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

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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
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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
  2 DTNB-Based Quantification of In Vitro Enzymatic N-Terminal
Acetyltransferase Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Håvard Foyn, Paul R. Thompson, and Thomas Arnesen
  3 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
  4 Profiling of Protein N-Termini and Their Modifications in Complex Samples . . . . .
Fatih Demir, Stefan Niedermaier, Jayachandran N. Kizhakkedathu,
and Pitter F. Huesgen
  5 Protease Substrate Profiling by N-Terminal COFRADIC . . . . . . . . . . . . . . . . .
An Staes, Petra Van Damme, Evy Timmerman, Bart Ruttens,
Elisabeth Stes, Kris Gevaert, and Francis Impens
  6 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
  7 Multidimensional Analysis of Protease Substrates and Their Cellular
Origins in Mixed Secretomes from Multiple Cell Types . . . . . . . . . . . . . . . . . .
Pascal Schlage and Ulrich auf dem Keller
  8 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
  9 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

1

9

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


xii

Contributors

Joanna Tucher  •  AG Systematic Proteome Research & Bioanalytics,
Institute for Experimental Medicine, Christian-Albrechts-Universität zu Kiel, Kiel,
Germany
Steven H.L. Verhelst  •  Leibniz Institute for Analytical Sciences ISAS, e.v.,

Dortmund, Germany; Department of Cellular and Molecular Medicine,
KU Leuven—University of Leuven, Leuven, Belgium
Robert Vidmar  •  Department of Biochemistry and Molecular and Structural Biology,
Jožef Stefan Institute, Ljubljana, Slovenia; International Postgraduate School Jožef
Stefan, Ljubljana, Slovenia
Matej Vizovišek  •  Department of Biochemistry and Molecular and Structural Biology,
Jožef Stefan Institute, Ljubljana, Slovenia
Benoit Westermann  •  BioOrganic Mass Spectrometry Laboratory (LSMBO), IPHC,
CNRS—UdS, UMR 7178, University of Strasbourg, Strasbourg, France
Guoqiang Xu  •  Jiangsu Key Laboratory of Translational Research and Therapy
for Neuro-Psycho-Diseases and College of Pharmaceutical Sciences,
Soochow University, Suzhou, Jiangsu, P.R., China; Jiangsu Key Laboratory of
Preventive and Translational Medicine for Geriatric Diseases, Soochow University,
Suzhou, Jiangsu, P.R., China
Yong Xu  •  Department of Electrical and Computer Engineering, Wayne State University,
Detroit, MI, USA


Chapter 1
[14C]-Acetyl-Coenzyme A-Based In Vitro
N-Terminal Acetylation Assay
Adrian Drazic and Thomas Arnesen
Abstract
N-terminal acetylation is one of the most abundant co- and posttranslational protein modifications,
conserved from prokaryotes to eukaryotes. The functional consequences of this modification are manifold,
ranging from protein folding, stability, and interaction to subcellular localization. We describe here an
isotope-labeled [14C]-acetyl-Coenzyme A-based acetylation assay, allowing the determination of weak
catalytic activities of NATs in vitro. It allows the use of purified recombinant enzymes from Escherichia coli,
or co-immunoprecipitated enzymes from various organisms, as well as the determination of the in vitro
activity of various cell lysates. Although marked as an old-fashioned biochemical approach, it is the ideal

method to hunt for catalytic activities and defining peptide specificities of new potential N-terminal acetyltransferase candidates.
Key words N-terminal acetylation, N-terminal acetyltransferase (NAT), Acetyl-CoA (Ac-CoA),
[Acetyl-1-14C]-coenzyme A ([14C]-Ac-CoA), P81 filter disks, Oligopeptide, Peptide acetylation,
Catalytic activity

1  Introduction
N-terminal (Nt-) acetylation is among the most common protein
modifications in eukaryotes [1]. It is conserved from prokaryotes
to humans, whereby the abundance increases with the complexity
of the organism [1–5]. In human cells 80–90% of all newly synthesized and soluble polypeptides become co- or posttranslationally
Nt-acetylated [1]. The transfer of an acetyl moiety from acetylCoenzyme A (Ac-CoA) to the N-terminus is catalyzed by several
N-terminal acetyltransferases (NATs). Six different enzymes,
NatA–NatF, have been identified so far in humans with a sequencespecific activity [3]. Recently, a seventh NAT, NatG, was identified
in the plant Arabidopsis thaliana [6]. The NAT activity is based on
one catalytic subunit and up to two auxiliary subunits, responsible
for ribosome association and substrate specificity [7]. NatA–NatE
are conserved from Saccharomyces cerevisiae to humans. NatF,

