Development and bioanalytical application of affinity-mass spectrometry for identification and structural characterisation of protein-ligand interactions

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mass spectrometry for identification and structural characterisation of protein-ligand interactions

Dissertation

zur Erlangung des akademischen Grades des Doktors der Naturwissenschaften

(Dr. rer. nat.)

vorgelegt von

Mihaela Stumbaum

an der

Mathematisch-Naturwissenschaftliche Sektion Fachbereich Chemie

Konstanz, 2014

Tag der mündlichen Prüfung: 29.05.2013

1. Referent: Prof. Dr. Dr. h.c. Michael Przybylski

2. Referent: Prof. Dr. Jörg Hartig

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Science, properly understood, heals the man of his pride;

she shows him his limits.

Albert Schweitzer

I dedicate this work to my wonderful parents Atena and Pavel Drăguşanu, and to my loving husband Jörg.

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The present work has been performed in the time frame from January 2007 to March 2011 in the Laboratory of Analytical Chemistry and Biopolymer Structure Analysis, Department of Chemistry, University of Konstanz, under the supervision of Prof. Dr.

Dr. h. c. Michael Przybylski.

I would like to express my deep gratitude and appreciation to Professor Michael Przybylski for the interesting research topic and scientific discussions, guiding my steps throughout these years and for his continuous encouragement during my research. His expert knowledge, enthusiasm, dedication to research, and education inspired me and will continue to influence me in the future.

My collaborators involved in my reserach projects are acknowledged:

Prof. Dr. M. L. Gross and Dr. T. Tu for the interesting collaboration and scientific discussions during my visits in St. Louis, Washington University, within a DAAD Programme.

Prof. Dr. J. Hoheisel and his co-worker M. Dauber from the Functional Genome Analysis Laboratory, DKFZ Heidelberg for providing me the Ets1 peptides library and DNA samples, and the fascinating scientific discussions.

Prof. Dr. M. Glocker and his co-workers from the Proteome Center Rostock for providing me with patients samples in the rheumatoid arthritis project and for interesting scientific disscusions.

Thanks to all members of the group for the nice and sociable atmosphere, but most of all I want to thank to Dr. Alina Petre, Dr. Marilena Manea, Dr. Camelia Vlad, Kathrin Lindner and Adrian Moise for enlightening scientific discussions.

Special thanks to Nicole Engel for the dedicated work during her Master thesis in the development of the online coupling of SAW biosensor with high resolution mass

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Special thanks to Dr. Lisa Jones for her support and the wonderful time spent together in St. Louis.

Special thanks to all my friends in Konstanz for their support and the wonderful time spent together.

Last but not least, my deepest gratitude is dedicated to my beloved family. I could never have made it so far without the love and encouragement of my parents and my sister LuminiŃa. Thank you with all my heart to my husband and friend, Jörg, for his encouragement, support, help and patience.

Furthermore, I need to appreciate the funding by the German Academic Exchange Service (DAAD, Bonn, Germany, PP 502/09).

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This dissertation has been partially published in the following articles and presented at the following Conferences:

Publications

1. Petre B.A., Drăguşanu M., Przybylski M. Molecular recognition specificity of anti-3-nitrotyrosine antibodies revealed by affinity-mass spectrometry and immunoanalytical methods in „Applications of Mass Spectrometry in Life Safety”, Springer Science 2008, 55-67.

2. Drochioiu G., Manea M., Drăguşanu M., Murariu M., Dragan E.S., Petre B.A., Mezo G., Przybylski M. “Interaction of beta-amyloid (1-40) peptide with pairs of metal ions: An electrospray ion trap mass spectrometric model study”, Biophys. Chem. 2009, 144(1-2): 9-20.

3. Tu T., Drăguşanu M., Petre B.A., Rempel D.L., Przybylski M. and Gross M.L.

Protein-Peptide Affinity Determination Using an H/D Exchange Dilution Strategy: Application to Antigen-Antibody Interactions, J. Am. Soc. Mass.

Spectrom. 2010, 21, 1660-1667.

4. Drăguşanu M., Petre B.A., Slamnoiu S., Vlad C., Tu T., Przybylski M., Online bioaffinity-electrospray mass spectrometry for simultaneous detection, identification and quantification of protein-ligand interactions, J. Am. Soc., Mass. Spectrom. 2010, 21, 1643-1648.

5. Drăguşanu M., Petre B.A., Przybylski M., Epitope motif of an anti- nitrotyrosine antibody specific for nitrotyrosine-modified peptides revealed by affinity-mass spectrometry, J. Peptide Sci. 2011, 17: 184-191.

6. Stumbaum M., Gronewold T. and Przybylski M., Biosensor-Electrospray Mass Spectrometry, GEN 2011, 31(4).

7. Petre B.A., Ulrich M., Stumbaum M., Bernevic B., Moise A., Döring G., Przybylski M., When is Mass Spectrometry Combined with Affinity Approaches Essential? A Case Study of Tyrosine Nitration in Proteins, J. Am. Soc. Mass.

Spectrom. 2012, 23 (11), 1831-1840.

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Conference presentations

1 Oxidative Post-Translational Modifications of Proteins in Cardiovascular Disease Conference, Boston 2008, USA, “Cysteinyl-nitrosylated peptides:

synthesis, structure and immunoanalytical characterisation”.

2 DGMS-Workshop “Affinity-Mass Spectrometry-Methods & Bioanalytical Applications", Konstanz 2009, “Biosensor-affinity & mass spectrometry”.

Conference poster presentations

1 Drăguşanu M., Petre B.-A., Weber R. and Przybylski M. (2007) “Mass spectrometric epitope identification of tyrosine nitrated peptides recognized by a monoclonal 3-nitrotyrosine antibody”, Lausanne, Swiss Proteomics Society (SPS).

2. Drăguşanu M., Petre B.-A., Döring G. and Przybylski M. (2008), “Recognition epitope of an anti-nitrotyrosyl-antibody specific for protein nitration revealed by affinity-mass spectrometry”, Denver, Colorado, USA, ASMS 56th Annual Conference.

3. Drăguşanu M., Petre B.-A., Tu T., Rempel D., Gross M. L. and Przybylski M.

(2008), “Online Bioaffinity-Ion Trap mass spectrometry: combining molecular identification and bioaffinity quantification in biopolymer interaction”, Boston USA, "Oxidative Post-Translational Modifications of Proteins in Cardiovascular Disease" Conference.

