Methods in Molecular Biology 1574 Oliver Schilling Editor Protein Terminal Profiling Methods and Protocols Methods in Molecular Biology Series Editor John M. Walker School of Life and Medical Sciences University of Hertfordshire Hatfield, Hertfordshire, AL10 9AB, UK For further volumes: http://www.springer.com/series/7651 Protein Terminal Profiling Methods and Protocols Edited by Oliver Schilling Institute of Molecular Medicine and Cell Research, University of Freiburg, Freiburg, Germany; BIOSS Centre of Biological Signaling Studies, University of Freiburg, Freiburg, Germany; German Cancer Consortium (DKTK) and German Cancer Research Center (DKFZ), Heidelberg, Germany Editor Oliver Schilling Institute of Molecular Medicine and Cell Research University Freiburg, Freiburg, Germany BIOSS Centre of Biological Signaling Studies University of Freiburg, Freiburg, Germany German Cancer Consortium (DKTK) and German Cancer Research Center (DKFZ) Heidelberg, Germany ISSN 1064-3745 ISSN 1940-6029 (electronic) Methods in Molecular Biology ISBN 978-1-4939-6849-7 ISBN 978-1-4939-6850-3 (eBook) DOI 10.1007/978-1-4939-6850-3 Library of Congress Control Number: 2017931661 © Springer Science+Business Media LLC 2017 This work is subject to copyright All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed The use of general descriptive names, registered names, trademarks, service marks, etc in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use The publisher, the authors and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication Neither the publisher nor the authors or the editors give a warranty, express or implied, with respect to the material contained herein or for any errors or omissions that may have been made The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations Printed on acid-free paper This Humana Press imprint is published by Springer Nature The registered company is Springer Science+Business Media LLC The registered company address is: 233 Spring Street, New York, NY 10013, U.S.A Preface Amino and carboxy-termini of proteins are subject to a variety of enzymatically catalyzed, post-translational modifications with diverse biological functions Generally, protein terminal sequences may determine protein function, localization, and turnover Endoproteases generate stable cleavage products with novel N- or C-termini while exoproteases yield stepwise truncations In addition, there are modifications such as N-terminal acetylation and pyroglutamate formation, which contribute to protein functionality and stability Over the last years, a number of techniques for N- and C-terminal profiling have been developed To a large extent, these encompass proteomic techniques that are based on liquid chromatography–tandem mass spectrometry This book presents detailed protocols for several of these novel strategies together with approaches for their annotation in order to enable an improved functional understanding of protein N- and C-terminal biology Protein termini are often generated by proteolytic truncations thus placing proteases and (limited) proteolysis in a central position when studying N- and C-terminal biology and biochemistry Accordingly, a large proportion of this book addresses topics of proteolysis research Its topics include protease specificity profiling, N-terminal acetylation, assays to probe protease activity (and its possible inhibition) in cellular systems, proteomic techniques to explore protein N- and C-termini on a proteome-wide scale, computational approaches to correlate cleavage sequences with candidate proteases, design of activity-based probes for proteolytic enzymes, and biochemical approaches to deconvolute extracellular protease activities The book targets researchers who focus on biochemistry and cell biology and who share a broad interest in protein functionality and protein modifications I sincerely thank all authors for their valuable contributions—it was a privilege to compile this edition of Methods in Molecular Biology I also want to thank the series editor, John Walker, for his continuous support Freiburg, Germany Oliver Schilling v Contents Preface v Contributors ix 1 [14C]-Acetyl-Coenzyme A-Based In Vitro N-Terminal Acetylation Assay Adrian Drazic and Thomas Arnesen DTNB-Based Quantification of In Vitro Enzymatic N-Terminal Acetyltransferase Activity Håvard Foyn, Paul R Thompson, and Thomas Arnesen SILProNAQ: A Convenient Approach for Proteome-Wide Analysis of Protein N-Termini and N-Terminal Acetylation Quantitation Willy V Bienvenut, Carmela Giglione, and Thierry Meinnel Profiling of Protein N-Termini and Their Modifications in Complex Samples Fatih Demir, Stefan Niedermaier, Jayachandran N Kizhakkedathu, and Pitter F Huesgen Protease Substrate Profiling by N-Terminal COFRADIC An Staes, Petra Van Damme, Evy Timmerman, Bart Ruttens, Elisabeth Stes, Kris Gevaert, and Francis Impens Doublet N-Terminal Oriented Proteomics for N-Terminomics and Proteolytic Processing Identification Benoit Westermann, Alvaro Sebastian Vaca Jacome, Magali Rompais, Christine Carapito, and Christine Schaeffer-Reiss Multidimensional Analysis of Protease Substrates and Their Cellular Origins in Mixed Secretomes from Multiple Cell Types Pascal Schlage and Ulrich auf dem Keller System-Wide Profiling of Protein Amino Termini from Formalin-Fixed, Paraffin-Embedded Tissue Specimens for the Identification of Novel Substrates Zon W Lai and Oliver Schilling Identification of Carboxypeptidase Substrates by C-Terminal COFRADIC Sebastian Tanco, Francesc Xavier Aviles, Kris Gevaert, Julia Lorenzo, and Petra Van Damme 10 ProC-TEL: Profiling of Protein C-Termini by Enzymatic Labeling Wenwen Duan and Guoqiang Xu 11 Determining Protease Substrates Within a Complex Protein Background Using the PROtein TOpography and Migration Analysis Platform (PROTOMAP) R.A Fuhrman-Luck, L.M Silva, M.L Hastie, J.J Gorman, and J.A Clements vii 17 35 51 77 91 105 115 135 145 viii Contents 12 Multiplexed Protease Specificity Profiling Using Isobaric Labeling Joanna Tucher and Andreas Tholey 13 FPPS: Fast Profiling of Protease Specificity Matej Vizovišek, Robert Vidmar, and Marko Fonović 14 Profiling of Protease Cleavage Sites by Proteome-Derived Peptide Libraries and Quantitative Proteomics Chia-yi Chen, Bettina Mayer, and