Oliver Schilling (ed.), Protein Terminal Profiling: Methods and Protocols, Methods in Molecular Biology, vol. 1574,
DOI 10.1007/978-1-4939-6850-3_1, © Springer Science+Business Media LLC 2017

1


2

Adrian Drazic and Thomas Arnesen

NatG, and further NAT isoforms exist additionally in distinct
multicellular eukaryotes [3, 6]. The molecular consequences of

being Nt-acetylated are manifold for polypeptides. Nt-acetylation
can affect protein folding and stability, subcellular localization, and
protein-protein interaction [7]. Recently determined structures of
Naa40 (NatD), Naa50 (NatE), and the Naa10–Naa15 (NatA)
complex, combined with mutational activity studies, have given
an insight into the catalytic mechanisms that stand behind
Nt-acetylation [8–10].
There are several different methods available to determine the
catalytic activity of NATs. One approach is the “classical” molecular
biological use of antibodies, specifically detecting and binding to the
acetylated N-termini of the protein of interest [11]. Only few specific antibodies for detecting Nt-acetylation have been used to date
in studies, most probably due to lack of specificity and potential high
costs of purchasing them. Nevertheless, the big advantage of an antibody-based approach is the possibility of detecting the acetylation
status of a protein or peptide in vivo. Another detection method uses
the high-performance liquid chromatography (HPLC). The advantage of this method is that the differential Nt-acetylation status of
the investigated peptide can be quantitatively determined. In parallel, the ratio of the substrate Ac-CoA and the product CoA can also
be determined spectrophotometrically, giving two independent ways
of calculating the amount of Nt-acetylated product and therefore
the catalytic activity of a NAT [12]. The disadvantages are that the
HPLC approach is time-consuming, taking around 1 h for analyzing
one sample, and it is limited to in vitro acetylation. The DTNB assay
detects spectrophotometrically Nt-acetylation and is suitable for
high-throughput screening. The DTNB (5,5′-dithiobis-(2-nitrobenzoic acid)) assay is described in detail in this volume [13]. In
brief, the DTNB assay can be performed in a 96-well plate allowing
a high number of samples in a short time. It also allows the use of
small molecules as substrates and is not limited to peptides or proteins. Nevertheless, it depends on high enzyme and substrate concentrations and therefore lacks sensitivity. A compromise between
the HPLC and DTNB assay is a [14C]-Ac-CoA-based acetylation
assay. It allows scanning through a lot of samples in a short time with
precise and reproducible results. It also allows the quantitative analysis of the product formation. Further, whole cell lysates can be used
as starting material. The biggest advantage of this approach is the

high sensitivity with the possibility of detecting very weak acetylation events. However, some drawbacks are that the [14C]-Ac-CoAbased acetylation assay is not suitable for small molecules as substrates
and the necessity of special waste treatment for the radioactive
material.


Radioactive Nt-acetylation assay

3

2  Materials
The acetylation reaction requires a thermomixer. Determining the
protein concentration for recombinant purified enzymes requires a
spectrophotometer. The radioactivity/concentration of the incorporated [14C] is determined by a Perkin Elmer Tri-Carb 2900TR
Liquid Scintillation Analyzer.
1.Acetylation buffer: 50 mM Tris/HCl, pH 7.4, 1 mM dithiothreitol (DTT), 1 mM EDTA, and 10% (v/v) glycerol. The buffer is stored at 4 °C without DTT and DTT is freshly added
before use (see Note 1).
2.
Acetyl coenzyme A (“hot”), [acetyl-1-14C]: 50 μCi
(1.85 Mbq) (see Note 2).
3.Acetyl coenzyme A (“cold”), nonisotope labeled (see Note 3).
4.Oligopeptides: 12–24-mers (has to contain at least one positively charged amino acid) synthesized at least 90% purity, dissolved in H2O to a final concentration of 1–5 mM.
5.NAT/enzyme (see Note 4).
6.P81 Phosphocellulose filter squares (see Note 5).
7.P81 Filter washing buffer: 10 mM HEPES, pH 7.4.
8.Alpha/beta liquid scintillation cocktail: Ultima Gold F.
9.Scintillation vials: 6 mL high-density polyethylene (HDPE)
Omni-Vials with polypropylene caps.

