Morris M. New mass spectrometric assay for angiotensin-converting enzyme 2 activity. Hypertension 2006;47:1010-1017. https://doi.org/10.1161/01.HYP.0000215588.38536.30

Erich K, Reinle K, Müller T, Munteanu B, Sammour DA, Hinsenkamp I, Gutting T, Burgermeister E, Findeisen P, Ebert MP, Krijgsveld J, Hopf C. Spatial distribution of endogenous tissue protease activity in gastric carcinoma mapped by MALDI mass spectrometry imaging. Mol. Cell. Proteomics 2018;18:151-161. https://doi.org/10.1074/mcp.RA118.000980

Filandr F, Man P, Halada P, Chang H, Ludwig R, Kracher D. The H2O2-dependent activity of a fungal lytic polysaccharide monooxygenase investigated with a turbidimetric assay. Biotechnol. Biofuels 2020;13:37. https://doi.org/10.1186/s13068-020-01673-4

Finehout EJ, Lee KH. An introduction to mass spectrometry applications in biological research. Biochem. Mol. Biol. Edu. 2004;32:93-100. https://doi.org/10.1002/bmb.2004.494032020331

Fleming A. On the antibacterial action of cultures of a penicillium, with special reference to their use in the isolation of B. influenzae. Br. J. Exp. Pathol. 1929;10:226-236.

Galuszka P, Šebela M, Luhová L, Zajoncová L, Frébort I, Strnad M, Peč P. Cytokinins as inhibitors of plant amine oxidase. J. Enzym. Inhib. 1998;13:457-463. https://doi.org/10.3109/14756369809020549

Gao H, Leary JA. Multiplex inhibitor screening and kinetic constant determination for yeast hexokinase using mass spectrometry based assays, J. Am. Soc. Mass Spectrom. 2003;14:173-181. https://doi.org/10.1016/S1044-0305(02)00867-X

Gardner QA, Younas H, Akhtar M. Studies on the regioselectivity and kinetics of the action of trypsin on proinsulin and its derivatives using mass spectrometry. Biochim. Biophys. Acta 2013;1834:182-190. https://doi.org/10.1016/j.bbapap.2012.09.004

Gaucher-Wieczorek F, Guérineau V, Touboul D, Thétiot-Laurent S, Pelissier F, Badet-Denisot MA, Badet B, Durand P. Evaluation of synthase and hemisynthase activities of glucosamine-6-phosphate synthase by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Anal. Biochem. 2014;458:61-65. https://doi.org/10.1016/j.ab.2014.04.033

Ge X, Sirich TL, Beyer MK, Desaire H, Leary JA. A strategy for the determination of enzyme kinetics using electrospray ionization with an ion trap mass spectrometer. Anal. Chem. 2001;73:5078-5082. https://doi.org/10.1021/ac0105890

Gil-Serrano J, Cardona V, Luengo O, Guilarte M, Sala-Cunill A, Galvan-Blasco P, Labrador-Horrillo M. Anaphylactic shock to meropenem with ertapenem tolerance: a case report. J. Allergy Clin. Immunol. Pract. 2019;7:2057-2058. https://doi.org/10.1016/j.jaip.2019.01.052

Goddard JP, Raymond JL. Recent advances in enzyme assays. Trends Biotechnol. 2004;22:363-370. https://doi.org/10.1016/j.tibtech.2004.04.005

Greis KD. Mass spectrometry for enzyme assays and inhibitor screening: an emerging application in pharmaceutical research. Mass Spectrom. Rev. 2007;26:324-339. https://doi.org/10.1002/mas.20127

Greis KD, Zhou S, Burt TM, Carr AN, Dolan E, Easwaran V, Evdokimov A, Kawamoto R, Roesgen J, Davis GF. MALDI-TOF MS as a label-free approach to rapid inhibitor screening. J. Am. Soc. Mass Spectrom. 2006;17:815-822. https://doi.org/10.1016/j.jasms.2006.02.019