4. Drăguşanu M., Petre B.-A., Tu T., Gross M. L. and Przybylski M. (2009)

“Molecular recognition specificity and bioaffinity quantification in biopolymer interaction of anti-3-nitrotyrosine antibody revealed by SAW-ESI-MS and PLIMSTEX dilutions strategy”, Philadelphia, Pennsylvania, 57^th ASMS Conference on Mass Spectrometry.

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1 Introduction

1.1 Physiological and pathophysiological protein-ligand interactions

Physiological and pathophysiological protein-ligand interactions have created many challenges and raised many questions in the field of biochemistry and molecular biology, such as in drug discovery [1-4].

In protein-ligand interactions, the study of the biological and physical manifestations of disease has been of interest as of late since they correlate with the underlying abnormalities and physiological disturbances. Molecular interactions play a most important role in biological processes, including DNA replication, transcription and translation, gene splicing, protein secretion, cell cycle control, signal transduction, drug binding, antigen recognition and enzyme-substrate interactions [3, 5-11].

Therefore, it is crucial to identify and characterise components and structures of protein-ligand complexes for definition and identification of protein functions [8, 9].

Fundamental in protein-ligand interactions is the recognition of a ligand at a unique binding site and/or surface such that it binds in a defined way in order to carry out its function. Often a ligand is a signal-triggering molecule that fits into specific receptor

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change in the receptor proteins structure can then activate or inhibit another biological mechanism linked to it. In order to understand the receptor proteins functions and molecular causes of malfunction, the receptor proteins interactions with themselves and/or with other biomolecules need to be characterised and analysed. For explaining such interactions, also called bioaffinities, a major paradigm has been introduced more than 100 years ago by the “lock-and-key” model, formulated for explaining the specificity of enzymatic hydrolysis of glycosides [12] and suggested to be strongly correlated with the threedimensional (3D) structure of a protein.

Molecular recognition is the result of interaction processes such as hydrophobic interactions, hydrogen bonding, electrostatic interactions and van der Waals forces [7].

Molecular structural changes during the formation of a protein-ligand complex can contribute to, or disturb the complex formation. To understand the biological functions of proteins, not only their structures have to be determined, but also the binding reactions have to be characterised. This includes such properties as ligand binding site flexibility, distortion energies, desolvation effects, entropy, molecular electrostatic field complementarity, and kinetics determinations. Therefore, it is of high general interest to identify the binding regions and, above all, to examine the binding kinetics and affinities [7, 8, 13]. Protein-ligand interactions have been studied by many scientists in the last years, and identified for hundreds of proteins and their interaction partners [1, 14, 15].

Protein-ligand interactions are integral to a wide range of biological processes, including hormone, neurotransmitter or drug binding, antigen recognition, and enzyme-substrate interactions. Fundamental to each of these interactions is the recognition by a ligand of a unique binding surface whereby it binds in a defined way in order to carry out its function. Through an understanding of these specific interactions, it may be possible to design or discover analogous ligands with altered binding properties and, therefore, to intervene in the chemical pathway in a specific manner. The ligand of interest may be an organic small molecule, a peptide or a carbohydrate [8].

Protein-ligand interactions can be disturbed in human diseases such that the disease is the result of, e.g. genetic alterations in physiologically important signalling

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pathways. Disease-derived protein variants have been frequently used to provide biomedical reference for the study of protein-ligand interactions. Therefore, it is important to delineate the structural characteristics, as well the affinity binding of the proteins, in order to develop a model of the binding interaction. One pathophysiological type of protein-protein interaction is oligomerisation and aggregation. Deposition of aggregates in the brain comprises diseases, including Alzheimer´s, Parkinson´s and Prion Disease [16-19]. Therefore, structural characterisation of these aggregates and the protein sequences which lead to the deposition of these aggregates in brain is very important. Mass spectrometry has been applied with success in the structural identification and characterisation of such proteins and their aggregation [16, 20, 21].

In addition to aggregation/oligomerization, post-translational modifications in proteins may also lead to disease and/or disturbed pathways occurring at physiological conditions [16, 22-24]. For example, oxidation of a protein can lead to aggregation, fragmentation, denaturation, and destruction of secondary and tertiary structure resulting in increased proteolytic susceptibility of the oxidized proteins. Free radicals can lead to oxidation of amino acid side chains, cleavage of peptide bonds, and formation of covalently cross-linked protein derivatives [25]. Oxidative modifications of proteins can result in functional inactivation or activation through the site-selective oxidative modification of specific amino acids. These are not only indicators of toxic and destructive processes in living systems, but can also serve to control enzyme activity [26].

One of the most studied oxidative post-translational modifications is tyrosine nitration which can occur under physiological conditions, including signal transduction [27], and may be substantially enhanced under various pathophysiological conditions associated with oxidative stress. For the molecular correlation of protein nitration with pathogenic mechanisms of human diseases and with animal or cellular models of diseases, it is essential to identify the protein targets of nitration and the specific individual modification sites. Researchers have been interested in the role of the tyrosine nitration for understanding the potential

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Parkinson’s disease [27-32]. Tyrosine nitration occurred in both in vitro and in vivo samples under physiological and pathophysiological conditions, and has been successfully characterised and analysed by mass spectrometry [33-38]. For example, nitration of prostacyclin synthase (PCS), upon treatment of bovine aortic microsomes by peroxynitrite (PN), has been identified at Tyr-430 residue using a combination of proteolytic fragmentation, HPLC detection and high resolution mass spectrometry [38]. The specificity of this single post-translational modification, nitration of the Tyr- 430 residue, producing inactivation of prostacycline synthase may be explained by heme catalysis. The Tyr-430 residue is located near a heme group and is also accessible to a reactive nitrogen species. Therefore, mutation of the Tyr-430 residue should give further informations to the importance of this amino acid (Figure 1a) [38].

In contrast, physiological tyrosine nitration in human eosinophil granule proteins (eosinophil peroxidase (EPO), eosinophil cationic protein (ECP), eosinophil neurotoxin protein (EDN)), isolated from patients with hypereosinophilia, has been successfullly identified by mass spectrometry [37]. The close relationship between eosinophilia and nitro-tyrosine formation suggested that the EPO itself is an important factor in promoting protein nitration [35, 37]. By using high resolution affinity-mass spectrometry specific single nitration sites at Tyr-349 in EPO and Tyr-33 in both ECP and EDN have been identified [37] (Figure 1b).