3  Methods
For determining the catalytic activity of a NAT or search for its

substrate specificities, the NAT has to be purified as recombinant
enzyme prior to the actual acetylation assay as described in [14].
NATs, endogenous or tagged isoforms, can also be pulled down
from transfected or untransfected cells by immunoprecipitation
(IP) via NAT/tag-specific antibodies and affinity beads [15]. The
protein concentration for recombinant purified enzymes is determined spectrophotometrically by measuring absorbance at 280 nm.
The estimated concentration of pulled-down enzyme via IP has to
be determined via Western blot analysis.
3.1  Calculation
of the [Acetyl-1-­14C]CoA Concentration

To determine the molarity of [acetyl-1-14C]-CoA one has to
count in its specific activity and its stock concentration.
1 Ci (Curie) = 3.7 × 1010 Bq (Bequerel);
1 Bq = 60 dpm (desintegrations per minute).


4

Adrian Drazic and Thomas Arnesen

Here is an example for the calculations:
1.Specific activity (according to the supplier): 60mCi/mmol.
2.Stock concentration: 0.02mCi/mL.
3.Total radioactivity: 50Ci (1.85MBq) in 2.5mL.
total radioactivity ( mCi ) or volume ( mL )
4. activity per L =
ổ mCi ử
concentration ỗ
ữ

ố mL ứ

= 20 nCi / L
(also defined by the stock concentration)
stock concentration 0.02 mCi / mL
=
specific activity
60 mCi / mmol
0.02 Ci / L
=
= 0.333 mmol / L = 333 M
60 Ci / mmol

5. Molarity =

3.2 The Compilation
oftheAcetylation
Reaction



All buffers and reaction components have to be completely thawed
before use. The typical reaction volume is 20L for a single time
point and concentration. Typical concentrations of the components for one reaction:
1.200M oligopeptide (see Note 6).
2.60nCi [14C]-Ac-CoA (60nCi=2220Bq=133200dpm).
3. 10nM NAT (for recombinant enzymes, the concentration can
vary between 10 and 100nM; enzyme concentrations are also
variable and sometimes not determinable for immunoprecipitated enzymes or whole cell lysates).
4. Addition of nonisotope labeled (cold) Ac-CoA to adjust the

total Ac-CoA concentration to the desired level. An example
for the calculation for a final Ac-CoA concentration of 200M
in 20L final volume.
Stock concentration hot Ac-CoA: 333M; 60nCi=3L.
Stock concentration cold Ac-CoA: 1000M.
Calculations:





c [ hot Ac - CoA] =

V ( final )

=

3 m L 333 m M
= 50 m M
20 m L

c [ total Ac - CoA] = c [ hot Ac - CoA] + c [ cold Ac - CoA]
đ c [ cold Ac - CoA] = 200 m M - 50 m M = 150 m M
c [ cold Ac - CoA] =



V ( hot ) c ( stock )

V ( cold ) c ( stock )

V ( final )

=





y m L 1000 m M
= 150 m M =
20 m L

đ y ộởV ( cold ) ựỷ = 3 m L







Radioactive Nt-acetylation assay

5

This means that a 1:1 mixed stock solution can be prepared
and 6 μL of the total Ac-CoA mix has to be added to a 20 μL reaction volume to get a final Ac-CoA concentration of 200 μM.
3.3  The Acetylation
Reaction

1.Add fresh DTT to the acetylation buffer.

2.Transfer the precalculated volume of acetylation buffer into a
1.5 mL Eppendorf tube. Total reaction reaction volume is
20 μL.
3.Add the oligopeptide (0.8 μL, 200 μM).
4.Add nonisotope labeled Ac-CoA, if desired (see Subheading
3.2, step 4).
5.Add [14C]-Ac-CoA (3 μL, 60 nCi = 50 μM).
6.Start the reaction by adding the enzyme (NAT) to a final concentration of 100 nM and a final reaction volume of 20 μL (see
Note 7).
7.Transfer tubes to a Thermomixer block.
8.Incubate at 37 °C for 1 h, shaking at 1000 rpm.
9.Transfer the reaction from the tube to a 1.5 × 1.5 cm prelabeled P81 phosphocellulose filter disk (reaction stop).
10.Transfer up to ten filter disks into a 50 mL Falcon tube filled
with 20 mL P81 filter washing buffer (10 mM HEPES, pH
7.4); only the positively charged oligopeptide is binding to the
P81 filter disks, unincorporated [14C]-Ac-CoA will be washed
off.
11.Gently shake or roll tubes for 5 min at room temperature (see
Note 8).
12.Remove carefully the washing buffer by decanting (see Note
9).
13.Add fresh washing buffer into the Falcon tube.
14. Repeat steps 11–13 twice and remove washing buffer.
15.Take out filter papers and carefully separate them with
tweezers.
16.Put filter disks on a dry paper and let them dry on air for ca.
20 min (see Note 10).
17. Transfer the filter disks into scintillation vials and push them to
the bottom of the vials (see Note 11).
18. Add 5 mL of Ultima Gold F scintillation cocktail into each vial

