Faculty of Sciences Department of Analytical Chemistry Tryptic cleavage of proteinaceous paint: a high-performance protein binder analytical technique Doctoral dissertation to meet the requirements to take the doctoral exam Doctor of Science: Chemistry Wim Fremout Academic year 2013-2014 Supervisor: Prof Dr Luc Moens Co-supervisor: Prof Dr Peter Vandenabeele Co-supervisor: Dr Steven Saverwyns Co-supervisor: Dr Jana Sanyova Cover: chicken ovalbumin on canvas. It represents the fusion of the (often considered incompatible) artistic and (bio)analytical worlds; the boundary that was explored in this doctoral dissertation. Ovalbumin is the main protein in egg white, an ingredient of many historical paint recipes and as such a frequently encountered analyte. The photograph of the canvas and the 3D structure of ovalbumin are public domain. Members of the jury Prof Dr Frank Vanhaecke Ghent University, faculty of Sciences, department of Analytical Chemistry Chairman Prof Dr Luc Moens Ghent University, faculty of Sciences, department of Analytical Chemistry Supervisor Prof Dr Peter Vandenabeele Ghent University, faculty of Arts and Philosophy, department of archaeology Co-supervisor Dr Steven Saverwyns Royal Institute for Cultural Heritage, Laboratory department Co-supervisor Dr Jana Sanyova Royal Institute for Cultural Heritage, Laboratory department Co-supervisor Dr Eleni Kouloumpi National Gallery & Alexandros Soutzos Museum (Athens, Greece), laboratory of Physicochemical Research Dr Stepanka Kuckova Institute of Chemical Technology (Prague, Czech Republic), department of Biochemistry and Microbiology Dr Hilde De Clercq Royal Institute for Cultural Heritage, Laboratory department Prof Dr Dieter Deforce Ghent University, faculty of Pharmaceutical Sciences, department of Pharmaceutics Prof Dr Frederic Lynen Ghent University, faculty of Sciences, department of Organic Chemistry Prof Dr Laszlo Vincze Ghent University, faculty of Sciences, department of Analytical Chemistry This is a joint research project of the following partners Ghent University Royal Institute for Cultural Belgian Science Policy Faculty of Sciences Heritage Department of Analytical Laboratory department Chemistry TABLE OF CONTENTS Abbreviations.........................................................................................................................9 I Introduction........................................................................................................................13 II Proteinaceous binders in painted works of art.............................................................19 II.1 Artists© paints...............................................................................................................19 II.1.1 Pigments..............................................................................................................20 II.1.2 Binding medium.................................................................................................20 II.1.3 Additives.............................................................................................................22 II.2 Introduction to protein theory.................................................................................23 II.2.1 Amino acids........................................................................................................23 II.2.2 Protein synthesis.................................................................................................27 II.2.3 Important protein properties............................................................................29 II.2.4 Homologous protein in different species.......................................................30 II.3 Protein binders in painting........................................................................................30 II.3.1 Animal glue........................................................................................................31 II.3.2 Egg based binders............................................................................................33 II.3.3 Milk-based binders............................................................................................36 II.3.4 Other protein sources in paint.........................................................................37 II.4 Chapter epilogue......................................................................................................38 III Protein binder analysis.....................................................................................................39 III.1 General overview of the analytical techniques..................................................39 III.1.1 Amino acid analysis (AAA)..............................................................................42 III.1.2 Pyrolysis GC-MS.................................................................................................45 III.1.3 Immunostaining.................................................................................................46 III.1.4 Peptide analysis................................................................................................49 III.2 Sample pretreatment in peptide analysis.............................................................50 III.2.1 Dissolving and denaturation...........................................................................50 III.2.2 Proteolysis...........................................................................................................55 III.2.3 Clean-up and preparation for detection method......................................57 5 III.3 Peptide chromatogram fingerprinting using HPLC-DAD....................................59 III.3.1 Principles............................................................................................................59 III.3.2 Experimental set-up..........................................................................................60 III.4 Peptide mass fingerprinting using MALDI-TOF-MS................................................60 III.4.1 Principle of MALDI-TOF-MS...............................................................................60 III.4.2 Experimental set-up..........................................................................................63 III.5 Peptide identification using HPLC-ESI-QTOF-MS/MS............................................63 III.5.1 Measurement....................................................................................................63 III.5.2 Data treatment.................................................................................................69 III.6 Chapter epilogue.....................................................................................................74 IV Identification of protein binders in works of art by high-performance liquid chromatography diode array detector analysis of their tryptic digests......................77 IV.1 Introduction..............................................................................................................78 IV.2 Experimental.............................................................................................................80 IV.2.1 Reagents...........................................................................................................80 IV.2.2 Samples.............................................................................................................80 IV.2.3 Apparatus and chromatographic conditions.............................................82 IV.2.4 Analytical procedures.....................................................................................83 IV.3 Results and discussion.............................................................................................85 IV.3.1 Pure protein samples.......................................................................................85 IV.3.2 Paint models and historical paint samples...................................................87 IV.4 Conclusions..............................................................................................................91 V Classification of protein binders in artists© paints by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry: an evaluation of principal component analysis and soft independent modelling of class analogy....................93 V.1 Introduction...............................................................................................................94 V.2 Experimental.............................................................................................................96 V.2.1 Reagents............................................................................................................96 V.2.2 Samples..............................................................................................................96 V.2.3 Sample pretreatment.......................................................................................97 V.2.4 Instrumentation.................................................................................................98 V.2.5 PCA and SIMCA................................................................................................98 V.3 Results and discussion..............................................................................................98 V.3.1 Determination of the main protein binder class..........................................98 V.3.2 Species determination of animal glues.......................................................107 V.3.3 St Margaret of Antioch..................................................................................108