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a b

Figure 1: (a) Reconstructed 3D structural model of human PCS based on the P450BM-3 X-ray structure [39]. The red structure denotes heme and the arrow indicates the probable localisation of nitrated fragment 427-430 in a tight fold around the heme binding site. (b) Diagram representation of ECP. Nitrated Tyr-33 is represented as a ball-and-stick model [35, 37-39].

Oxidation represents only one type of protein modifications which occurs under physiological and pathophysiological conditions. Once one or more protein-ligand interactions have been identified, it is desirable to investigate the interaction(s) for (i) understanding the mechanism in which the proteins are involved at molecular level;

(ii) understanding the interactions functional significance in vivo and (iii) developing methods to specifically disturb the interaction in vivo [40]. Through an understanding or identification of these specific interactions it may be possible to design or discover analogous ligands/drug with altered binding properties and, therefore, to intervene in the biochemical pathway in a highly specific manner.

Proteins regulate all biological processes in cells, including gene expression, cell growth, morphology, mobility, intercellular communication and apoptosis. Therefore, structural characterisation and the affinity binding of the protein-ligand interaction is of crucial importance for understanding the functions of the proteins as well their mechanism. The proteins that are used to complete specific functions may not always be expressed or activated, or they are expressed in a cell type-dependent manner.

Therefore, highly sensitive and specific approaches are needed in the structural identification and characterisation of proteins. In recent years, it has been

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characterisation and identification of protein modifications and complex formation in physiological and pathophysiological conditions [33, 36, 37, 41, 42].

1.2 Analytical methods for characterisation of protein-ligand interactions

A large number of bioanalytical methods have been developed over the years for structure characterisation and determination of protein-ligand complexes. These methods have provided a pool of information regarding protein structure and function.

Each of these approaches has its strengths and weaknesses, especially with regard to the sensitivity and specificity [1, 2, 10]. A summary of analytical methods applied for identification and characterisation of protein-ligand interactions is summarised in Table 1. These methods enable the definition and analysis of protein structure, both in native form as well as while interacting with molecular partners, and some thermodynamic characteristics.

The primary structure of proteins is crucial since the specific amino acid sequences determine structural characteristics of the proteins such as the formation of the disulphide bridges and accessibility to post-translational modifications. Tandem mass spectrometry fragmentation directly enables the identification of amino acid sequences and consequently the elucidation of unknown protein sequences. The protein terminal groups can be determined by N- or C-terminal analysis, while partial sequences can be determined by chemical or enzymatic degradation in combination with Edman-sequencing [43, 44].

Approaches to determine secondary structure elements of proteins are spectroscopic methods, such as IR spectroscopy [45] and circular dichroism spectroscopy (CD) [46, 47]. CD spectroscopy is frequently used for proteins with α-helical structure, while IR spectroscopy is employed for the characterisation of proteins with ß-sheet or ß-turn structures.

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Table 1: Analytical methods applied for structural and thermodynamic protein characterisation and identification.

Analytical methods Ref. Characteristics

Edman sequencing [43, 44]

Circular Dichroism (CD) [46, 47]

IR Spectroscopy [45]

HDX [48-51]

Nuclear Magnetic Resonance (NMR) [52, 53]

X-Ray co-crystalography [54-56]

Mass Spectrometry (MS) [57-59]

Epitope mapping [60-63]

Structural

Cross-linking [64-66]

primary, secondary,

tertiary structure

Quartz Crystal Microbalance

(QCM) [67-69]

Surface plasmon resonance

(SPR) [70-72]

Surface acoustic wave (SAW)

[73-80]

Biosensors

Bio-Layer Interferometry [81, 82]

Analytical Ultracentrifugation (AUC) [8, 83, 84]

Fluorescence Resonance Energy Transfer

(FRET) [85]

Thermodynamic

equilibrium constants,

kinetic constants (KD,

kon, koff), binding energies

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Nuclear magnetic resonance (NMR) and X-ray crystallography methods are applied for the characterisation of the tertiary and quaternary structure in proteins.

High resolution 2D and 3D NMR provides structure analysis in solution and information about the dynamics of protein molecules. However, this method is limited to small proteins (<30 kDa). Previous studies [58] have shown tertiary structure- selective modification of charged residues as an efficient approach for the structural characterisation of proteins; X-ray crystallography and mass spectrometry have been shown to be complementary analytical tools for defining precisely chemically modified structures.

Amide hydrogen/deuterium exchange mass spectrometry has become in recent years a powerful method for high-resolution analysis of protein dynamics, structure and function [48-51]. Hydrogen/deuterium exchange approaches can provide information that augments and refines information derived from high-resolution structural studies, and can provide detailed information on native protein structure when structural information is unavailable. Structural studies using mass spectrometry coupled with hydrogen/deuterium exchange can be carried out in a number of physiologically relevant contexts, including ligand binding, self-association, and conformational switching. Advancements in other techniques such as Raman spectroscopy also hold promise for use in high-resolution and high-throughput protein structure and dynamics studies [48-50, 89].

Chemical crosslinking is based on formation of covalent bonds between different molecules (intermolecular) or parts of a molecule (intramolecular) and has been successfully applied in protein-ligand interactions analysis in combination with mass spectrometry [90, 91] as a tool for structure determination [92]. It is a fast procedure with low material consumption offering the opportunity to gain insight into 3D structures of proteins or protein complexes under native conditions. The aim of performing intramolecular chemical crosslinking of a protein is to get information on its three dimensional structure [93], whereas the approach of intermolecular crosslinking is focused on the elucidation of interaction between different protein molecules [66].

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Thermodynamic approaches enable the determination of specific thermodynamic and energetic parameters of protein-ligand interactions (e.g.

equilibrium constants, kinetic constants and binding energies). Most of the analytical methods used in determination of thermodynamic characteristics of protein-ligand interactions are applied on a surface at which one of the binding partners is immobilised. Several types of biosensors, such as Surface Plasmon Resonance (SPR), Surface Acoustic Wave (SAW), Quartz Crystal Microbalance (QCM) and Bio-Layer Interferometry (BLI), have been applied in the analysis of protein-ligand interactions.

Each of these bioaffinity techniques has its strengths and weaknesses with regard to the detection approach. However, all biosensors independent of the detection method are measuring the same type of binding curve (Figure 2).