and lock it with pre-labeled caps.
19.Determine the amount of incorporated [14C]-acetyl by measuring the radioactivity via a liquid scintillation analyzer (measuring time: 2 min for satisfying signal-to-noise ratio) (see
Note 12).


6

Adrian Drazic and Thomas Arnesen

4  Notes
1.The acetylation buffer is variable. Its composition and the pH
are dependent on the stability of the enzyme in the respective
buffer and the optimal reaction conditions can differ between
different NAT enzymes. The optimal conditions have to be
experimentally determined. However, the buffer described
here works for most known NATs purified from E. coli.
2.Radiolabeled Ac-CoA can also be purchased as [3H]-Ac-CoA
in case your radioactive license restricts you to this isotope. Of
note, 3H has a lower energy compared to 14C and thus a lower
efficiency in liquid scintillation counting.
3. In principle, it is also possible to increase the amount [14C]-AcCoA added to the reaction to increase the total Ac-­CoA concentration. Due to the high cost of radiolabeled Ac-CoA, this
is not recommended. If “hot” and “cold” Ac-CoA are simultaneously used in the assay, they should be both blended in a
mastermix. This assures the same relative amount of detectable
“hot” Ac-CoA in all samples and allows a calculation of the
incorporated acetyl.
4.The NAT enzyme can originate from purified enzyme from E.
coli expression systems or immunoprecipitated enzyme from
various cell systems. The NAT enzyme can also be contained in
whole cell lysates or a particular subcellular fraction of a cell
lysate, which can be used as input for the acetylation reaction.

5.Be aware that the amount of suppliers for P81 phosphocellulose filter disks has dramatically decreased in the last years. The
production has been stopped by most producers. Nevertheless,
a few suppliers have the filters still in stock.
6.The concentration of oligopeptide is variable. Some oligopeptides are aggregation prone in certain acetylation conditions
(pH, salt concentration). A dilution 1:10 is in most cases helpful (20 μM final concentration). Due to the high sensitivity of
the method a reliable detection is still possible.
7.In case whole cell lysates are used as starting material, which
already contain the enzyme of interest, the order of components added to the reaction mixture has to be adjusted. Then,
Ac-CoA is added in the final step to start the acetylation
reaction.
8.It is absolutely sufficient to keep the individual washing steps
to 5 min shaking or rolling. The shaking or rolling should be
gentle, otherwise one risks to tear the filter disks apart.
Extended washing steps should be avoided due to disintegration of the filter disks. It is also important not to put too many
filter disks in one Falcon tube. This diminishes the washing
efficiency and increases the background signal.


Radioactive Nt-acetylation assay

7

9. Be aware that your washing buffer contains all unincorporated
[14C]-Ac-CoA and thus has to be treated as special radioactive
waste.
10.Older protocols state the use of acetone to dry the filters after
the last washing step. This is possible, but we observed in our
hands an increased disintegration of the filter disks. Thus, we
prefer to take the time and air dry the filters spread out on a
paper before adding the scintillation cocktail.

11. The scintillation counter detects light pulses that are indirectly
generated by ionizing radiation. Therefore, it is important that
the vials are not labeled on the side for full counting efficiency.
The vials can be marked on the top of the caps.
12.As it applies for all experiments, the results stand and fall with
the controls. This also holds true for the radiolabeled Ac-CoA
assay. The controls should include samples without enzyme, as
well as samples without the oligopeptide. Weak and spontaneous acetylation reaction (Nt-acetylation; lysine acetylation) can
occur in certain conditions, especially in lower pH ranges (<6).
The determined control values should then be subtracted as
background from the actual samples. To determine the maximal possible radioactivity/dpm value (100% incorporation), a
vial with scintillation cocktail and 60 nCi [14C]-Ac-CoA without filter disk can be measured.