Oliver Schilling 15 Prediction of Proteases Involved in Peptide Generation Mercedes Arguello Casteleiro, Robert Stevens, and Julie Klein 16 Live-Cell Imaging of Protease Activity: Assays to Screen Therapeutic Approaches Anita Chalasani, Kyungmin Ji, Mansoureh Sameni, Samia H Mazumder, Yong Xu, Kamiar Moin, and Bonnie F Sloane 17 Protein Translocation Assays to Probe Protease Function and Screen for Inhibitors Angelina Hahlbrock, Dorothée Gößwein, and Roland H Stauber 18 Simultaneous Detection of Metalloprotease Activities in Complex Biological Samples Using the PrAMA (Proteolytic Activity Matrix Assay) Method Catharina Conrad, Miles A Miller, Jörg W Bartsch, Uwe Schlomann, and Douglas A Lauffenburger 19 Synthesis and Application of Activity-Based Probes for Proteases Tim Van Kersavond, Minh T.N Nguyen, and Steven H.L Verhelst 171 183 197 205 215 227 243 255 Index 267 Contributors Thomas Arnesen • Department of Molecular Biology, University of Bergen, Bergen, Norway; Department of Surgery, Haukeland University Hospital, Bergen, Norway Francesc Xavier Aviles • Institut de Biotecnologia i Biomedicina (IBB), Departament de Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona, Barcelona, Spain Jörg W. Bartsch • Department of Neurosurgery, Marburg University, Marburg, Germany Willy V. Bienvenut • Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Univ Paris-Sud, Université Paris Saclay, Gif-sur-Yvette cedex, France Christine Carapito • BioOrganic Mass Spectrometry Laboratory (LSMBO), IPHC, CNRS—UdS, UMR 7178, University of Strasbourg, Strasbourg, France Mercedes Arguello Casteleiro • School of Computer Science, University of Manchester, Manchester, UK Anita Chalasani • Department of Pharmacology, School of Medicine, Wayne State University, Detroit, MI, USA Chia-yi Chen • Institute of Molecular Medicine and Cell Research, University of Freiburg, Freiburg, Germany J.A. Clements • Australian Prostate Cancer Research Centre—Queensland, Institute of Health and Biomedical Innovation, Queensland University of Technology, Brisbane, QLD, Australia; Translational Research Institute, Brisbane, QLD, Australia Catharina Conrad • Department of Neurosurgery, Marburg University, Marburg, Germany; Department of Anesthesiology and Intensive Care Medicine, University Hospital, Münster, Germany Petra Van Damme • VIB-UGent Center for Medical Biotechnology, Ghent, Belgium; Department of Biochemistry, Ghent University, Ghent, Belgium Fatih Demir • Central Institute for Engineering, Electronics and Analytics, ZEA-3, Forschungszentrum Jülich, Jülich, Germany Adrian Drazic • Department of Molecular Biology, University of Bergen, Bergen, Norway Wenwen Duan • Jiangsu Key Laboratory of Translational Research and Therapy for Neuro-Psycho-Diseases and College of Pharmaceutical Sciences, Soochow University, Suzhou, Jiangsu, P.R China Marko Fonović • Department of Biochemistry and Molecular and Structural Biology, Jožef Stefan Institute, Ljubljana, Slovenia; Centre of Excellence for Integrated Approaches in Chemistry and Biology of Proteins, Ljubljana, Slovenia Håvard Foyn • Department of Molecular Biology, University of Bergen, Bergen, Norway R.A. Fuhrman-Luck • Australian Prostate Cancer Research Centre—Queensland, Institute of Health and Biomedical Innovation, Queensland University of Technology, Brisbane, QLD, Australia; Translational Research Institute, Brisbane, Queensland, Australia Kris Gevaert • VIB-UGent Center for Medical Biotechnology, Ghent, Belgium; Department of Biochemistry, Ghent University, Ghent, Belgium ix x Contributors Carmela Giglione • Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Univ Paris-Sud, Uni Paris Saclay, Gif-sur-Yvette cedex, France J.J. Gorman • Protein Discovery Centre, QIMR Berghofer Medical Research Institute, Brisbane, QLD, Australia Dorothée Gösswein • Molecular and Cellular Oncology, ENT/University Medical Center Mainz, Mainz, Germany Angelina Hahlbrock • Molecular and Cellular Oncology, ENT/University Medical Center Mainz, Mainz, Germany M.L. Hastie • Protein Discovery Centre, QIMR Berghofer Medical Research Institute, Brisbane, Queensland, Australia Pitter F. Huesgen • Central Institute for Engineering, Electronics and Analytics, ZEA-3, Forschungszentrum Jülich, Jülich, Germany Francis Impens • VIB Proteomics Core, Ghent, Belgium; VIB-UGent Center for Medical Biotechnology, Ghent, Belgium; Department of Biochemistry, Ghent University, Ghent, Belgium Alvaro Sebastian Vaca Jacome • BioOrganic Mass Spectrometry Laboratory (LSMBO), IPHC, CNRS—UdS, UMR 7178, University of Strasbourg, Strasbourg, France Kyungmin Ji • Department of Pharmacology, School of Medicine, Wayne State University, Detroit, MI, USA Ulrich auf dem Keller • Department of Biology, Institute of Molecular Health Sciences, ETH Zurich, Zurich, Switzerland Tim Van Kersavond • Leibniz Institute for Analytical Sciences ISAS, e.v., Dortmund, Germany Jayachandran N. Kizhakkedathu • Centre for Blood Research, Department of Pathology & Laboratory Medicine, University of British Columbia, Vancouver, Canada; Department of Chemistry, University of British Columbia, Vancouver, Canada Julie Klein • Institute of Cardiovascular and Metabolic Disease, INSERM U1048, Toulouse, France; Université Toulouse III Paul-Sabatier, Toulouse, France Zon W. Lai • Department of Genetics and Complex Diseases, Harvard T. H Chan School of Public Health, Boston, MA, USA; Institute of Molecular Medicine and Cell Research, University of Freiburg, Freiburg, Germany Douglas A. Lauffenburger • Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA Julia Lorenzo • Institut de Biotecnologia i Biomedicina (IBB), Departament de Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona, Barcelona, Spain Bettina Mayer • Institute of Molecular Medicine and Cell Research, University of Freiburg, Freiburg, Germany Samia H. Mazumder • Department of Pharmacology, School of Medicine, Wayne State University, Detroit, MI, USA Thierry Meinnel • Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Univ Paris-Sud, Univ Paris Saclay, Gif-sur-Yvette Cedex, France Miles A. Miller • Center for Systems Biology, Massachusetts General Hospital, Boston, MA, USA Contributors xi Kamiar Moin • Department of Pharmacology, School of Medicine, Wayne State University, Detroit, MI, USA Minh T.N. Nguyen • Leibniz Institute for Analytical Sciences ISAS, e.v., Dortmund, Germany Stefan Niedermaier • Central Institute for Engineering, Electronics and Analytics, ZEA-3, Forschungszentrum Jülich, Jülich, Germany Magali Rompais • BioOrganic Mass Spectrometry Laboratory (LSMBO), IPHC, CNRS—UdS, UMR 7178, University of Strasbourg, Strasbourg, France Bart Ruttens • VIB-UGent Center for Medical Biotechnology, Ghent, Belgium; Department of Biochemistry, Ghent University, Ghent, Belgium Mansoureh Sameni • Department of Pharmacology, School of Medicine, Wayne State University, Detroit, MI, USA Christine Schaeffer-Reiss • BioOrganic Mass Spectrometry Laboratory (LSMBO), IPHC, CNRS—UdS, UMR 7178, University of Strasbourg, Strasbourg, France Oliver Schilling • Institute of Molecular Medicine and Cell Research, University of Freiburg, Freiburg, Germany; BIOSS Centre of Biological Signaling Studies, University of Freiburg, Freiburg, Germany; German Cancer Consortium (DKTK) and German Cancer Research Center (DKFZ), Heidelberg, Germany Pascal Schlage • Department of Biology, Institute of Molecular Health Sciences, ETH Zurich, Zurich, Switzerland Uwe Schlomann • Department of Neurosurgery, Marburg University, Marburg, Germany L.M. Silva • Translational Research Institute, Brisbane, QLD, Australia; Cancer Program, Institute of Health and Biomedical Innovation, Queensland University of Technology, Brisbane, Queensland, Australia Bonnie F. Sloane • Department of Pharmacology, School of Medicine, Wayne State University, Detroit, MI, USA An Staes • VIB Proteomics Core, Ghent, Belgium; VIB-UGent Center for Medical Biotechnology, Ghent, Belgium; Department of Biochemistry, Ghent University, Ghent, Belgium Roland H. Stauber • Molecular and Cellular Oncology, ENT/University Medical Center Mainz, Mainz, Germany Elisabeth Stes • VIB-UGent Center for Medical Biotechnology, Ghent, Belgium; Department of Biochemistry, Ghent University, Ghent, Belgium Robert Stevens • School of Computer Science, University of Manchester, Manchester, UK Sebastian Tanco • VIB-UGent Center for Medical Biotechnology, Ghent, Belgium; Department of Biochemistry, Ghent University, Ghent, Belgium Andreas Tholey • AG Systematic Proteome Research & Bioanalytics, Institute for Experimental Medicine, Christian-Albrechts-Universität zu Kiel, Kiel, Germany Paul R. Thompson • Department of Biochemistry and Molecular Pharmacology, UMASS Medical School, Worcester, MA, USA Evy Timmerman • VIB Proteomics Core, Ghent, Belgium; VIB-UGent Center for Medical Biotechnology, Ghent, Belgium; Department of Biochemistry, Ghent University, Ghent, Belgium Simultaneous Detection of Metalloprotease Activities in Complex Biological Samples… 253 References Kleiner DE, Stetler-Stevenson WG (1994) Quantitative zymography: detection of picogram quantities of gelatinases Anal Biochem 218(2):325–329 Lauer-Fields JL, Nagase H, Fields GB (2004) Development of a solid-phase assay for analysis of matrix metalloproteinase activity J Biomol Tech 15(4):305–316 Gosalia DN, Denney WS, Salisbury CM, Ellman JA, Diamond SL (2006) Functional phenotyping of human plasma using a 361-fluorogenic substrate biosensing microarray Biotechnol Bioeng 94(6):1099–1110 Sieber SA, Niessen S, Hoover HS, Cravatt BF (2006) Proteomic profiling of metalloprotease activities with cocktails of active-site probes Nat Chem Biol 2(5):274–281 Miller MA, Barkal L, Jeng K et al (2011) Proteolytic Activity Matrix Analysis (PrAMA) for simultaneous determination of multiple protease activities Integr Biol (Camb) 3(4):422–438 Dang M, Armbruster N, Miller MA et al (2013) Regulated ADAM17-dependent EGF family ligand release by substrate-selecting signaling pathways Proc Natl Acad Sci U S A 110(24):9776–9781 Schlomann U, Koller G, Conrad C et al (2015) ADAM8 as a drug target in pancreatic cancer Nat Commun 6:6175 Chen CH, Miller MA, Sarkar A et al (2013) Multiplexed protease activity assay for low- volume clinical samples using droplet-based microfluidics and its application to endometriosis J Am Chem Soc 135(5):1645–1648 Miller MA, Meyer AS, Beste MT et al (2013) ADAM-10 and -17 regulate endometriotic cell migration via concerted ligand and receptor shedding feedback on kinase signaling Proc Natl Acad Sci U S A 110(22): E2074–E2083 10 Miller MA, Oudin MJ, Sullivan RJ et al (2016) Reduced proteolytic shedding of receptor tyrosine kinases is a post-translational mechanism of kinase inhibitor resistance Cancer Discov 6(4):382–399 11 Miller MA, Moss ML, Powell G et al (2015) Targeting autocrine HB-EGF signaling with specific ADAM12 inhibition using recombinant ADAM12 prodomain Sci Rep 5:15150 12 Ng EX, Miller MA, Jing T, Lauffenburger DA, Chen CH (2015) Low-volume multiplexed proteolytic activity assay and inhibitor analysis through a pico-injector array Lab Chip 15(4):1153–1159 13 Ng EX, Miller MA, Jing T, Chen CH (2016) Single cell multiplexed assay for proteolytic activity using droplet microfluidics Biosens Bioelectron 81:408–414 14 Moss ML, Rasmussen FH (2007) Fluorescent substrates for the proteinases ADAM17, ADAM10, ADAM8, and ADAM12 useful for high-throughput inhibitor screening Anal Biochem 366(2):144–148 Chapter 19 Synthesis and Application of Activity-Based Probes for Proteases Tim Van Kersavond, Minh T.N. Nguyen, and Steven H.L. Verhelst Abstract The detection, visualization, and identification of active proteases can be facilitated by activity-based probes, which covalently bind to a catalytic residue of the target protease The synthesis of activity-based probes can be challenging We here outline a simple protocol for probe synthesis based on standard solid phase peptide synthesis followed by capping of the N-terminus with a reactive electrophile as a warhead The applicability of the probes is illustrated by labeling cysteine proteases in cell and tissue lysates with Western blotting or fluorescence scanning as a readout Key words Activity-based probes, Activity profiling, Chemical proteomics, Click chemistry, Proteases, Solid phase peptide synthesis 1 Introduction Peptidases (often referred to as proteases) are responsible for cleavage of peptide bonds in proteins and polypeptides This process can activate or deactivate the cleaved substrate, resulting in a downstream biological effect Because of the irreversible nature