Figure 2: Association and dissociation curve of a protein-ligand interaction. A protein binds to the covalently immobilised ligand during sample injection, resulting in an increase in signal (response). At the end of the injection, the sample is replaced by a continuous flow of buffer, and the decrease in signal now reflects dissociation of the protein [3].

One of the first biosensors introduced was the surface plasmon resonance (SPR) biosensor. The SPR method measures changes in the resonant angle of a laser

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limitation that it is difficult to detect protein from blood or serum, and on cell surfaces [70-72].

Another optical label free technology, Bio-Layer Interferometry (BLI), analyses the interference pattern of white light reflected from two surfaces: a layer of immobilised protein on the biosensor tip and an internal reference layer. Any change in the number of molecules bound to the biosensor tip causes a shift in the interference pattern that can be measured in real-time. The binding between an immobilised ligand on the biosensor tip surface and an analyte in solution produces an increase in optical thickness at the biosensor tip which results in a wavelength shift, ∆λ. This wavelength shift is a direct measure of the change in thickness of the biological layer [81, 82].

The Quartz Crystal Microbalance (QCM) technology is a sensitive approach capable of measuring changes in mass at the molecular level. An applied AC-potential causes the quartz crystal to vibrate at a low resonance frequency. The frequency will change as molecules bind to the immobilised ligand on the crystal surface and this frequency change is used to characterise label-free molecular interactions in real time.

In addition to measuring the frequency, the dissipation can be measured and is related to the protein viscoelastic properties [67-69].

In the early 19^th century, the first use of the surface acoustic wave (SAW) technology was reported by the brothers Paul-Jacques and Pierre Curie who discovered the piezoelectric effect [94] and by Lord Rayleigh who described surface waves by examining earthquakes [95]. In 1979 the first gas sensing application was perfomed based on Rayleigh waves with a sensitive polymer layer [73-76]. The surface acoustic wave created and used for the detection of protein-ligand interaction is a mechanical acoustic wave, called a Love wave. The development and characterisation of a Love wave based biosensor was described by Schlensog et. al.

[77]. The Love waves travel along the surface of a piezoelectric crystal and the interdigital transducers (IDTs) deposited on the surface of the piezoelectric substance guide the waves between two electrodes. The wave changes in both, phase and amplitude, in response to proteins binding to an immobilised ligand on the crystal surface. The piezoelectric crystals can be made to vibrate at a specific high frequency

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with the application of an electrical signal, e.g. 150 MHz [79]. These oscillations are mechanical waves that travel through the bulk matter. Their frequency is dependent on the electrical frequency applied to the crystal as well as the crystals mass. Therefore, when the mass increases due to binding of proteins, the oscillation frequency changes and the resulting change can be measured electrically and used to determine the additional mass of the crystal. This is the function principle of a QCM [67]. If the oscillation is confined in a thin layer on the surface of the crystal, one can speak of SAW [78-80, 96]. Different types of acoustic waves can be employed in a SAW device, but Love waves [77] offer particularly high sensitivities due to the confinement of the acoustic energy to the sensing surface. The Love waves are in fact horizontally polarized guided waves [97].

All biosensors enable the determination of kinetics data such as on- and off- rates and a KD value. The differences between all biosensors relate to the detection and type of applications. QCM is somewhat less sensitive compared to SAW due to the low frequency used for detection, SPR is limited to gold surfaces and only BLI provides high-throughput analysis capability.

Enzyme-linked immunosorbent assay (ELISA) is a biochemical technique used to detect the presence, and determine the affinity of an antibody or an antigen in a sample. Performing an ELISA involves at least one antibody with specificity for a particular antigen. The sample with an unknown amount of antigen or antibody is immobilised on a solid support (usually a polystyrene microtiter plate) either non- specifically (via adsorption to the surface) or specifically (via capture by another antibody or a sample-specific antigen). After the sample is immobilised, the detection antibody is added, forming a complex with the antigen. The detection antibody can be covalently linked to an enzyme or can itself be detected by a secondary antibody that is linked to an enzyme through bio-conjugation. Between each step, the plate is typically washed with a mild detergent solution to remove any proteins or antibodies that are not specifically bound. After the final wash step, the plate is developed by adding an enzymatic substrate to produce a visible signal which indicates the quantity

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Fluorescence Resonance Energy Transfer (FRET) is a fluorescence based approach in which the photon energy is transferred from an excited fluorophore labelled protein (the donor) to another fluorophore labelled protein (the acceptor) when both are located within close proximity (1-10 nm). By using fluorescence digital imaging microscopy, one can visualise the location of green fluorescent proteins within a living cell and thereby follow the time course of the changes in FRET corresponding to cellular events at a millisecond time resolution. The observation of such dynamic molecular events in vivo provides vital insight into the action of biological molecules [85].

Analytical ultracentrifugation (AUC) is a classical method for the characterisation of interactions between purified proteins in solution. Protein complexes can be characterised with regard to their stoichiometry and the thermodynamic binding constants of complex formation. Sedimentation techniques can distinguish among multiple coexisting complexes of different stoichiometries and also provide information on self-associated properties and on mixed self- and hetero- association [8, 83].

For all these bioaffinity techniques, the combination of different approaches for the simultaneous structural identification, characterisation and kinetics determination of protein-ligand complexes has not yet been previously reported. The direct instrumental combination of a biosensor technique with mass spectrometry has been first developed in the present dissertation.

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1.3 Bioaffinity-mass spectrometry for protein structure and interaction analysis

In recent years, combined bioaffinity-mass spectrometry methods have been shown to be powerful methods, particularly in the identification of epitopes and paratopes in antigen-antibody complexes.

Antibodies recognise sequences of amino acids located at the surface of an antigen, called epitopes or antigenic determinants. To form an immune complex, non- covalent interactions take place between the surfaces of antigen and antibody molecules, complementary in their topology. The paratope represents the amino acid sequence in the antibody that is recognised by the antigen.