Gros L, Saparbaev MK, Laval J. Enzymology of the repair of free radicals-induced DNA damage. Oncogene 2002;21:8905-8925. https://doi.org/10.1038/sj.onc.1206005

Guilbault GG, Brignac Jr. P, Zimmer M. Homovanillic acid as a fluorometric substrate for oxidative enzymes. Analytical applications of the peroxidase, glucose oxidase, and xanthine oxidase systems. Anal. Chem. 1968;40:190-196. https://doi.org/10.1021/ac60257a002

Guillarme D, Ruta J, Rudaz S, Veuthey JL. 2010. New trends in fast and high-resolution liquid chromatography: a critical comparison of existing approaches. Anal. Bioanal. Chem. 2010;397:1069-1082. https://doi.org/10.1007/s00216-009-3305-8

Guitot K, Scarabelli S, Drujon T, Bolbach G, Amoura M, Burlina F, Jeltsch A, Sagan S, Guianvarc'h D. Label-free measurement of histone lysine methyltransferases activity by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Anal. Biochem. 2014;456:25-31. https://doi.org/10.1016/j.ab.2014.04.006

Guitot K, Drujon T, Burlina F, Sagan S, Beaupierre S, Pamlard O, Dodd RH, Guillou C, Bolbach G, Sachon E, Guianvarc'h D. A direct label-free MALDI-TOF mass spectrometry based assay for the characterization of inhibitors of protein lysine methyltransferases. Anal. Bioanal. Chem. 2017;409:3767-3777. https://doi.org/10.1007/s00216-017-0319-5

Halim VA, Muck A, Hartl M, Ibáñez AJ, Giri A, Erfurth F, Baldwin IT, Svatoš A. A dual fluorescent/MALDI chip platform for analyzing enzymatic activity and for protein profiling. Proteomics 2009;9:171-181. https://doi.org/10.1002/pmic.200800390

Hassiepen U, Eidhoff U, Meder G, Bulber JF, Hein A, Bodendorf U, Lorthiois E, Martoglio B. A sensitive fluorescence intensity assay for deubiquitinating proteases using ubiquitin-rhodamine110-glycine as substrate. Anal. Biochem. 2007;371:201-207. https://doi.org/10.1016/j.ab.2007.07.034

Havlicek V, Lemr K, Schug KA. Current trends in microbial diagnostics based on mass spectrometry. Anal. Chem. 2013;15:790-797. https://doi.org/10.1021/ac3031866

Heeb MJ, Gabriel O. Enzyme localization in gels. Methods Enzymol. 1984;104:416-439. https://doi.org/10.1016/s0076-6879(84)04109-4

Herraiz T, Flores A, Fernández L. Analysis of monoamine oxidase (MAO) enzymatic activity by high-performance liquid chromatography-diode array detection combined with an assay of oxidation with a peroxidase and its application to MAO inhibitors from foods and plants. J. Chromatogr. B 2018;1073:136-144. https://doi.org/10.1016/j.jchromb.2017.12.004

Hooff GP, van Kampen JJA, Meesters RJW, van Belkum A, Goessens WHF, Luider TM. Characterization of β-lactamase enzyme activity in bacterial lysates using MALDI-mass spectrometry. J. Proteome Res. 2012;11:79-84. https://doi.org/10.1021/pr200858r

Hou TY, Chiang-Ni C, Teng SH. Current status of MALDI-TOF mass spectrometry in clinical microbiology. J. Food Drug Anal. 2019;27:404-414. https://doi.org/10.1016/j.jfda.2019.01.001

Hrabák J, Chudáčková E, Walková R. Matrix-assisted laser desorption ionization-time of flight (MALDITOF) mass spectrometry for detection of antibiotic resistance mechanisms: from research to routine diagnosis. Clin. Microb. Rev. 2013;26:103-114. https://doi.org/10.1128/CMR.00058-12

Hrabák J, Walková R, Študentová V, Chudáčková E, Bergerová T. Carbapenemase activity detection by matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) mass spectrometry. J. Clin. Microbiol. 2011;49:3222-3227. https://doi.org/10.1128/JCM.00984-11