Acknowledgments
The authors gratefully acknowledge support by research grants from
the Deutsche Forschungsgemeinschaft DFG (grant DR998/2-1 to
AD), the Norwegian Cancer Society (to T.A.), The Bergen Research
Foundation BFS (to T.A.), the Research Council of Norway (grant
230865 to T.A), and the Western Norway Regional Health Authority
(to T.A.).
References
1. Arnesen T, Van Damme P, Polevoda B, Helsens
K, Evjenth R, Colaert N, Varhaug JE,
Vandekerckhove J, Lillehaug JR, Sherman F,
Gevaert K (2009) Proteomics analyses reveal the
evolutionary conservation and divergence of
N-terminal acetyltransferases from yeast and
humans. Proc Natl Acad Sci USA 106(20):8157–
8162. doi:10.1073/pnas.0901931106
2. Soppa J (2010) Protein acetylation in archaea,

bacteria, and eukaryotes. Archaea 2010.
doi:10.1155/2010/820681

3.Van Damme P, Hole K, Pimenta-Marques A,
Helsens K, Vandekerckhove J, Martinho RG,
Gevaert K, Arnesen T (2011) NatF contributes
to an evolutionary shift in protein N-terminal
acetylation and is important for normal chromosome segregation. PLoS Genet 7(7):e1002169.
doi:10.1371/journal.pgen.1002169
4. Goetze S, Qeli E, Mosimann C, Staes A, Gerrits
B, Roschitzki B, Mohanty S, Niederer EM,
Laczko E, Timmerman E, Lange V, Hafen E,
Aebersold R, Vandekerckhove J, Basler K,


8

Adrian Drazic and Thomas Arnesen

Ahrens CH, Gevaert K, Brunner E (2009)
Identification and functional characterization of
N-terminally acetylated proteins in Drosophila
melanogaster. PLoS Biol 7(11):e1000236.
doi:10.1371/journal.pbio.1000236
5. Bienvenut WV, Sumpton D, Martinez A, Lilla
S, Espagne C, Meinnel T, Giglione C (2012)
Comparative large scale characterization of
plant versus mammal proteins reveals similar
and idiosyncratic N-alpha-acetylation features.
Mol Cell Proteomics 11(6):M111.015131.

doi:10.1074/mcp.M111.015131
6. Dinh TV, Bienvenut WV, Linster E, FeldmanSalit A, Jung VA, Meinnel T, Hell R, Giglione
C, Wirtz M (2015) Molecular identification
and functional characterization of the first
Nalpha-acetyltransferase in plastids by global
acetylome profiling. Proteomics 15(14):2426–
2435. doi:10.1002/pmic.201500025
7.Aksnes H, Hole K, Arnesen T (2015)
Molecular, cellular, and physiological significance of N-terminal acetylation. Int Rev Cell
Mol Biol 316:267–305. doi:10.1016/bs.
ircmb.2015.01.001
8. Liszczak G, Arnesen T, Marmorstein R (2011)
Structure of a ternary Naa50p (NAT5/SAN)
N-terminal acetyltransferase complex reveals
the molecular basis for substrate-specific acetylation. J Biol Chem 286(42):37002–37010.
doi:10.1074/jbc.M111.282863
9.Liszczak G, Goldberg JM, Foyn H, Petersson
EJ, Arnesen T, Marmorstein R (2013) Molecular
basis for N-terminal acetylation by the heterodimeric NatA complex. Nat Struct Mol Biol
20(9):1098–1105. doi:10.1038/nsmb.2636


10.Magin RS, Liszczak GP, Marmorstein R
(2015) The molecular basis for histone H4and H2A-specific amino-terminal acetylation
by
NatD. Structure
23(2):332–341.
doi:10.1016/j.str.2014.10.025
11. Hwang CS, Shemorry A, Varshavsky A (2010)
N-terminal acetylation of cellular proteins creates specific degradation signals. Science

327(5968):973–977. doi:10.1126/science.
1183147

12.Evjenth RH, Van Damme P, Gevaert K,
Arnesen T (2013) HPLC-based quantification of in vitro N-terminal acetylation.
Methods
Mol
Biol
981:95–102.
doi:10.1007/978-1-62703-305-3_7
13. Foyn H, Thompson PR, Arnesen T (in press)
DTNB-based quantification of in vitro enzymatic N-terminal acetyltransferase activity.
Methods Mol Biol
14. Van Damme P, Evjenth R, Foyn H, Demeyer K,
De Bock PJ, Lillehaug JR, Vandekerckhove J,
Arnesen T, Gevaert K (2011) Proteome-derived
peptide libraries allow detailed analysis of the substrate specificities of N(alpha)-acetyltransferases
and point to hNaa10p as the post-translational
actin N(alpha)-acetyltransferase. Mol Cell
Proteomics 10(5):M110.004580. doi:10.1074/
mcp.M110.004580
15. Hole K, Van Damme P, Dalva M, Aksnes H,
Glomnes N, Varhaug JE, Lillehaug JR, Gevaert
K, Arnesen T (2011) The human N-alphaacetyltransferase 40 (hNaa40p/hNatD) is conserved from yeast and N-terminally acetylates
histones H2A and H4. PLoS One 6(9):e24713.
doi:10.1371/journal.pone.0024713