The combined proteolytic-excision mass spectrometry approach, first developed by our laboratory [60], is now commonly used in the identification of epitopes from peptides and proteins antigens by many groups [60-63, 98-102]. This approach combines the advantages of the proteolytic stability of antibodies and antigen epitopes by shielding with the molecular identification provided by mass spectrometry [60, 61, 63, 102]. This method has been successfully applied by many scientists, resulting in the identification and characterisation of peptide epitopes such as the plaque-specific Aß(4-10)-epitope correlated with Alzheimer disease and carbohydrate-recognising epitopes using the Carbohydrate Recognition Domain Excision (CREDEX)-MS method [35, 60, 62, 63, 99, 103-108]. The general scheme illustrating this bioaffinity- mass spectrometry approach (epitope excision- and extraction-mass spectrometry) is shown in Figure 3.

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Figure 3: General scheme illustrating the two complementary approaches, epitope excision- and extraction-mass spectrometry. The ligand (an antibody in this example) is covalently bound on a sepharose column and the protein/peptide is passed over. The protein can be bound in digested form of peptide fragments (epitope extraction) or as whole protein with the digestion occuring outside of the binding sites (epitope excision) [60, 104].

Further development of bioaffinity-mass spectrometry with identification of post-translational modifications, such as identification of specific nitration sites in biological materials, have been reported in our laboratory [36, 37]. The principle is analogous to the epitope extraction-MS method whereby the protein containing the antigen is digested in solution; the proteolytic peptide mixture is then added to the antibody column and the resulting peptide epitope identified by mass spectrometry.

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Bioaffinity-mass spectrometry which is usually performed on affinity columns could also be applied on a biosensor surface. However, the bioaffinity-mass spectrometry methods developed have not been previously applied to the simultaneous structural identification, characterisation and kinetic analysis of the protein interactions [34-36, 60, 61, 98, 103, 104, 109, 110]. The combination of a mass spectrometric method with a surface based biosensor detection system for the analysis of a protein-ligand interaction allows for the determination of both kinetic data and structural characterisation. This will greatly improve the examination of compound mixtures and also contribute to the identification of binding regions.

Some combinations of mass spectrometry with biosensors have been described in the last years, but none of them has been able to provide simultaneous structural characterisation/identification and kinetic data determination. Among these approaches are offline combinations of MALDI-MS with SPR [111-131] and SAW [132, 133], as well as online combination of SPR with ESI-LC/MS [134-136]. The first offline combination of SPR with MALDI-MS was reported in the middle 1990´s and described as biomolecular interaction analysis MS (BIA–MS). Krone et al.

identified femtomole quantities of the peptide myotoxin by direct MALDI analysis from a sensor chip used for interaction studies of a polyclonal anti-myotoxin antibody.

The MALDI solution matrix was applied onto the sensor chip after affinity measurements, and used for disrupting the protein-ligand interaction, liberating the ligand into solution for subsequent mass spectrometric identification [137]. Several SPR-MS approaches include direct ionisation of analyte immobilised on the biosensor chip by adding a matrix solution after a on-chip digestion followed by elution, chromatographic concentration of the sample and mass spectrometric analysis [111- 131, 138], as well elution of the ligand from the sensor surface and mass spectrometric analysis with transfer of the ligand sample to an LC-MS/MS, or spotting on MALDI targets [138-140]. Strigter et al. developed a fully automated digestion combined with nano-LC-MS/MS for the quantification and identification of interferon in plasma [134]. However, none of these applications is capable to the simultaneous characterisation of a protein structure and kinetic parameters, particulary binding

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offline manner for the analysis of a protein complex consisting of human blood clotting cascade factor α-thrombin and human anti-thrombin [132, 133]. In contrast the online combination of an SAW biosensor with electrospray mass spectrometry provides for the first time simultaneous structure identification/characterisation by mass spectrometry and kinetic data determinations for protein-ligand complexes [141].

1.4 Mass spectrometric methods for protein structure determination

Mass spectrometry has proven in the last decade to be a powerful technique for structural characterisation of proteins and their interactions. Therefore, the application of mass spectrometry as an important tool in biochemical and biomedical science has rapidly increased over the last years. Proteins have been analysed by mass spectrometry for identification of amino acid sequences and post-translational modifications, as well for characterisation of their interactions with different ligands.

The capability to study extremely small quantities of molecules and mixtures of proteins with high sensitivity is a major advantage of mass spectrometric analyses over other analytical techniques.

In 2002, the Nobel Prize for Chemistry was awarded to John Fenn and Koichi Tanaka for the development of the two major “gentle ionisation” techniques, electrospray ionisation (ESI) and matrix-assisted laser desorption/ionisation (MALDI) mass spectrometry. Both ionisation processes have been shown to be able of the analysis of protein-ligand complexes.

A number of mass spectrometric methods [57, 59, 142] and combinations of mass spectrometry methods with H/D exchange [49, 50], Protein Ligand Interaction by Mass Spectrometry, Titration and H/D Exchange approach (PLIMSTEX) [143- 145], Stability of Unpurified Proteins from Rates of H/D Exchange (SUPREX) [146, 147] and bioaffinity [98, 103, 104] have been established and developed for detection and quantification of protein-ligand interactions. HDX takes advantage of the drastically slower kinetics of HD exchange rates in regions of a polypeptide antigen shielded by binding to an antibody [148, 149]. SUPREX makes use of the extent of protein HDX in various concentrations of denaturants to determine the affinities of

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protein-ligand complexes. PLIMSTEX, first developed by Zhu et. al. [143], tracks changes in the extent of protein HDX for various ligand/protein ratios to determine affinity, stoichiometry and conformational changes that occur upon ligand binding.

Thus far, there are no reports of extending these MS methods to antibody-antigen binding. Nevertheless, HDX is effective for epitope mapping to probe antibody- antigen interactions [148, 150].

HDX-MS is a powerful technique for characterisation of conformation and dynamics of proteins and protein complexes. The method can be applied in cases when conventional structural approaches such as NMR and X-ray crystallography cannot be applied due to low concentrations and impure samples. The application of mass spectrometry has therefore found high interest, particularly due to its high sensitivity (picomole-femtomole detection limits) [49, 50].

In H/D exchange, amide hydrogen atoms exchange for deuterium atoms when a protein is incubated in D₂O. The kinetics of exchange is sensitive to the hydrogen bonding of the amide backbone-amide hydrogens. In dynamic regions, the hydrogens will exchange quickly while tightly hydrogen-bonded amides exchange much more slowly. After subjecting a deuterated protein to rapid proteolysis under conditions that preserve the deuterium label, MS analysis reveals the extent of deuteration at a resolution of 5-20 residues (Figure 4). In protein-ligand complexes the binding domains are shielded from HDX and thus can be identified by mass spectrometry.