Hu L, Jiang G, Xu S, Pan C, Zou H. Monitoring enzyme reaction and screening enzyme inhibitor based on MALDI-TOF-MS platform with a matrix of oxidized carbon nanotubes. J. Am. Soc. Mass Spectrom. 2006;17:1616-1619. https://doi.org/10.1016/j.jasms.2006.07.005

Hu J, Liu F, Ju H. Peptide code-on-a-microplate for protease activity analysis via MALDI-TOF mass spectrometric quantitation. Anal. Chem. 2015;87:4409-4414. https://doi.org/10.1021/acs.analchem.5b00230

Hu J, Liu F, Feng N, Ju H. Peptide codes for multiple protease activity assay via high-resolution mass spectrometric quantitation. Rapid Commun. Mass. Spectrom. 2016;30:196-201. https://doi.org/10.1002/rcm.7631

Humeny A, Beck C, Becker CM, Jeltsch A. Detection and analysis of enzymatic DNA methylation of oligonucleotide substrates by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Anal. Biochem. 2003;313:160-166. https://doi.org/10.1016/s0003-2697(02)00568-7

Idelevich EA, Sparbier K, Kostrzewa M, Becker K. Rapid detection of antibiotic resistance by MALDI-TOF mass spectrometry using a novel direct-on-target microdroplet growth assay. Clin. Microbiol. Infect. 2018;24:738-743. https://doi.org/10.1016/j.cmi.2017.10.016

Jankowski J, Stephan N, Knobloch M, Fischer S, Schmaltz D, Zidek W, Schlüter H. Mass-spectrometry-linked screening of protein fractions for enzymatic activities-a tool for functional genomics. Anal. Biochem. 2001;290:324-329. https://doi.org/10.1006/abio.2001.5001

John RA. 2002. Photometric Assays. In: Eisenthal R, Danson MJ, editors. Enzyme Assays. 2nd Edition (The Practical Approach Series No. 257). New York: Oxford University Press. p 49-78.

Jung JS, Popp C, Sparbier K, Lange C, Kostrzewa M, Schubert S. Evaluation of matrix-assisted laser desorption ionization-time of flight mass spectrometry for rapid detection of β-lactam resistance in Enterobacteriaceae derived from blood cultures. J. Clin. Microbiol. 2014;52:924-930. https://doi.org/10.1128/JCM.02691-13

Kang MJ, Tholey A, Heinzle E. Application of automated matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for the measurement of enzyme activities. Rapid Commun. Mass Spectrom. 2001;15:1327-1333. https://doi.org/10.1002/rcm.376

Kang MJ, Tholey A, Heinzle E. Quantitation of low molecular mass substrates and products of enzyme catalyzed reactions using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Rapid Commun. Mass Spectrom. 2000;14:1972-1978. https://doi.org/10.1002/1097-0231(20001115)14:21%3C1972::AID-RCM119%3E3.0.CO;2-5

Komander D. The emerging complexity of protein ubiquitination. Biochem. Soc. Trans. 2009;37:937-953. https://doi.org/10.1042/BST0370937

Kong KF, Schneper L, Mathee K. Beta-lactam antibiotics: from antibiosis to resistance and bacteriology. APMIS 2010;118:1-36. https://doi.org/10.1111/j.1600-0463.2009.02563.x

Kostrzewa M, Sparbier K, Maier T, Schubert S. MALDI-TOF M: an upcoming tool for rapid detection of antibiotic resistance in microorganisms. Proteomics Clin. Appl. 2013;7:767-778. https://doi.org/10.1002/prca.201300042

Kruger NJ. Errors and artifacts in coupled spectrophotometric assays of enzyme activity, Phytochemistry 1995;38:1065-1071. https://doi.org/10.1016/0031-9422(94)00787-T

Kumar A, Christian GD. Assay of L-tyrosine in serum by amperometric measurement of tyrosinase-catalyzed oxygen consumption. Clin. Chem. 1975;21:325-329.