Chapter 2
DTNB-Based Quantification of In Vitro Enzymatic

N-Terminal Acetyltransferase Activity
Håvard Foyn, Paul R. Thompson, and Thomas Arnesen
Abstract
We here describe a quick and easy method to quantitatively measure in vitro acetylation activity of not only
N-terminal acetyltransferase (NAT) enzymes, but acetyltransferases using acetyl-coenzyme A as an acetyl
donor in general.
Key words N-terminal acetyltransferase (NAT), Acetyltransferase, Enzyme activity, Enzyme assay,
5,5′-dithiobis-(2-nitrobenzoic acid), Thiol quantification, Thiol detection, Ac-CoA

1  Introduction
N-terminal acetylation is one of the most common protein modifications in eukaryotes [1]. To date, seven different N-terminal acetyltransferases (NatA–NatG) have been shown to be responsible for this
modification. NatA–NatE are ribosome associated and conserved
from yeast to humans [2]. NatF is localized to the Golgi membrane
and acetylates transmembrane proteins in multicellular eukaryotes
[3, 4], whereas NatG is acting inside plant chloroplasts [5]. The
NATs have been linked to human diseases. Several NAT subunits
have been demonstrated to be mostly up-regulated in different cancer types [6] whereas mutations in the catalytic subunit of NatA may
lead to intellectual disabilities and Ogden syndrome [7, 8].
Almost every study of the NATs includes some sort of in vitro
assaying of enzyme activity, whether it is to determine substrate
specificity, activity of mutants, inhibition studies, or others.
Currently, there are a number of methods available for in vitro
quantification of N-terminal acetylation. The high-pressure liquid
chromatography (HPLC)-based method described by Evjenth
et al. [9, 10] is a sensitive method that is compatible with both
recombinant enzyme and immunoprecipitated enzymes. However,
each HPLC run is rather time-consuming and when facing large-­
Oliver Schilling (ed.), Protein Terminal Profiling: Methods and Protocols, Methods in Molecular Biology, vol. 1574,
DOI 10.1007/978-1-4939-6850-3_2, © Springer Science+Business Media LLC 2017


9


10

Håvard Foyn et al.

Fig. 1 Ac-CoA donates the acetyl group in the N-terminal acetylation reaction and a thiol is exposed. This thiol
is able to readily cleave DTNB that yield TNB−. TNB− ionizes to TNB2− in neutral or alkaline pH that yields a light
yellow color and absorbs light at 412 nm. As its formation is in 1:1 stoichiometry to acetylated peptide, it can
be used as an indirect quantification of N-terminal acetylation

scale or multiple assays it can easily take days and weeks until the
samples are analyzed. A radioactivity-based method is another very
sensitive method, but requires specialized labs [11].
Thus, there is a need for a fast, safe and cheap method for
in vitro quantification of N-terminal acetylation.
As the N-terminal acetylation process involves the transfer of
acetyl from acetyl-coenzyme A (Ac-CoA) to the N-termini of peptides, it exposes a thiol-group on CoA. After adding 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), it will readily react with the thiol
that yields 2-nitro-5-thiobenzoate (TNB−) that will ionize to
TNB2− in neutral or alkaline pH (Fig. 1) [12]. This is easily quantified by measuring the absorbance at 412 nm.
The only drawback to the method is lower sensitivity compared with other methods and its incompatibility with immunoprecipitated enzyme because of high background probably caused
by the cysteine-rich antibodies. However, since the consumption
of Acyl-CoA is measured, this method can be used not only for
N-terminal acetylation, but for any acylation reaction where Acyl-­
CoA is consumed.

2  Materials
The acetylation reaction requires purified recombinant enzyme (see
Note 1), a heating block and all samples are analyzed with a spectrophotometer. The samples from our lab are analyzed by an Epoch

microplate spectrophotometer from Biotek.
1.2× Acetylation buffer: 100 mM HEPES-HCl (pH 7.5), 200
mM NaCl, 2 mM EDTA. This is the buffer used in our lab for
hNaa50, but most buffers are compatible with the assay as
long as they contain as low concentration as possible of reducing agents such as DTT (see Note 1).