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H→DExchange D₂O incubation

H+, 0°C

Proteolysis

MS HPLC

Protein Amide hydrogens in

disordered regions exchange fast

H/DExchange is measured as m/z

shift by MS

Figure 4: Schematic representation of the HDX-MS approach. Protein is incubated with D₂O solution, reaction which is essentially quenched by shifting the pH to 2 at 0 °C. The HDX-exchanged protein is then proteolyzed with pepsin. The peptic fragments are then chromatographically separated and their masses determined by mass spectrometry. The experiment is repeated in the absence of deuterium and the weight gain of each fragment attributed to deuteration.

Despite the fact that mass spectrometry measures biopolymer ions in the gas phase, it has been established and widely applied to protein-ligand interactions, since non-covalent interactions of protein structures are generally preserved in electrospray mass spectra [59, 90, 142, 151-154]. Electrospray ionisation is a soft ionisation method for producing intact molecular ions of biopolymers which has found high general application in bioanalytical chemistry. ESI-MS produces gaseous ionised molecules from solution [90] by creating a fine spray of charged droplets in an electric field [154-156]. The basic steps for ion formation involve the production of charged droplets at the electrospray tip in the presence of an external electric field, successive droplet disintegration, solvent evaporation leading to highly charged smaller droplets, and finally the release of multiply charged gas phase ions from the smaller droplets.

An electric field and gas flow force ions created near the atmosphere-vacuum interface

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of a mass spectrometer to accelerate toward the inlet and enter the first vacuum pumping stage. An illustration of the ESI process is shown in Figure 5.

+ + - -

+ -

+ + - -

+ - +

+

+ ++ ++ - -

+ + + ++ +

++

Spray needle tip Tailor cone Aerosol of fine charged droplets

1 µm

+ +

+

+ Columbic

explosion +

+ ++ + +

++ + +

Solvated macromolecular ion

10 nM 1 nM

+

+ ++

Solvent + evaporation

Desolvated macromolecular ion a

b

Figure 5: Schematic representation of electrospray ionisation. (a) A high positive potential is applied to the capillary, causing positive ions in solution to drift towards the meniscus. Destabilization of the meniscus occurs, leading to the formation of a “Taylor cone” and a fine jet emitting droplets with excess positive charge. (b) Gas phase ions are formed from charged droplets in a series of solvent evaporation-Coulomb fission [59].

The repulsion between the charges on the surface causes intact ions to leave the droplet by a process known as a “Taylor cone” [157, 158]. The low flow rates of this technique provide significant advantages, including the use of buffers, detergents and other co-solvents required for the solubilisation of biopolymers. Multiple charging allows ions to be analysed based on a mass-to-charge (m/z) ratio, which greatly extends the mass range of the mass analyzer. Generally, ESI mass spectra show a steady increase of the charge state of ions with increasing molecular weight. The number of charges varies, depending on several parameters such as analyte, solvent, pH and temperature. For positive ion analysis of peptides and proteins in acidic solutions (pH < 4), the charges are normally associated with the most basic amino acids (Lys, Arg) of the molecule and the amino terminus.

During the last years, several types of non-covalent complexes have been analysed by ESI-MS. Example include protein-peptide, polypeptide-metal ion and protein-nucleic

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ESI-MS are illustrated in Figure 6. The first example (Figure 6a), the complex formation between a synthetic peptide (R16L) and calmodulin was used as a model to analyse the interactions between calmodulin (CaM) and the CaM-binding sequences of smooth-muscle myosin light chain kinase in the presence and absence of Ca²⁺. The results indicate the importance of electrostatic forces in interactions between CaM and targets, particularly in the presence of Ca²⁺, and the role of hydrophobic forces in contributing additional stability to the complexes regardless if Ca²⁺ is present or absent. The stoichiometry of peptide binding was observed to be one peptide per one CaM. [159]. A second example illustrating a non-covalent protein complex analysis by ESI-MS is the complex between a nucleotide and the protein binding regions in the EF-Tu from T. thermophilus [57]. The transition of EF-Tu from an ”inactive” GDP to the “active” GTP binding form upon interaction with the nucleotide exchange factor EF-Ts was characterised by selective chemical modification and direct mass spectrometric analysis of the EF-Tu/-Ts and GDP complexes, and the Lys residues have been identified for the distinct nucleotide binding region. As an example, ESI spectra of free and GDP-bound forms of EF-Tu are compared in Figure 6c vs. Figure 6d which revealed a homogeneous ion series of a ratio 1:2 EFTu/GDP complex under the conditions employed [57].

For the first time simultaneous structure identification/characterisation by mass spectrometry, and kinetic data determinations for CaM-peptides complexes and nucleotide-peptide library have been possible due to the online combination of an SAW biosensor with electrospray mass spectrometry.

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a

b

c

d

Figure 6: (a) ESI-FTICR mass spectrum of CaM with R16L peptide (concentration ratio 1:1.5) in 5 mM ammonium acetate buffer, pH 5.9. Insets show the expansions of the 7+ and 8+ charge-states. C represents CaM and P represents peptide, respectively [159]. (b) ESI mass spectra and structure model of (c) free EF-Tu; (d) EF-Tu complexed with GDP. The residue numbers denote lysine residues found shielded in the EF-Tu/GDP complex

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Compared to the continuous ionisation mode of ESI from solution, matrix-assisted laser desorption ionisation (MALDI) utilizes a pulsed laser for the desorption of analyte molecules from a target surface on which they are co-crystallized with an excess of specific wavelength-absorbing matrix molecules, such as α-cyano-4-hydroxy cinnamic acid [160]. Ions are produced by short laser pulses at 337 nm wave length and accelerated into the mass analyser by applying a high potential electric field between the sample and the orifice. Although the details of energy conversion for ion formation are not fully understood, a general scheme of the MALDI process can be formulated as shown in Figure 7.

c ++

matrix/analyt could hν

laser pulse matrix/analyt

cluster

+

macro-ion in cluster

macro-ion

Figure 7: Schematic representation of the ion formation in MALDI mass spectrometry. A matrix/analyte cloud is desorbed from the microcrystalline matrix/sample preparation by a laser pulse. Gas-phase proton-transfer with matrix ions is considered to be primarily responsible for the subsequent generation of analyte ions.