Kurien BT, Patel NC, Porter AC, Kurono S, Matsumoto H, Wang H, Scofield RH. Determination of prolidase activity using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Anal. Biochem. 2004;331:224-229. https://doi.org/10.1016/j.ab.2004.04.043

Lakowicz JR. 2006. Principles of Fluorescence Spectroscopy. 3rd Edition. Boston: Springer. 954 p.

Laloue M, Fox JE. Cytokinin oxidase from wheat: Partial purification and general properties. Plant Physiol. 1989;90:899-906. https://doi.org/10.1104/pp.90.3.899

Li J, Liu X, Chu H, Fu X, Li T, Hu L, Xing S, Li G, Gu J, Zhao ZJ. Specific dephosphorylation of Janus Kinase 2 by protein tyrosine phosphatases. Proteomics 2015;15:68-76. https://doi.org/10.1002/pmic.201400146

Li H, Luo W, Lin J, Lin Z, Zhang Y. Assay of plasma semicarbazide-sensitive amine oxidase and determination of its endogenous substrate methylamine by liquid chromatography. J. Chromatogr. B. 2004;810:277-282. https://doi.org/10.1016/j.jchromb.2004.08.011

Li Y, Ogata Y, Freeze HH, Scott CR, Tureček F, Gelb MG. Affinity capture and elution/electrospray ionization mass spectrometry assay of phosphomannomutase and phosphomannose isomerase for the multiplex analysis of congenital disorders of glycosylation types Ia and Ib. Anal. Chem. 2003;75:42-48. https://doi.org/10.1021/ac0205053

Liesener A, Karst U. Monitoring enzymatic conversions by mass spectrometry: a critical review. Anal. Bioanal. Chem. 2005;382:1451-1464. https://doi.org/10.1007/s00216-005-3305-2

Liigand J, Wang T, Kellogg J, Smedsgaard J, Cech N, Kruve A. Quantification for non-targeted LC/MS screening without standard substances. Sci. Rep. 2020;10:5808. https://doi.org/10.1038/s41598-020-62573-z

Ling L, Xiao C, Wang S, Guo L, Guo X. A pyrene linked peptide probe for quantitative analysis of protease activity via MALDI-TOF-MS. Talanta 2019;200:236-241. https://doi.org/10.1016/j.talanta.2019.03.055

Liu Y, Li Y, Liu J, Deng C, Zhang X. High throughput enzyme inhibitor screening by functionalized magnetic carbonaceous microspheres and graphene oxide-based MALDI-TOF-MS. J. Am. Soc. Mass Spectrom. 2011;22:2188-2198. https://doi.org/10.1007/s13361-011-0231-8

Lössl P, van den Waterbeemd M, Heck AJR. The diverse and expanding role of mass spectrometry in structural and molecular biology. EMBO J. 2016;35:2634-2657. https://doi.org/10.15252/embj.201694818

Lü S, Luo Q, Li X, Wu J, Liu J, Xiong S, Feng YQ, Wang F. Inhibitor screening of protein kinases using MALDI-TOF MS combined with separation and enrichment of phosphopeptides by TiO2 nanoparticle deposited capillary column. Analyst 2010;135:2858-2863. https://doi.org/10.1039/c0an00339e

Machálková M, Schejbal J, Glatz Z, Preisler Z. A label-free MALDI TOF MS-based method for studying the kinetics and inhibitor screening of the Alzheimer's disease drug target β-secretase. Anal. Bioanal. Chem. 2018;410:7441-7448. https://doi.org/10.1007/s00216-018-1354-6

Maillard LT, Guérinau V, Badet-Denisot MA, Badet B, Laprévote O, Durand P. Monitoring enzyme-catalyzed production of glucosamine-6P by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry: a new enzymatic assay for glucosamine-6P synthase. Rapid Commun. Mass Spectrom. 2006;20:666-672. https://doi.org/10.1002/rcm.2361

Manchenko GP. 2003. Handbook on the Detection of Enzymes on Electrophoretic Gels. 2nd Edition. Boca Raton: CRC Press. 553 p.