of proteolysis, the action of proteases needs to be tightly regulated to prevent undesired and uncontrolled cleavage of proteins The regulation is executed on several levels and includes protease substrate specificity, separation of protease and substrate in different compartments, and synthesis of proteases as inactive proenzymes, which are activated in a controlled manner Whereas various chapters in this book deal with the identification of protease substrates and substrate specificities, this chapter will focus on a method to detect the proteases themselves by making use of so-called activity-based probes (ABPs) ABPs are designed to react with active enzymes, thereby covalently coupling the enzyme target to a tag for detection or purification [1, 2] They have been particularly useful in protease research [3, 4] In general, Oliver Schilling (ed.), Protein Terminal Profiling: Methods and Protocols, Methods in Molecular Biology, vol 1574, DOI 10.1007/978-1-4939-6850-3_19, © Springer Science+Business Media LLC 2017 255 256 Tim Van Kersavond et al ABPs consist of three parts (1) A warhead, which often is an electrophile that binds to the catalytic residue(s) of the target protease in a mechanism-based reaction (2) A tag, which determines the possibilities for detection—e.g., a fluorophore for fluorescence detection or a biotin for Western blot or enrichment (3) A spacer or recognition element, which not only separates the warhead from the tagging moiety, but may also influence the probe selectivity Interestingly, information about substrates of proteases or substrate specificities can be utilized to design probes that are selective toward one specific protease or protease family [5] Probe synthesis can form a bottleneck for the widespread application of ABPs While some warheads are commercially available or easily obtainable from commercial starting materials, others require complicated multistep syntheses Perhaps the easiest probe construction takes place on solid support by standard solid phase peptide synthesis (SPPS) followed by capping of the N-terminus with a reactive electrophile as a warhead It has been implemented for several types of warheads, including epoxysuccinates [6], chloroacetamides [7], and phosphoramidates [8] In this chapter, we highlight this strategy by describing the synthesis and application of two epoxysuccinate ABPs that target clan CA cysteine proteases One comprises the well-known DCG-04 [6], the other an alkyne analog (SV38) (Fig. 1) Both are based on the natural product E-64, which was discovered as a cysteine protease inhibitor in 1978 [9] The selectivity of E-64 and derived probes originates from the leucine residue that fits into the S2 pocket of clan CA proteases, as demonstrated by crystallography [10] The tags comprise a biotin in the case of DCG-04 and an alkyne in the case of SV38 Both Fig Structures of the natural clan CA cysteine protease inhibitor E-64, and the ABPs DCG-04 and SV38 with indication of tag, spacer, and warhead Note that both probes have a C-terminal amide derived from the linkage to the solid support and an N-terminal epoxysuccinate as a reactive warhead for attachment to the active site cysteine Activity-Based Probes for Proteases 257 have the possibility for radio-iodination at the tyrosine residue [6] The alkyne in SV38 can be functionalized by copper-assisted “click”-chemistry [11] using any tag that contains an azide As a result, the same probe can be used for different detection purposes We will here illustrate the usage of the probes by the detection of cysteine proteases in cell and tissue lysates by Western blot and in-gel fluorescence scanning 2 Materials 2.1 Construction of Probes 2.1.1 Synthesis of Ethyl (2S, 3S)-Epoxysuccinate (2S, 3S)-diethyl-2,3-epoxysuccinate Potassium hydroxide (KOH) Ethanol (EtOH) Hydrochloric acid (HCl) Equipment: ventilated hood, magnetic stir plate, rotary evaporator 2.1.2 Solid Phase Peptide Synthesis of the Probes DCG-04 and SV38 Rink resin Piperidine Solvents: dimethyl formamide (DMF), methanol (MeOH), dichloromethane (DCM), dimethyl sulfoxide (DMSO) Deprotection solution: 20% piperidine in DMF Kaiser test solution A: 49 mL pyridine, mL of a solution of 16.5 mg KCN in 25 mL water Kaiser test solution B: g ninhydrin in 20 mL EtOH Kaiser test solution C: 40 mL phenol, 10 mL EtOH Fmoc-amino acids: Fmoc-Lys(biotin)-OH, Fmoc-Pra-OH (propargylglycine), Fmoc-Ahx-OH (aminohexanoic acid), Fmoc-Tyr-OH, Fmoc-Leu-OH Ethyl (2S, 3S)-2,3-epoxysuccinate 10 Coupling reagents: diisopropylcarbodiimide (DIC), hydroxybenzotriazole hydrate (HOBt.H2O), 2-(1H-benzotriazol-1yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), and N,N-diisopropyl-N-ethylamine (DIEA) 11 For manual peptide synthesis: fritted syringes with plastic caps and stopcocks 12 Equipment for manual peptide synthesis: ventilated hood, vacuum erlenmeyer, vacuum manifold, vacuum pump, tumbling shaker 13 Equipment for automated peptide synthesis: peptide synthesis robot 258 Tim Van Kersavond et al 2.2 Biochemical Usage of Probes Lysis buffer A (for RAW264.7 cells): 50 mM sodium acetate, mM DTT, mM MgCl2, and 0.1% Triton-X100, pH 5.5 Lysis buffer B (for rat liver tissue): 50 mM sodium acetate, mM DTT, mM MgCl2, and 250 mM sucrose, pH 5.5 Bradford reagent (Bio-Rad) 4× Laemmli buffer: 200 mM Tris, pH 6.8, 40% glycerol, 20% beta-mercaptoethanol, 12% SDS (w/v), 0.04% bromophenol blue (w/v) Tris-glycine SDS-PAGE running buffer: 75 g Tris base, 360 g glycine, 25 g SDS in 5 L deionized water for a 5× stock solution Tris-glycine transfer buffer with 20% methanol PBST as wash buffer: standard phosphate-buffered saline supplemented with 0.05% Tween-20 Blocking buffer: 3% skim milk in PBST Streptavidin-HRP (Sigma-Aldrich, mg/mL in phosphate- buffered saline with 50% glycerol) 10 Luminescent substrate, such as Pierce™ ECL Western blotting substrate 11 Click chemistry solution I: 50 mM CuSO4 in water (prepare fresh by adding 125 μL water per mg of CuSO4) 12 Click chemistry solution II: 50 mM sodium ascorbate in water (prepare fresh by adding 100 μL water per mg of sodium ascorbate) 13 Click chemistry solution III: mM tris(benzyltriazolylmethyl) amine (TBTA) in DMSO (store at −20 °C) 14 Click chemistry solution IV: mM of an azide-fluorophore derivative in DMSO (store at −20 °C), such as azide-fluor 545 (Sigma-Aldrich) or TAMRA-azide (Carl Roth, used here) 15 Equipment: standard gel running and Western blot transfer equipment, Odyssey® Fc Imaging system (LI-COR Biosciences) for luminescence detection, Typhoon™ FLA9500 fluorescent scanner 3 Methods 3.1 Construction of Probes 3.1.1 Synthesis of (2S, 3S)-Ethyl-2,3- Epoxysuccinate The epoxysuccinate warhead can be obtained in two ways: (1) in four steps starting from diethyl d-tartrate according to the procedure described by Chehade et al [12]; (2) in one step starting from the (more expensive) (2S, 3S)-diethyl-2,3-epoxysuccinate (Fig. 2a) This step comprises a simple saponification of one of the ethyl esters with 1 eq KOH in ethanol following the protocol below Activity-Based Probes for Proteases 259 Fig (a) One-step synthesis of (2S, 3S)-ethyl-2,3-epoxysuccinate from commercial starting material (b) Construction of the probe DCG-04 on solid support Chain elongation is achieved by subsequent repetition of an Fmoc-deprotection and peptide coupling using an amino acid derivative or warhead, respectively Cleavage from the resin is performed in 95% TFA 1.88 g of (2S, 3S)-diethyl-2,3-epoxysuccinate (10 mmol) is added to a 250 mL round-bottom flask, equipped with a magnetic stir bar, and 50 mL anhydrous EtOH is added The flask is cooled to °C in an ice bath 0.56 g KOH is dissolved in 15 mL anhydrous EtOH (see Note 1) It is then added dropwise to the epoxysuccinate solution—either making use of a large plastic syringe or a dropping funnel The solution is stirred for h (see Note 2), while slowly warming to room temperature TLC analysis (developed with 40% ethyl acetate in petroleum ether eluent and stained with cerium ammonium molybdate stain) shows complete disappearance of the starting material The solvent is evaporated using a rotary evaporator The residue is dissolved in 20 mL water, extracted with 20 mL of DCM and acidified with concentrated HCl until the pH is lower than (approximately 0.5–1 mL) The aqueous phase is then extracted with EtOAc (3 × 20 mL), dried with MgSO4, filtered and concentrated under reduced pressure using a rotary evaporator The identity and purity of the product may be checked by 1H NMR or LC-MS (negative ionization mode) (see Note 3) 3.1.2 Solid Phase Peptide Synthesis of the Probes DCG-04 and SV38 Typical yields are around 85% Probes DCG-04 and SV38 are synthesized on solid support using standard Fmoc-based solid phase peptide chemistry (Fig. 2b) 260 Tim Van Kersavond et al Fig Pictures of the different possible setups for solid phase peptide synthesis (a) A vacuum erlenmeyer capped with a septum and a needle on which the fritted syringe can be placed, (b) a vacuum manifold allowing the parallel manual synthesis of multiple peptides, (c) example of a peptide synthesis robot—the Biotage Syro I, which can be programmed to synthesize multiple peptides in parallel (d) Example of a positive (left, dark blue) and negative (right, clear) Kaiser test Different setups can be used for this, depending on the available equipment Three possibilities with increasing complexity are listed in Fig. 3a–c (1) A vacuum Erlenmeyer capped with a septum and a needle This is the most basic and inexpensive setup, and works well when no specific peptide synthesis equipment is available (2) A vacuum manifold This allows the parallel synthesis of multiple probes (3) An automated peptide synthesizer Although this may not be available in most laboratories, it is often present at facilities of universities and research institutes The peptide-like probes are constructed from the C- to the N-terminus and the solid phase of choice is a Rink amide resin This ensures that an uncharged C-terminal amide is obtained after Activity-Based Probes for Proteases 261 cleavage from the resin in contrast to, e.g., a Wang resin, which would yield a C-terminal carboxylic acid We here describe the synthesis at a 50 μmol scale using a manual setup (Fig. 3b), but it can easily be scaled up or down, or adapted to automated setups Weigh the resin (50 μmol, 84 mg with a loading of 0.59 mmol/g) in a fritted syringe, fitted with a stopcock Treat the resin with approximately mL deprotection solution Cap the syringe and let it shake on a tumbling shaker for 15 min Drain the solution by vacuum filtration and wash with DMF three times For confirmation of deprotection, a Kaiser test may be performed Wash the resin prior to the Kaiser test two times with DCM to remove residual DMF, which may contain traces of free amines For the Kaiser test, add two drops each of test solutions A, B, and C to a small glass test tube (alternatively, a clear 1.5 mL eppendorf tube may be used) Add a tiny amount of the resin to the solution and heat in a heatblock of 95 °C for 2 min A positive result (blue solution) indicates the successful deprotection (Fig. 3d) For the amino acid coupling 3 eq of the respective amino acid and 3 eq of HOBt hydrate are added to the resin and dissolved in DMF (final concentration of approximately 0.2 M) Next, 3 eq of DIC are added (see Notes and 5) The reactor is shaken for h after which the solution is drained The resin is washed three times each with DMF and DCM. With manual solid phase synthesis, the completion of the coupling can be evaluated with a Kaiser test, where the result should be negative (clear solution—Fig 2d) If a positive result is obtained, the coupling needs to be repeated by incubating again with 3 eq of the amino acid and the coupling reagents In this synthesis, elongation of the molecules took place by using Fmoc-Lys(biotin)-OH, Fmoc-Ahx-OH (see Note 6), Fmoc-Tyr(but)-OH, and Fmoc-Leu-OH (for DCG-04); Fmoc-Pra-OH (see Note 7), Fmoc-Ahx-OH, FmocTyr(but)-OH, and Fmoc-Leu-OH (for SV38) by repeating steps 3–4 for each amino acid The Fmoc group is removed by using 20% piperidine as in step The warhead ((2S, 3S)-ethyl-2,3-epoxysuccinate) is attached in a similar manner as the amino acids, but by making use of different coupling reagents: 3 eq HBTU and 6 eq DIEA. To this end, 24 mg of the warhead is mixed with 54 mg of HBTU in 0.75 mL DMF. To this mixture, 53 μL DIEA is added to preactivate the carboxylic acid After min, the mixture is added to the resin The final cleavage is performed in 95/2.5/2.5 TFA/TIS/ H2O (see Note 8) Add the