DTNB-Based Quantification of Acetyltransferase Activity

11

2.Acetyl-CoA: Acetyl-CoA trilithium salt, dissolved in H2O.
3.Acetyl-acceptor: We use custom-made 24-mer oligopeptides
to at least 90% purity, dissolved in H2O.
4. Quenching buffer: 3.2 M guanidinium-HCl, 100 mM sodium
phosphate dibasic (pH 6.8).
5.DTNB buffer: 100 mM sodium phosphate dibasic (pH 6.8),
10 mM EDTA, fresh DTNB is added prior to analysis. 10 mg/
mL of DTNB is added yielding a final concentration of around
3 M which is ample for most assays (see Note 2).

3  Methods
3.1  Substrate Screen

The DTNB assay has many applications, but as an example it may
be used to test if an enzyme has enzymatic activity toward a range
of specific substrates. In this case, the enzymatic activity of hNaa50
against the N-termini of 24-mer oligopeptides containing the start
sequences (one letter amino acid code) MLGP, MLGT, MDEL,
and SESS is tested (see Note 3).

1.Prepare samples with 500 μM substrate peptide and acetyl-­
CoA, 25 μL 2× acetylation buffer and H2O up to a total reaction volume of 50 μL. Three replicates are prepared along with
two negative controls of each assay condition (toward each
substrate peptide). Recombinant hNaa50 is then added to the
three replicates to a concentration of 300 nM while enzyme is
omitted in the negative controls.
2.Immediately after adding enzyme, quickly vortex the sample
and place on a heating block at 37 °C (see Note 4). The negative controls are also placed on a heating block.
3.After 30 min, stop the assay by adding 100 μL quenching
buffer (2-fold volume relative to the reaction volume) and vortex the samples.
4. When all samples are stopped, add the same amount of enzyme
to your negative controls as in the positive replicates.
5.Prepare DTNB buffer by adding 5 mg (10 mg/mL) fresh
DTNB to 500 μL DTNB buffer (see Note 2).
6.Add 20 μL of DTNB buffer to each sample and vortex.
7.Of the 170 μL (reaction volume + quenching buffer + DTNB
buffer), transfer 150 μL per sample to a microplate before
analysis by the spectrophotometer at 412 nm (see Note 5).

3.2  Quantification
of Acetylation

1.Average the negative controls for each substrate peptide and
subtract it from the absorbance of the positive replicates.
2. Given that the absorbance has been corrected for path length at
the microplate spectrophotometer, l is equal 1 and it is sufficient


12


Håvard Foyn et al.

Table 1
Absorbance values obtained after acetylation assay with hNaa50 with
MLGP, MLGT, MDEL, and SESS oligopeptides

Positive replicates

Negative controls

MLGP

MLGT

MDEL

SESS

1.268

1.084

1.011

0.961

1.242

1.121


0.995

0.972

1.214

1.073

0.987

0.952

0.973

0.983

0.978

0.920

1.002

0.991

0.997

0.929

Fig. 2 Using the values in Table 1, the product formation of hNaa50 with MLGP,
MLGT, MDEL, and SESS oligopeptides was calculated. The results show high

activity toward MLGP (63.0 ± 6.7 μM), some activity toward MLGT (26.2 ± 6.2 μM),
and only minor activity toward MDEL and SESS (2.5 ± 3.0 and 9.2 ± 2.5 μM)

to insert ΔAbs and the extinction factor of TNB− (13,700/M
cm) (see Note 6) into the Lambert Beer equation (Eq. 1)


c=

Abs
e *l

(Eq. 1)

3. Multiply the result with the dilution factor (170/50) to obtain
the concentration of enzymatic product formation.
Using the values obtained in Table 1, hNaa50 yielded 63.0 ±
6.7, 26.2 ± 6.2, 2.5 ± 3.0, and 9.2 ± 2.5 μM acetylated MLGP,
MLGT, MDEL, and SESS oligopeptides respectively (Fig. 2).