Mayerhöfer TG, Popp J. Beer's law-why absorbance depends (almost) linearly on concentration. ChemPhysChem 2019;20:511-515. https://doi.org/10.1002/cphc.201801073

McKee CJ, Hines HB, Ulrich RG. Analysis of protein tyrosine phosphatase interactions with microarrayed phosphopeptide substrate using. Anal. Biochem. 2013;442:62-67. https://doi.org/10.1016/j.ab.2013.07.031

Min DH, Su J, Mrksich M. Profiling kinase activities by using a peptide chip and mass spectrometry. Angew. Chem. Int. Ed. Engl. 2004;43:5973-5977. https://doi.org/10.1002/anie.200461061

Mirande C, Canard I, Buffet Croix Blanche S, Charrier JP, van Belkum A, Welker M, Chatellier, S. Rapid detection of carbapenemase activity: benefits and weaknesses of MALDI-TOF MS. Eur. J. Clin. Microbiol. Infect. Dis. 2015;34:2225-2234. https://doi.org/10.1007/s10096-015-2473-z

Mörsky P. Turbidimetric determination of lysozyme with Micrococcus lysodeikticus cells: reexamination of reaction conditions. Anal. Biochem. 1983;128:77-85. https://doi.org/10.1016/0003-2697(83)90347-0

Negelein E, Haas E. Über die Wirkungweise des Zwischenferments. Biochem. Z. 1935;282:206-220.

O'Rourke MB, Djordjevic SP, Padula MP. The quest for improved reproducibility in MALDI mass spectrometry. Mass Spectrom. Rev. 2016;37:217-228. https://doi.org/10.1002/mas.21515

Ouyang F, Yu T, Gu C, Wang G, Shi R, Lv R, Wu E, Ma C, Guo R, Li J, Zaczek A, Liu J. Sensitive detection of caspase-3 enzymatic activities and inhibitor screening by mass spectrometry with dual maleimide labelling quantitation. Analyst 2019;144:6751-6759. https://doi.org/10.1039/c9an01458f

Oviaño M, Bou G. Matrix-assisted laser desorption ionization-time of flight mass spectrometry for the rapid detection of antimicrobial resistance mechanisms and beyond. Clin. Microbiol. Rev. 2019;32:e00037-18. https://doi.org/10.1128/CMR.00037-18

Park JM, Kim JI, Noh JY, Kim M, Kang MJ, Pyun JC. A highly sensitive carbapenemase assay using laser desorption/ionization mass spectrometry based on a parylene-matrix chip. Enzyme Microb. Technol. 2017;104:56-68. https://doi.org/10.1016/j.enzmictec.2017.05.010

Patrie SM, Roth MJ, Plymire DA, Maresh E, Zhang J. Measurement of blood protease kinetic parameters with self-assembled monolayer ligand binding assays and label-free MALDI-TOF MS. Anal. Chem. 2013;85:10597-10604. https://doi.org/10.1021/ac402739z

Petković M, Müller J, Müller M, Schiller J, Arnold K, Arnhold J. Application of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for monitoring the digestion of phosphatidylcholine by pancreatic phospholipase A2. Anal. Biochem. 2002;308:61-70. https://doi.org/10.1016/s0003-2697(02)00236-1

Pourceau G, Chevolot Y, Goudot A, Giroux F, Meyer A, Moulés V, Lina B, Cecioni S, Vidal S, Yu H, Chen X, Ferraris O, Praly JP, Souteyrand E, Vasseur JJ, Morvan F. Measurement of enzymatic activity and specificity of human and avian influenza neuraminidases from whole virus by glycoarray and MALDI-TOF mass spectrometry. ChemBioChem 2011;12:2071-2080. https://doi.org/10.1002/cbic.201100128