cleavage cocktail to the resin so 262 Tim Van Kersavond et al that it just covers the resin Let it sit for h without shaking Drain the solution in a 10 mL round-bottom glass flask Wash the resin twice with a small amount of 95% TFA cleavage solution and collect in the glass flask The combined fractions may be precipitated in cold diethyl ether This can yield a sufficiently pure product for further usage 10 Purification by HPLC is preferred For this, the precipitation step may be skipped and the eluted cleavage solution is concentrated using a rotary evaporator 11 The crude product is dissolved in DMSO and purified by reversed phase HPLC (C18 column) The combined fractions are dried by lyophilization The product will appear as a fluffy white solid Yields may vary, but in our hands, yields up to 35% after purification have been achieved 3.2 Biochemical Usage of the Probes In principle, a cell lysate or tissue homogenate in any buffer of pH 5.5 and mM DTT can be used as long as it is compatible with the protease of interest (see also Note 9) Here, we use lysates from the macrophage cell line RAW264.7 and homogenates of rat liver Incubate RAW264.7 cells with 5–10 volumes of lysis buffer A on ice for 30 min with occasional vortexing Cut rat liver in small pieces and lyse by douncing on ice in lysis buffer B Centrifuge both lysates in a microcentrifuge at 15,000 × g at °C for 20 min to clear the lysate Collect the supernatant and determine the protein concentration by a Bradford or similar assay In a typical experiment, lysate is diluted to mg/mL and 50 μg total protein is used for a labeling reaction Add DCG-04 or SV38 from a 100 μM DMSO stock (1 μM final concentration, 1% DMSO) A control reaction without probe or pretreated with appropriate protease inhibitors should be used Incubate at room temperature for 30 min (see Note 9) For probe SV38, a buffer exchange is necessary, as the introduction of a fluorophore by click chemistry occurs best at neutral pH. This can be done by a protein precipitation step (e.g., acetone precipitation or CHCl3/MeOH precipitation) or by a small gel filtration column, used according to the procedure provided by the manufacturer and equilibrated with a buffer compatible with click chemistry (e.g., PBS) To initiate the click chemistry, add to your eluate of the spin column or to your resolubilized protein pellet: 0.25 μL click Activity-Based Probes for Proteases 263 chemistry solution IV, 0.5 μL click chemistry solution III, μL click chemistry solution I, and μL click chemistry solution II. Mix and incubate in the dark at room temperature for h 10 After the labeling step, add Laemmli buffer to the samples, boil at 90 °C for min, and separate by SDS-PAGE 11 For detection of biotinylated probes by biotin blot, follow steps 12–17 For fluorescent detection, proceed to step 18 12 Transfer the proteins in the gel to nitrocellulose or PVDF membrane 13 Block with blocking buffer 14 Wash the blot three times several minutes with washing buffer 15 Incubate with streptavidin-HRP (1:3500 dilution in PBS; see Note 10) 16 After h incubation, wash the blot three times for 15 min with washing buffer 17 Incubate the blot with ECL substrate and develop using any method that detects luminescence—this can be traditional photographic film or an imaging system such as the Odyssey® Fc (LI-COR Biosciences) as used here 18 Detection of fluorescently labeled proteins can be done directly from the gel—either in the glass or from the wet gel slab using an appropriate fluorescent scanner, such as a Typhoon™ FLA9500 as used here 19 Typical results are depicted in Fig. 4 Some lysates or cells will have endogenously biotinylated proteins, which can be easily detected in the control samples without probe incubation (Fig. 4a, lane 2) The active cathepsin proteases typically appear as multiple bands between 20 and 35 kDa Naturally, with fluorescent detection, the endogenous biotinylated proteins will not be visible However, click chemistry can give rise to some background due to the excess of fluorophore reagent Note that the click chemistry at pH 5.5 does not yield visible cathepsin bands (Fig. 4b, lane 1), illustrating the need for a buffer exchange In conclusion, this chapter describes a simple synthetic protocol toward ABPs using building blocks that are all commercially available The synthesis itself should be feasible for many bioorganic or biochemistry laboratories or for any research institute with a peptide synthesis facility We here illustrated the applicability of ABPs by gel-based detection, which allows a simple visualization of active proteases using basic biochemistry equipment More sophisticated analyses are also possible and include the use of tandem mass spectrometry for protease target identification, 264 Tim Van Kersavond et al Fig (a) Western Blot detection of DCG-04-labeled cathepsins in rat liver homogenate (lanes and 2) and lysate of the mouse macrophage RAW 264.7 cell line (lanes and 4) Cathepsin bands between 22 and 35 kDa can be observed only in samples treated with probe while some background of endogenous biotinylated- proteins with higher molecular weight can be seen regardless of probe addition For both lysates, a similar pattern was obtained as in Yang et al [13] (b) Fluorescence detection of SV38-labeled cathepsins in rat liver homogenate after click chemistry Prior to click chemistry, lanes and have been subjected to a buffer exchange to phosphate-buffered saline (pH 7.4) using a desalting column fluorescent microscopy, inhibitor screening using fluorescent polarization, and detection using protein microarray More information and background about such applications can be found in some recent reviews [3, 4] 4 Notes KOH does not readily dissolve in dry EtOH and may need vigorous stirring for an extended amount of time The reaction turns into a viscous, white mixture In our experience, the product only ionizes well in the negative mode of electrospray-ionization due to the presence of a free carboxylic acid Values for 1H NMR can be found in Chehade et al [12] Fmoc-Lys(biotin)-OH does not readily dissolve in DMF. As an alternative solvent, N-methyl pyrrolidone (NMP) may be used as well as slight heating in a glass tube prior to addition to the resin Other coupling reagents than DIC/HOBt