The laser causes a rapid heating of the matrix crystals leading to the sublimation of matrix and analyte molecules. Ions are formed through gas-phase proton-transfer reactions with photoionized matrix molecules, generally producing low charge state ions [91, 161]. MALDI offers the advantages of intact protein ionisation, low sample requirements and high throughput analysis. Moreover, it has a higher tolerance towards salts compared to ESI analysis. MALDI-MS has also been applied to the study of protein complexes, DNA duplexes and anionic compounds binding to polybasic peptides [152, 162, 163], although with the limitation of uncertain structural states. Figure 8 illustrates the mass spectrum of heparin disaccharides complexed with a synthetic peptide.

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a

b

[P+2H]²⁺

[P+D1+2H]²⁺

[P+H]⁺ 1442.8

[P+D1+H]⁺ 1981.8

[P+2D1+H]⁺

[P+2H]²⁺

[P+H]⁺ 1442.8

[P+D2+H]⁺ 2019.5

-SO3 1940.4

Figure 8: IR-MALDI mass spectra of heparin disaccharides D1 and D2 mixed with the synthetic peptide SP1. The lability of N-sulfate group(s) is obvious from spectrum (b) [163].

Even though the MALDI technique is a soft ionisation process, solvent evaporation during sample preparation may affect the complex stability and represents a limiting factor for the observation of non-covalent complexes. Furthermore, during evaporation, pH and ionic strength of the solution may be altered, leading to altered structure and interactions, thus presenting limitations for the use of MALDI-MS as a general tool for studying protein-ligand interactions.

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1.5 Scientific goals of the dissertation

Protein-ligand interactions are crucially important to a wide range of biological processes and pathophysiological modifications may result in disease conditions.

Reasoning that disease states often result from genetic alterations in physiologically important signalling pathways, disease derived protein variants have frequently been used to study protein-ligand interactions. Therefore, it is important to delineate structural characteristics of the bound species in biopolymer interactions. Developing new methods for elucidating kinetic studies together with mass spectrometric characterization has been an important goal in the protein-ligand interaction studies and in drug discovery.

The major goals of this dissertation were the development and bioanalytical applications of affinity-mass spectrometry methods as a tool for investigation of protein-ligand interactions. Within these goals, the following major objectives were pursued:

• Development of an online coupling instrumentation of a SAW biosensor with electrospray mass spectrometry.

Online coupling of (i) an electrospray ion trap mass spectrometer and (ii) electrospray-Fouriertransform-ion cyclotron resonance-mass spectrometer with an SAW biosensor was developed for structural identification, characterisation and kinetic determination of protein-ligand complexes. Therefore, corresponding interfaces were developed for sample desalting and concentration in combination with the mass spectrometric analysis of the protein.

• Application of online bioaffinity-mass spectrometry for the identification of a recognition epitope motif for tyrosine-nitrated peptides by anti-3- nitrotyrosine antibodies.

An anti-3-nitrotyrosine antibody was applied in the study to identify a recognition epitope motif of nitrated peptides, using (i) proteolytic epitope-excision/extraction -mass spectrometry, (ii) ELISA methods, (iii) online combination SAW

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biosensor-ESI-MS and (iv) the combination of protein ligand titration and HD- exchange-MS.

• Bioanalytical applications of the bioaffinity-mass spectrometry combination (i) to the mass spectrometric structural characterisation and kinetic determination of peptide complexes of a Ca-binding protein;

(ii) to the study of a nucleotide-transcription factor interaction with a small binding peptide library to the dsDNA;

(iii) to the elucidation of an anti-Rheumatoid Arthritis autoantibody (RA-33) interaction with epitope peptides of a ribonucleoprotein epitope peptides. In addition to the kinetic evaluation of an epitope peptide, to a monoclonal anti- RA33 antibody, a major goal was to characterise peptides derived from RA33 protein sequences with autoantibodies from patients with Rheumatoid Arthritis.

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2 Results and discussion

2.1 Development of online bioaffinity-mass spectrometry instrumentation

Simultaneous structural characterisation/identification and kinetic analysis of protein-ligand interactions at the molecular level require the application of complementary analytical methods and the combination of different approaches. Each of the approaches presented in chapter 1.2 has its own strengths and weaknesses, especially with regard to sensitivity and specificity.

Previous studies in our laboratory have been focused on the development of selective and high resolution mass spectrometric approaches for the identification of antigen-antibody recognition structures. Affinity-mass spectrometry methods, in combination with proteolytic digestion, have been previously developed and employed for epitope identification and affinity-proteomics approaches [60, 61, 98, 102-104, 164]. These methods also provide direct protein identification from biological material with high selectivity, through analysis of the epitope. However, a kinetic evaluation has previously not been possible by affinity-mass spectrometry.

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The surface acoustic wave (SAW) biosensor is a label free method for rapid kinetic studies by determination of association/dissociation constants of small sample amounts [77-80, 132, 165, 166]. While providing sensitive and accurate determinations of association/dissociation constants (k_on, k_off) and affinity binding constants (KD), a major limitation of all bioaffinity methods is the lack of chemical structure identification of the affinity-bound protein. In contrast, the combination of biosensor detection and mass spectrometry could enable both the structure identification and quantification of bioaffinity interactions of biopolymers. Therefore, the combination of these two methods was a major goal of this thesis in developing methods for the simultaneous identification and quantification of bioaffinity interactions using online coupling of an SAW biosensor with an electrospray mass spectrometer.

2.1.1 Development of online bioaffinity-electrospray ion trap mass spectrometry Two strategies were pursued in the development of an online bioaffinity-mass spectrometric approach (Figure 9): (i) direct coupling of the waste capillary of the SAW biosensor with an electrospray mass spectrometer and (ii) online coupling of the SAW biosensor with an electrospray mass spectrometer via an interface used for desalting and sample concentration (Figure 9).

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SAW - Biosensor ESI-MS

Kinetics protein-ligand

Structural characterisation protein-ligand

a.

b. SAW - Biosensor ESI-MS

Interface

Figure 9: Strategies used in the development of the online bioaffinity-mass spectrometry approach. (a) Direct coupling of the waste capillary of SAW biosensor with an electrospray mass spectrometer; (b) online coupling of the SAW biosensor with an electrospray mass spectrometer via an interface used for desalting and sample concentration.

Several important aspects were taken into account during the development of the online coupling: (i) the need for physiological pH 7.4 in the analysis of protein-ligand interactions; (ii) suitable flow rate for the ESI-MS analysis; and (iii) buffers containing low salt concentrations which enable good ionisation process.