Price NJ, Naumann TA. A high-throughput matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry-based assay of chitinase activity. Anal. Biochem. 2011;411:94-99. https://doi.org/10.1016/j.ab.2010.12.027

Puapaiboon U, Jai-nhuknan J, Cowan JA. Characterization of a multi-functional metal-mediated nuclease by MALDI-TOF mass spectrometry. Nucleic Acids Res. 2001;29:3652-3656. https://doi.org/10.1093/nar/29.17.3652

Ritorto MS, Ewan R, Perez-Oliva AB, Knebel A, Buhrlage SJ, Wightman M, Kelly SM, Wood NT, Virdee S, Gray NS, Morrice NA, Alessi DR, Trost M. Screening of DUB activity and specificity by MALDI-TOF mass spectrometry. Nat. Commun. 2014;5:4673. https://doi.org/10.1038/ncomms5763

Sabbagh B, Costina V, Buchheidt D, Reinwald M, Neumaier M, Findeisen P. Functional protease profiling for laboratory based diagnosis of invasive aspergillosis. Int. J. Oncol. 2015;47:143-150. https://doi.org/10.3892/ijo.2015.2984

Sanchez-Ruiz A, Serna S, Ruiz N, Martin-Lomas M, Reichardt NC. MALDI-TOF mass spectrometric analysis of enzyme activity and lectin trapping on an array of N-glycans. Angew. Chem. Int. Ed. Engl. 2011;50:1801-1804. https://doi.org/10.1002/anie.201006304

Savary BJ, Vasu P, Cameron RG, McCollum TG, Nuñez A. 2013. Structural characterization of the thermally tolerant pectin methylesterase purified from Citrus sinensis fruit and its gene sequence. J. Agric. Food. Chem. 61:12711-12719. https://doi.org/10.1021/jf403914u

Schlüter H, Jankowski J, Rykl J, Thiemann J, Belgardt S, Zidek W, Wittmann B, Pohl T. Detection of protease activities with the mass-spectrometry-assisted enzyme-screening (MES) system. Anal. Bioanal. Chem. 2003;377:1102-1107. https://doi.org/10.1007/s00216-003-2211-8

Scholle MD, Liu C, Deval J, Gurard-Levin ZA. Label-free screening of SARS-CoV-2 NSP14 exonuclease activity using SAMDI mass spectrometry. SLAS Discov. 2021;26:766-774. https://doi.org/10.1177/24725552211008854

Šebela M, Luhová L, Brauner F, Galuszka P, Radová A, Peč P. Light microscopic localisation of aminoaldehyde dehydrogenase activity in plant tissues using nitroblue tetrazolium-based staining method. Plant Physiol. Biochem. 2001;39:831-839. https://doi.org/10.1016/S0981-9428(01)01304-3

Šebela M, Štosová T, Havliš J, Wielsch N, Thomas H, Zdráhal Z, Shevchenko A. Thermostable trypsin conjugates for high-throughput proteomics: synthesis and performance evaluation. Proteomics 2006;6:2959-2963. https://doi.org/10.1002/pmic.200500576

Simon RP, Winter M, Kleiner C, Ries R, Schnapp G, Heimann A, Li J, Zuvela-Jelaska L, Bretschneider T, Luippold AH, Reindl W, Bischoff D, Büttner FH. MALDI-TOF mass spectrometry-based high-throughput screening for inhibitors of the cytosolic DNA sensor cGAS. SLAS Discov. 2020;25:372-383. https://doi.org/10.1177/2472555219880185

Singhal N, Kumar M, Kanaujia PK, Virdi JS. MALDI-TOF mass spectrometry: an emerging technology for microbial identification and diagnosis. Front. Microbiol. 2015;6:791. https://doi.org/10.3389/fmicb.2015.00791

Šlachtová V, Šebela M, Torfs E, Oorts L, Cappoen D, Berka K, Bazgier V, Brulíková L. Novel thiazolidinedione-hydroxamates as inhibitors of Mycobacterium tuberculosis virulence factor Zmp1. Eur. J. Med. Chem. 2020;185:111812. https://doi.org/10.1016/j.ejmech.2019.111812