may be used as well We here chose for this combination, since it is one of the least expensive Activity-Based Probes for Proteases 265 We experienced that Fmoc-Ahx-OH does not couple as efficiently as other amino acids and may therefore need a double coupling step Even with a free amino group, the propargylglycine (Pra) residue results in a negative Kaiser test Hence, the success of the next coupling (Fmoc-Ahx-OH) cannot be monitored by Kaiser test and we therefore recommend performing a double coupling of Fmoc-Ahx-OH (see also Note 6) Caution: TFA is corrosive and contact with the skin should be avoided Note that this step, as all other steps with volatile solvents and chemicals, should be performed in a ventilated chemistry hood For labeling of other proteases by different types of probes, use a lysate buffer in which your protease targets are active Avoid the usage of Tris buffer as well as the detergent CHAPS and high concentrations of SDS, as these inhibit detection by click chemistry See Yang et al [14] Also make sure to use a sufficient sample amount to reach the detection limit of your readout method (either Western blot or fluorescence detection) When other probes are used, the labeling conditions may need to be optimized, including probe concentration, labeling time, pH, and lysis method 10 Streptavidin-HRP from other vendors could be more or less sensitive and the dilution may need to be optimized Acknowledgment We acknowledge funding by the Deutsche Forschungsgemeinschaft DFG and the Ministerium für Innovation, Wissenschaft und Forschung des Landes Nordrhein-Westfalen References Cravatt BF, Wright AT, Kozarich JW (2008) Activity-based protein profiling: from enzyme chemistry to proteomic chemistry Annu Rev Biochem 77:383–414 Heal WP, Dang THT, Tate EW (2011) Activity-based probes: discovering new biology and new drug targets Chem Soc Rev 40(1):246–257 Serim S, Haedke U, Verhelst SHL (2012) Activity-based probes for the study of proteases: recent advances and developments ChemMedChem 7(7):1146–1159 Sanman LE, Bogyo M (2014) Activity-based profiling of proteases Annu Rev Biochem 83:249–273 Haedke U, Kuttler EV, Vosyka O, Yang Y, Verhelst SHL (2013) Tuning probe selectivity for chemical proteomics applications Curr Opin Chem Biol 17(1):102–109 Greenbaum D, Medzihradszky KF, Burlingame A, Bogyo M (2000) Epoxide electrophiles as activity-dependent cysteine protease profiling and discovery tools Chem Biol 7(8):569–581 Barglow KT, Cravatt BF (2004) Discovering disease-associated enzymes by proteome reactivity profiling Chem Biol 11(11):1523–1531 Haedke UR, Frommel SC, Hansen F, Hahne H, Kuster B, Bogyo M, Verhelst SH (2014) Phosphoramidates as novel activity-based 266 Tim Van Kersavond et al probes for serine proteases Chembiochem 15(8):1106–1110 Hanada K, Tamai M, Yamagishi M, Ohmura S, Sawada J, Tanaka I (1978) Isolation and characterization Of E-64, a new thiol protease inhibitor Agric Biol Chem 42(3):523–528 10 Yamamoto D, Matsumoto K, Ohishi H, Ishida T, Inoue M, Kitamura K, Mizuno H (1991) Refined x-ray structure of papain.E-64-c complex at 2.1-A resolution J Biol Chem 266(22):14771–14777 11 Speers AE, Cravatt BF (2004) Profiling enzyme activities in vivo using click chemistry methods Chem Biol 11(4):535–546 12 Chehade KAH, Baruch A, Verhelst SHL, Bogyo M (2005) An improved preparation of the activity-based probe JPM-OEt and in situ applications Synthesis-Stuttgart 2:240–244 13 Yang Y, Hahne H, Kuster B, Verhelst SH (2013) A simple and effective cleavable linker for chemical proteomics applications Mol Cell Proteomics 12(1):237–244 14 Yang Y, Yang X, Verhelst SHL (2013) Comparative analysis of click chemistry mediated activity-based protein profiling in cell lysates Molecules 18(10):12599–12608 Index A F Acetylation yield quantification�������������������� 9–14, 18, 19, 27 [Acetyl-1-14C]-coenzyme A ([14C]-Ac-CoA)����������������2–7 Acetyl-coenzymeA (Ac-CoA)�������������� 1, 2, 4–7, 10, 11, 13, 14 Acetyltransferase�������������������������������������������������������� 1, 9–14 Activity-based probes (ABPs)�������������������������� 244, 255–265 Activity profiling��������������������������������������������������������������255 ADAM10��������������������������������������������������������� 174, 245, 251 Fluorescence microscopy������������������������������������������ 229, 232 Fluorescent imaging���������������������������������������������������������245 Formalin-fixed, paraffin-embedded (FFPE)��������� 105–111, 113 B Biocytinamide�����������������������������������137, 139, 140, 142, 143 Biomarkers��������������������������������������������������������������� 205, 206 C Carboxypeptidases������������������������������������ 115–132, 137, 139 Catalytic activity������������������������������������������������� 2, 3, 18, 243 Chemical biology�������������������������������������������������������������229 Chemical proteomics��������������������������������������������������������255 Cleavage sites�����������������������������������36, 54, 70, 106, 109, 113, 130, 135, 148, 168, 171–174, 179–181, 184, 190, 191, 197–204, 206–210, 212, 228–230, 244 Cleavage site specificities��������������������������106, 171, 172, 174, 179–181, 191 Click chemistry���������������������������������������� 257, 258, 262–265 Combined fractional diagonal chromatography (COFRADIC)��������������������18, 51–74, 115–132, 184 Collagenase����������������������������������������������������������������������243 Confocal microscopy���������������������������������������� 218, 221, 222 C-terminomics�����������������������������������������������������������������131 D Databases�������������������������������������20, 27, 36, 44, 45, 69, 70, 77, 83, 89, 99–102, 127, 128, 131, 156, 157, 167, 178, 179, 181, 190, 200, 203, 204, 206, 207, 210 Degradomics������������������������������������������������ 54, 62, 148, 171 5,5’-Dithiobis-(2-nitrobenzoic acid) (DTNB)���������������2, 9–14 Doublet N-terminal oriented proteomics (dN-TOP) approach����������������������������������������������� 77–85, 87, 89 E EnCOUNTer��������������������������������������������������������� 26–28, 31 Enzymatic labeling���������������������������������������������������135–143 Enzyme activity�������������������������������������������������������� 9, 64, 77 Enzyme assay�����������������������������������������������������������������9, 13 H Heat maps�����������������������������������������174, 179, 190, 192, 194 High content imaging�������������������������������������� 218, 224, 237 High content screening (HCS)���������������� 232–233, 235–238 I In-solution labeling����������������������������������������������������������185 Intact protein-based cleavage site discovery������������� 158, 159 Isobaric labeling��������������������������������������������������������171–181 iTRAQ��������������������������������������������19, 71, 92–101, 165, 181 L LC-MS See Liquid chromatography-mass spectrometry (LC-MS) Liquid chromatography-mass spectrometry (LC-MS)�������������������20, 37, 47, 59–61, 66–69, 74, 78, 81–83, 87, 98, 109, 112, 113, 116–117, 120, 125–126, 146, 147, 149, 155, 164, 171, 173–176, 178, 179, 181, 184–186, 189, 197, 198, 200, 202–203, 259 M Mascot distiller�������������������� 20, 26–28, 31, 69, 120, 126, 127 Mass spectrometry (MS)������������������������� 19, 20, 25–28, 30, 37, 38, 43, 44, 47, 59–61, 66–69, 78, 81–83, 87, 109, 113, 116–118, 120, 125–126, 128, 136, 142, 143, 145–147, 149, 155–158, 164, 165, 171, 173–176, 178, 179, 181, 184–186, 188, 189, 194, 197, 198, 200, 202–203, 205, 259 Mature protein N-terminus������������������������������������������26, 28 Michaelis-Menten������������������������������������������������������������245 MS See Mass spectrometry (MS) Multiplexed measurement���������������������������������������� 244, 245 N Neo-N-termini������������������������������������� 35, 36, 53, 70, 74, 93, 106, 129, 173, 184, 185, 190, 193 Network analysis��������������������������������������������������������������243 NTA yield��������������������������������������������������������������� 26, 27, 31 N-terminal acetylation������������2–7, 9, 10, 17–31, 35, 44, 113, 156 Oliver Schilling (ed.), Protein Terminal Profiling: Methods and Protocols, Methods in Molecular Biology, vol 1574, DOI 10.1007/978-1-4939-6850-3, © Springer Science+Business Media LLC 2017 267 Protein Terminal Profiling: Methods and Protocols 268 Index N-terminal acetyltransferase (NAT)����������������������� 1–6, 9–14 N-terminal modification����������������������������17, 18, 27, 28, 45, 48, 51, 77, 78, 89 N-termini��������������������������������� 1, 10, 17, 35, 51, 77, 92, 106, 116, 135, 172, 184, 203, 256 N-terminome����������������������������������������������18, 19, 39, 47, 79 N-terminomics��������������������������������51, 52, 77–85, 87, 89, 99 O Oligopeptides������������������������������������������������������� 3–7, 11–13 Open source�������������������������������������������������� 88, 99, 156, 206 P Peptide acetylation������������������������������������������������� 1, 2, 6, 69 Peptides������������������������������������ 2, 10, 18, 35, 51, 78, 92, 106, 115, 135, 146, 172, 184, 197, 205, 244, 255 P81 filter disks������������������������������������������������������������������5, Positional proteomics���������������������������������������������������������36 Profiling of protein C-termini by enzymatic labeling (ProC-TEL)�������������������������������������������������135–143 Proteases����������������������������������������� 36, 51, 91, 115, 136, 145, 171, 183, 197, 206, 215, 228, 243, 255 Protease specificity profiling���������������54, 171–181, 184–190, 192–194, 207–208 Protease substrates������������������������������������37, 51–74, 92–102, 145–169, 171, 183, 184, 255 Protein C-termini�������������� 115, 116, 118, 128, 131, 135–143 Protein modification��������������������������������������1, 9, 51, 66, 106 Protein-protein interaction (PPI)����������������� 2, 228–230, 236 Proteogenomics������������������������������������������������������������77, 79 Proteolysis����������������������������������������������48, 93, 102, 106, 115, 118, 145–148, 150, 156–158, 163, 180, 187, 191, 197, 205, 207, 216, 217, 222, 223, 255 Proteome-derived library�������������������������� 172–174, 197–204 Proteomics���������������������������������� 30, 36, 70, 77, 95, 109, 116, 135, 163, 171, 184, 197, 205 S Screening assays�������������������������216–222, 224, 227, 236–238 Secretomes������������������������������������������������������������������92–102 Sheddase���������������������������������������������������������������������������243 Singular value decomposition�������������������������������������������243 Solid phase peptide synthesis (SPPS)���������������������� 256, 257, 259–262 Stable isotope labeling of amino acids in cell culture (SILAC)�����������������������������������19, 53, 55–56, 64, 71, 92–96, 98–101, 129, 146, 159–161, 165 Stable-isotope protein N-terminal acetylation quantification (SILProNAQ)��������������������������������������������������17–31 Strong cation exchange (SCX)���������������������������������18, 22, 25, 26, 30, 47, 53, 57–58, 66, 70, 71, 73, 74, 113, 116–117, 119, 122, 128–131 Substrates����������������������������������� 1, 9, 18, 36, 51, 78, 91, 116, 136, 145, 171, 184, 197, 206, 217, 229, 244, 255 T TACE�������������������������������������������������������������������������������243 Terminal amine isotope labeling of substrates (TAILS)������������������������������18, 36–38, 43–45, 47, 48, 52, 92–100, 106, 203 Terminal modifications������������������������������17, 18, 27, 28, 45, 48, 51, 77, 78, 89, 143 Thiol detection�������������������������������������������������������������������10 Thiol quantification������������������������������������������������������������10 TMPP derivatization��������������������������������������� 78, 80–82, 89 Translocation�����������������������������227–230, 232–236, 238, 239 ... NatF, Oliver Schilling (ed.), Protein Terminal Profiling: Methods and Protocols, Methods in Molecular Biology, vol 1574, DOI10.1007/978-1-4939-6850-3_1, â Springer Science+Business Media LLC 2017... N -terminal acetylation assay In: Schilling O (ed) Protein terminal profiling: methods and protocols, Methods in molecular biology, vol 1574 Springer Science+Business Media LLC, New York, NY 12 Ellman... post-translational Oliver Schilling (ed.), Protein Terminal Profiling: Methods and Protocols, Methods in Molecular Biology, vol 1574, DOI10.1007/978-1-4939-6850-3_3, â Springer Science+Business Media LLC 2017