In the first strategy (Figure 9a) the waste capillary of an S-Sens K5 Biosensor (SAW Instruments GmbH) was connected directly with the inlet capillary of the electrospray mass spectrometer (Bruker Esquire 3000 ion trap mass spectrometer). A peptide-anti- 3-nitrotyrosine antibody was used as model system to study the applicability of the direct coupling of the biosensor with an electrospray mass spectrometry. The antibody was covalently immobilised on the SAM-COOH surface after a pre-activation of the free COOH-groups (Figure 10a, 1). This was followed by affinity binding of the synthetic peptide H-GKRLKNY(NO₂)SLPWGA-OH (Figure 10a, 4) and a dissociation step (6) as illustrated in Figure 10b (5).

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0 500 -30

-20 -10 0

Elution of PCS3a

Phase [°]

Time [s]

Affinity binding PCS3a

9.9697 ° 0.01935 µg cm^-2 12.6197 pmol cm^-2

0 250 500 750

Time (s)

0

-10

-20

-30

Phase [°]

0 1000 2000 3000

0 10 20 30

Immobilisation of m anti 3-NTAb

Phase [°]

Time [s]

EDC/

NHS

Ethanolamin blocking

92.8837 ° 0.1803 µg cm^-2

0 500 1000 1500 2000 2500 3000

Time (s)

30

20

10

0

Phase [°]

a b

1 2 2 3 4 5

Figure 10: Sensogram of (a) Immobilisation of an anti-3-nitrotyrosine antibody and (b) binding affinity of H-GKRLKNY(NO₂)SLPWGA-OH peptide to the antibody. The left sensogram (a) illustrate injections of EDC/NHS (1) for activation of COOH-groups followed by the immobilisation of the anti-3- nitrotyrosine antibody (2), and capping by ethanolamine (3). The right sensogram represents the association curve (4) and elution curve (5) of Tyr-nitrated peptide [H-GKRLKNY(NO₂)SLPWGA-OH]. The elution or total dissociation of the peptide was done by pH shift using a 50 mM Glycine pH 2 as described in Experimental part (Chapter 3.5.7.1). An increasing of the phase signal was observed for association of the PCS peptide (4) and a decreasing by elution (5).

The synthetic peptide (relative amount 1 pmol) bound to the monoclonal anti-3- nitrotyrosine antibody could be identified with very low relative intensity by electrospray mass spectrometer (Bruker Esquire 3000 ion trap mass spectrometer) (Figure 11).

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767.90

1534.76

0 200 400 600 800 1000 Intens.

400 600 800 1000 1200 1400 1600 1800 m/z

[M+H]⁺ [M+2H]²⁺

H-GKRLKNY⁴³⁰(NO₂)SLPWGA-OH

Figure 11: Mass spectrum of the dissociated peptide [H-GKRLKNY(NO₂)SLPWGA- OH] bound to a monoclonal anti-3-nitrotyrosine antibody. Coupling was achieved by connecting the waste capillary of SAW biosensor directly with the capillary for direct infusion into the electrospray mass spectrometer (Esquire 3000 ion trap mass spectrometer).

The relatively low flow rate, 20-40 µL min^-1, of the SAW biosensor leads to significant dilution of the eluted peptide solution, if no pre-concentration of the sample solution for mass spectrometric analysis was performed. Also, the buffer containing high salts concentrations represents a significant disadvantage for the ionisation of the eluted protein solution in the mass spectrometer. Therefore, an interface was developed and applied in a second strategy (Figure 12). The interface developed for online coupling of SAW biosensor with an ESI-Ion trap mass spectrometer utilises a six-port valve micro-column and micro-injector for desalting and in-situ concentration of protein samples dissociated from the protein-ligand complex [141, 167] as illustrated in Figure 12¹.

1 The interface developed herein has been used for interactions studies of amyloid beta and synuclein by Stefan Slamnoiu in his diploma thesis [167].

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2

6

1 4

5

3 _{5 µm frit}

1.5 cm x 1 mm packed 40 µm particle size 5 µm frit

1.5 cm x 1 mm packed 40 µm particle size

Guard column

a b

Figure 12: Schematic representation of the six-port-valve micro-column interface (a) and of the guard column (b) used for desalting and in-situ concentration of protein samples dissociated from the protein-ligand complex [141].

The online coupling of SAW biosensor with an electrospray ion trap mass spectrometer was performed manually via two flow systems (Figure 13). After SAW detection of the protein-ligand association and elution of the protein(s) into the guard column using a by pH shift (Figure 13a, flow 1), the protein(s) sample was washed with 0.3 % aqueous HCOOH to remove buffer salts. Elution of the protein(s) from the guard column with aqueous 0.3 % HCOOH/80 % acetonitrile into a manual microvalve-injector transferred the sample into the ESI source (Figure 13b, flow 2). In this mode the online coupling was carried out manually by switching the multi valve injection unit. An HPLC-pumping system was used for performing the elution. The interface connections were made using a standard fused silica capillary (ID = 153 µm).

This interface system provided routine mass spectrometric analysis of real time protein association from the biosensor surface, and showed long term operation stability with minimal contamination of the ESI-MS system. [141].

Development and bioanalytical application of affinity-mass spectrometry for identification and structural characterisation of protein-ligand interactions

mass spectrometry for identification and structural characterisation of protein-ligand interactions

Dissertation

zur Erlangung des akademischen Grades des Doktors der Naturwissenschaften

(Dr. rer. nat.)

vorgelegt von

Mihaela Stumbaum

an der

Mathematisch-Naturwissenschaftliche Sektion Fachbereich Chemie

Konstanz, 2014

Tag der mündlichen Prüfung: 29.05.2013

1. Referent: Prof. Dr. Dr. h.c. Michael Przybylski

2. Referent: Prof. Dr. Jörg Hartig

Table of contents

1 Introduction

1.1 Physiological and pathophysiological protein-ligand interactions

1.2 Analytical methods for characterisation of protein-ligand interactions

1.3 Bioaffinity-mass spectrometry for protein structure and interaction analysis

1.4 Mass spectrometric methods for protein structure determination

a

b

c

d

1.5 Scientific goals of the dissertation

2 Results and discussion

2.1 Development of online bioaffinity-mass spectrometry instrumentation