Sparbier K, Schubert S, Weller U, Boogen C, Kostrzewa M. Matrix-assisted laser desorption ionization-time of flight mass spectrometry-based functional assay for rapid detection of resistance against β-lactam antibiotics. J. Clin. Microbiol. 2012;50:927-937. https://doi.org/10.1128/JCM.05737-11

Su J, Bringer MR, Ismagilov RF, Mrksich M. Combining microfluidic networks and peptide arrays for multi-enzyme assays. J. Am. Chem. Soc. 2005;127:7280-7281. https://doi.org/10.1021/ja051371o

Swartz M. HPLC detectors: a brief review. J. Liq. Chrom. Relat. Tech. 2010;33:1130-1150. https://doi.org/10.1080/10826076.2010.484356

Syal K, Mo M, Yu H, Iriya R, Jing W, Guodong S, Wang S, Grys TE, Haydel SE, Tao N. Current and emerging techniques for antibiotic susceptibility tests. Theranostics 2017;7:1795-1805. https://doi.org/10.7150/thno.19217

Szájli E, Fehér T, Medzihradszky K. Investigating the quantitative nature of MALDI-TOF MS. Mol. Cell. Proteomics 2008;7:2410-2418. https://doi.org/10.1074/mcp.M800108-MCP200

Tipton KF. 2002. Principles of enzyme assay and kinetic studies. In: Eisenthal R, Danson MJ, editors. Enzyme Assays. 2nd Edition (The Practical Approach Series No. 257). New York: Oxford University Press. p 1-47.

Torsi G, Chiavari G, Laghi C, Asmundsdottir AM, Fagioli F, Vecchietti R. Determination of the absolute number of moles ofan analyte in a flow-through system from peak-area measurements. J. Chromatogr. A. 1989;482:207-214. https://doi.org/10.1016/S0021-9673(01)93221-2

van Leeuwen HC, Klychnikov OI, Menks MA, Kuijper EJ, Drijfhout JW, Hensbergen PJ. Clostridium difficile sortase recognizes a (S/P)PXTG sequence motif and can accommodate diaminopimelic acid as a substrate for transpeptidation. FEBS Lett. 2014;588:4325-4333. https://doi.org/10.1016/j.febslet.2014.09.041

van Munster JM, Thomas B, Riese M, Davis AL, Gray CJ, Archer DB, Flitsch SL. Application of carbohydrate arrays coupled with mass spectrometry to detect activity of plant-polysaccharide degradative enzymes from the fungus Aspergillus niger. Sci. Rep. 2017;7:43117. https://doi.org/10.1038/srep43117

Verchot LV, Borelli T. Application of para-nitrophenol (pNP) enzyme assays in degraded tropical soils. Soil Biol. Biochem. 2005;37:625-633. https://doi.org/10.1016/j.soilbio.2004.09.005

Versari A, Parpinello GP, Fabiani A. Comparison of two quantitation methods in HPLC: Standardless versus calibration with external standards. Application to the analysis of amino acids in fruit juices. J. Chrom. Sci. 2007;45:515-518. https://doi.org/10.1093/chromsci/45.8.515

Vosyka O, Vinothkumar KR, Wolf EV, Brouwer AJ, Liskamp RMJ, Verhelst SHL. Activity-based probes for rhomboid proteases discovered in a mass spectrometry-based assay, Proc. Natl. Acad. Sci. U.S.A. 2013;110:2472-2477. https://doi.org/10.1073/pnas.1215076110

Wang D, Baudys J, Barr JR, Kalb SR. Improved sensitivity for the qualitative and quantitative analysis of active ricin by MALDI-TOF mass spectrometry. Anal. Chem. 2016;88:6867-6872. https://doi.org/10.1021/acs.analchem.6b01486