DTNB-Based Quantification of Acetyltransferase Activity

13

4  Notes
1. Recombinant enzymes may be purified in the presence of DTT
or other reducing agents as the normally high dilution of
enzymes into each sample renders the concentration negligible.
Up to 500 μM thiols present may still produce meaningful

results although the background noise is significantly increased.
2.Make a stock DTNB buffer (without DTNB) that can be
stored at room temperature. When analyzing samples, transfer
the amount of buffer needed (20 μL per sample plus a little
extra) to a new Eppendorf tube. Subsequently add 10 mg/mL
DTNB to the buffer that ensures an ample concentration of
3 M in the final 170 μL sample.
3.The MLGP, MLGT, MDEL, and SESS oligopeptides used as
substrates were custom made (Biogenes). The peptides contain seven unique amino acids at their N-termini, as these are
the
major
determinants
influencing
Nt-acetylation
(MLGPEGG, MLGTGPA, MDELDLD, SESSSKS). The next
17 amino acids are essentially identical to the adrenocorticotropic hormone peptide sequence (RWGRPVGRRRRPVRVYP);
however, lysines were replaced by arginines to minimize any
potential interference by Nε-acetylation.
4.The optimal enzyme concentration, assay temperature, and
timeframe must be experimentally determined by timecourse
assays. Ensure that the assay setup is within the timeframe of
the enzymes linear range. Experience from our lab indicates
that it is easier to obtain small standard deviations with product formations of 10 μΜ and higher, so if experimentally possible, set up the assay conditions to aim for at least 10 μΜ
product formation.
5.Remember to correct for path length at the microplate
spectrophotometer.
6.The extinction factor of TNB at 412 nm is 14,150/M cm in
phosphate buffer at pH 7.27, but the spectrum is shifted slightly
in the presence of guanidinium HCl to 13,700/M cm [13].
7. Being an absorbance-based assay there will be numerical variations in the results. As a test 25 μM CoA was aliquoted into ten

samples and analyzed along with ten negative controls (25 μM
Ac-CoA). The absorbance measured is given in Table 2. The
average concentration measured was 26.26 ± 1.52 μM. Given
the small variations, the lower the concentration measured the
higher the uncertainty of the data. Thus, to minimize the risk
of misinterpreting the data, the assay should be designed in
such a way that the sample with maximum activity (e.g., the
sample without inhibitor in an IC50 assay) has at least a product formation of 25 μM.


14

Håvard Foyn et al.

Table 2
Absorbance values obtained after analysis of 25 μM CoA
(positive replicates) or 25 μM Ac-CoA (negative replicates)
Absorbance at 412 nm
Positive replicates

Negative controls

0.711

0.712

0.715

0.724


0.728

0.717

0.725

0.713

0.724

0.717

0.623

0.614

0.619

0.613

0.618

0.628

0.619

0.598

0.603


0.593

Acknowledgments
These studies were supported by grants from the Norwegian
Cancer Society (to T.A.), The Bergen Research Foundation BFS
(to T.A.), the Research Council of Norway (grant 230865 to T.A),
and the Western Norway Regional Health Authority (to T.A.).
References
1. Arnesen T, Van Damme P, Polevoda B, Helsens
K, Evjenth R, Colaert N, Varhaug JE,
Vandekerckhove J, Lillehaug JR, Sherman F,
Gevaert K (2009) Proteomics analyses reveal
the evolutionary conservation and divergence
of N-terminal acetyltransferases from yeast
and humans. Proc Natl Acad Sci USA
106(20):8157–8162.
doi:10.1073/pnas.
0901931106 pii:0901931106
2.Aksnes H, Hole K, Arnesen T (2015) Molecular,
cellular, and physiological significance of
N-terminal acetylation. Int Rev Cell Mol Biol
316:267–305. doi:10.1016/bs.ircmb.2015.01.001
3.Van Damme P, Hole K, Pimenta-Marques A,
Helsens K, Vandekerckhove J, Martinho RG,
Gevaert K, Arnesen T (2011) NatF contributes
to an evolutionary shift in protein N-terminal
acetylation and is important for normal chromosome segregation. PLoS Genet 7(7):e1002169.
doi:10.1371/journal.pgen.1002169
pii:PGENETICS-D-11-00127
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KK, Marie M, Stove SI, Hoel C, Kalvik TV,
Hole K, Glomnes N, Furnes C, Ljostveit S,
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(2015) An organellar nalpha-acetyltransferase,
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5.Dinh TV, Bienvenut WV, Linster E, Feldman-­
Salit A, Jung VA, Meinnel T, Hell R, Giglione C,
Wirtz M (2015) Molecular identification and
functional characterization of the first Nalphaacetyltransferase in plastids by global acetylome
profiling. Proteomics 15(14):2426–2435.
doi:10.1002/pmic.201500025
6.
Kalvik TV, Arnesen T (2013) Protein
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A, Arnesen T, Reis A (2015) De novo missense mutations in the NAA10 gene cause
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