Wang S, Xiao C, Guo L, Ling L, Li M, Li H, Guo X. Rapidly quantitative analysis of γ-glutamyltranspeptidase activity in the lysate and blood via a rational design of the molecular probe by matrix-assisted laser desorption ionization mass spectrometry. Talanta 2019;205:120141. https://doi.org/10.1016/j.talanta.2019.120141

Wang L, Zhang Y, Xiao S, Hu G, Che B, Qing H, Li Y, Zhuang L, Deng Y. LC-MS method for determining the activity of semicarbazide-sensitive amine oxidase in rodents. Anal. Methods 2012;4:1383-1388. https://doi.org/10.1039/c2ay05914b

Watson DS, Jambunathan K, Askew DS, Kodukula K, Galande AK. Robust substrate profiling method reveals striking differences in specificities of serum and lung fluid proteases. Biotechniques 2011;51:95-104. https://doi.org/10.2144/000113717

Wilkinson JH, Baron DN, Moss DW, Walker PG. Standardization of clinical enzyme assays: a reference method for aspartate and alanine transaminases. J. Clin. Path. 1972;25:940-944. https://doi.org/10.1136/jcp.25.11.940

Winter M, Bretschneider T, Thamm S, Kleiner C, Grabowski D, Chandler S, Ries R, Kley JT, Fowler D, Bartlett C, Binetti R, Broadwater J, Luippold AH, Bischoff D, Büttner FH. Chemical derivatization enables MALDI-TOF-based high-throughput screening for microbial trimethylamine (TMA)-lyase inhibitors. SLAS Discov. 2019;24:766-777. https://doi.org/10.1177/2472555219838216

Wise R. The carbapenems and penem antibiotics-a brief review. Antimicrob. Newsletter 1990;7:73-78. https://doi.org/10.1016/0738-1751(90)90045-E

Woodard SL, Mayor JM, Bailey MR, Barker DK, Love RT, Lane JR, Delaney DE, McComas-Wagner JM, Mallubhotla HD, Hood EE, Dangott LJ, Tichy SE, Howard JA. Maize (Zea mays)-derived bovine trypsin: characterization of the first large-scale, commercial protein product from transgenic plants. Biotechnol. Appl. Biochem. 2003;38:123-130. https://doi.org/10.1042/BA20030026

Yamamoto H, Sawaguchi Y, Kimura M. The determination of protease specificity in mouse tisssue extracts by MALDI-TOF mass spectrometry: manipulating pH to cause specificity changes. JoVE 2018;135:e57469. https://doi.org/10.3791/57469

Yang H, Chan AL, LaVallo V, Cheng Q. Quantitation of alpha-glucosidase activity using fluorinated carbohydrate array and MALDI-TOF MS. ACS Appl. Mater. Interfaces 2016:8:2872-2878. https://doi.org/10.1021/acsami.5b12518

You SH, Lim HD, Cheong DE, Kim ES, Kim GJ. Rapid and sensitive detection of NADPH via mBFP-mediated enhancement of its fluorescence. PLoS ONE 2019;14:e0212061. https://doi.org/10.1371/journal.pone.0212061

Zhou Y, Kang L, Niu X, Wang J, Liu Z, Yuan S. Purification, characterization and physiological significance of a chitinase from the pilei of Coprinopsis cinerea fruiting bodies. FEMS Microbiol. Lett. 2016;363:fnw120. https://doi.org/10.1093/femsle/fnw120

Zhou X, Turecek F, Scott CR, Gelb MH. Quantification of cellular acid sphingomyelinase and galactocerebroside beta-galactosidase activities by electrospray ionization mass spectrometry. Clin. Chem. 2001;47:874-881. https://doi.org/10.1093/clinchem/47.5.874

Zhou G, Yan X, Wu D, Kron SJ. Photocleavable peptide-conjugated magnetic beads for protein kinase assays by MALDI-TOF MS. Bioconjugate Chem. 2010;21:1917-1924. https://doi.org/10.1021/bc1003058

A New Activity Assay Method for Diamine Oxidase Based on Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry