Photoionization and excitation processes in proteins and peptides

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(1)University of Groningen. Photoionization and excitation processes in proteins and peptides Egorov, Dmitrii. IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below.. Document Version Publisher's PDF, also known as Version of record. Publication date: 2018 Link to publication in University of Groningen/UMCG research database. Citation for published version (APA): Egorov, D. (2018). Photoionization and excitation processes in proteins and peptides. Rijksuniversiteit Groningen.. Copyright Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons). Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim.. Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum.. Download date: 17-07-2021.

(2) Photoionization and excitation processes in proteins and peptides. Dmitrii Egorov. 517030-L-sub01-bw-Egorov Processed on: 8-2-2018. PDF page: 1.

(3) Zernike Institute PhD thesis number: ISSN: ISBN (printed version): ISBN (electronic version):. Cover: Printed by:. 2018:10 1570-1530 978-94-034-0436-3 978-94-034-0436-6. Liana Egorova reviewing chapter 5 of this thesis Ipskamp Printing, Enshede, February 2018. The research presented in this PhD thesis was performed in the research group Quantum Interactions and Structural Dynamics (QISD) which is part of the Zernike Institute for Advanced Materials at the University of Groningen, the Netherlands. The research leading to these results has received funding from the European Community's Seventh Framework Programme (FP7/2007-2013) under grant agreement n.°312284. The authors would like to acknowledge the contribution of the COST Action CM1204 "XUV/X-ray light and fast ions for ultrafast chemistry" (XLIC). We thank HZB for the allocation of synchrotron radiation beamtime.. 517030-L-sub01-bw-Egorov Processed on: 8-2-2018. PDF page: 2.

(4) Photoionization and excitation processes in proteins and peptides. Proefschrift. ter verkrijging van de graad van doctor aan de Rijksuniversiteit Groningen op gezag van de rector magnificus prof. dr. E. Sterken en volgens besluit van het College voor Promoties. De openbare verdediging zal plaatsvinden op maandag 12 maart 2018 om 14.30 uur. door. Dmitrii Egorov geboren op 25 oktober 1988 te 6LQW3HWHUVEXUJ, 5XVODQG. 517030-L-sub01-bw-Egorov Processed on: 8-2-2018. PDF page: 3.

(5) Promotor Prof. dr. ir. R.A. Hoekstra. Copromotor Dr. T.A. Schlathölter. Beoordelingscommissie 3URIGU/$YDOGL Prof. dr. B. Noheda Pinuaga Prof. dr. J. Oomens. 517030-L-sub01-bw-Egorov Processed on: 8-2-2018. PDF page: 4.

(6) Table of Contents INTRODUCTION.........................................................................................1 1.1 Biological damage ..............................................................................................2 1.2 Astrobiology .......................................................................................................3 1.3 X-ray microscopy and crystallography ...........................................................3 1.4 Photon absorption experiments .......................................................................6 1.5 Peptides and proteins ........................................................................................8 1.6 Thesis outline.................................................................................................... 11 Bibliography .................................................................................................................. 12 CHAPTER 2 EXPERIMENT ............................................................................................. 15 2.1 PAULTJE ........................................................................................................... 16 2.2 Nanocluster trap setup .................................................................................... 18 2.3 Optimization of the high-fluence ESI source ............................................... 20 2.3.1 Ion funnel basics .......................................................................................... 20 2.3.2 Ion inlet system improvement ................................................................... 21 2.3.3 Ion funnel and octupole ion transmission experiments ......................... 21 2.3.4 RF ion funnel SIMION simulation ............................................................ 26 2.4 Microchannel plate sensitivity ....................................................................... 30 2.5 BESSY II synchrotron facility.......................................................................... 31 Bibliography .................................................................................................................. 34 NEAR-EDGE X-RAY ABSORPTION MASSSPECTROMETRY OF GAS-PHASE PROTEINS: THE INFLUENCE OF PROTEIN SIZE................................................................................................................... 37 3.1 Introduction ...................................................................................................... 38 3.2 Experiment ........................................................................................................ 39 3.3 Results and discussion .................................................................................... 43 3.3.1 Photoabsorption and auger ionization ..................................................... 43 3.3.2 Mass spectra ................................................................................................. 44 3.3.3 Electronic excitation and internal temperature ....................................... 53 3.3.4 Partial ion yields .......................................................................................... 56 3.4 Conclusions....................................................................................................... 61 Acknowledgements ...................................................................................................... 62 Bibliography .................................................................................................................. 63. 517030-L-sub01-bw-Egorov Processed on: 8-2-2018. PDF page: 5.

(7) COMPARATIVE VUV ABSORPTION MASSSPECTROSCOPY STUDY ON PROTONATED PEPTIDES OF DIFFERENT SIZE .............................................................................................................. 65 4.1 Introduction ...................................................................................................... 66 4.2 Experiment ........................................................................................................ 67 4.3 Results and discussion .................................................................................... 70 4.3.1 Valence ionization and excitation.............................................................. 70 4.3.2 Mass spectra ................................................................................................. 74 4.3.3 [YG10F+H]+ .................................................................................................... 74 4.3.4 [Gramicidin A+2H]2+ ................................................................................... 75 4.3.5 [Melittin+3H]3+.............................................................................................. 75 4.3.6 [YG5F+H]+ ...................................................................................................... 78 4.3.7 [Angiotensin I+2H]2+.................................................................................... 79 4.3.8 [PK26-P+3H]3+............................................................................................... 79 4.3.9 [Melittin+4H]4+.............................................................................................. 80 4.4 Peptide size effects ........................................................................................... 82 4.5 Conclusion ........................................................................................................ 88 Acknowledgements ...................................................................................................... 89 Bibliography .................................................................................................................. 90 NEAR-EDGE SOFT X-RAY MASS SPECTROMETRY OF PROTONATED MELITTIN ............................................................................................. 93 5.1 Introduction ...................................................................................................... 94 5.2 Experimental..................................................................................................... 95 5.2.1 The Groningen tandem mass-spectrometer ............................................. 96 5.2.2 The NanoClusterTrap at HZB .................................................................... 96 5.2.3 Electrospray .................................................................................................. 97 5.3 Results and discussion .................................................................................... 97 5.3.1 Soft X-ray spectra for non-dissociative and double ionization ............. 97 5.3.2 Small neutral losses ................................................................................... 104 5.3.3 Formation of sequence ions ...................................................................... 106 5.4 Conclusion ...................................................................................................... 112 Acknowledgements .................................................................................................... 113 Bibliography ................................................................................................................ 114 ...................................................................................................................... 117 SAMENVATTING........................................................................................................... 121 LIST OF PUBLICATIONS .............................................................................................. 125. 517030-L-sub01-bw-Egorov Processed on: 8-2-2018. PDF page: 6.

(8) INTRODUCTION. Introduction The investigation of radiation action upon peptides and proteins is not only of fundamental scientific interest but it is also important for applications to the field of radiation therapy. Here, molecular damage induced by ionizing radiation is the first and defining step of the ultimate biological radiation action. It is also relevant to astrobiology and to the entire field of X-ray based microscopy and analysis techniques, where accumulated radiation damage to the researched sample can distort the experimental results. In this introductory chapter I will underline the importance of the subject for different research areas, review the field of gas-phase peptide and protein photoabsorption spectroscopy and describe basics of protein and peptide fragmentation.. 1. 517030-L-sub01-bw-Egorov Processed on: 8-2-2018. PDF page: 7.

(9) INTRODUCTION. 1.1. BIOLOGICAL DAMAGE. The application of ionizing radiation for investigation of biological tissues started shortly after the discovery of X-rays by Röntgen in 1895 [1]. Their clinical use begun already in 1896, well before the potential threat X-rays pose to human health was known. First cases of radiation-induced cancer were reported in 1902 and subsequently numerous cases of cancer in radiation workers were observed. The first report on the occurrence of leukemia in radiation workers was published in 1912 [2]. After this, intense research on radiation damage was conducted which has shown that consequences of ionizing radiation doses exceeding 1 Sv can be both acute and chronical. For comparison it is of note that typical natural background radiation doses are on the order of 2.5 mSv per year. For radiation dose rates exceeding 0.1 Sv/h, the damage to the human organism can usually be classified as acute radiation poisoning and manifest as various syndromes in all sorts of human tissues. The most common syndromes are: hematopoietic (doses>2–3 Sv), gastrointestinal (doses 5–12 Sv) and cerebrovascular (doses 10–20 Sv). On the level of single biological cells, acute radiation damage usually leads to cell death at the time of mitosis unless the dose was extreme (>8 Sv). Therefore, damage manifests first in the fast-dividing cell lines, for instance those present in blood cells, bone marrow and, to lesser extent, intestinal cells. It is thus the average lifespan of fast-dividing cells that defines the onset of severe consequences for the entire organism. For this reason 50% lethality for the radiation dose is usually calculated after 60 days of exposure and labeled as LD50/60. LD50/60 for photon radiation accumulated during a short period of time is 3 Sv without treatment and 6 Sv with treatment. Doses higher than 8 Sv are usually 100% lethal. Acute consequences of radiation accumulated over longer periods of time are usually less severe, though, doses of radiation accumulated over long time intervals contribute significantly to the additional cancer risk: 5.5% per Sv [3]. Another possible longterm effect of ionizing radiation is an increased risk of central nervous system diseases [4]. On Earth, the magnetosphere reduces radiation exposure due to energetic solar wind ions. Radiation levels sharply increase with height: the daily dose at sea level averages to 4 μSv, while astronauts on the international space station accumulate about 500 μSv/day at an altitude of 500 km. Occasional events, such as 1972 solar storm [5] may even result in much higher doses. Cosmic radiation thus is one of the main obstacles for manned human space exploration. Radiobiological research on the influence of the ionizing radiation dose on colony forming of biological cells has already proven very early that cellular survival falls exponentially with the dose [6]. This is entirely different in comparison to other cell killing agents. Cellular exposure to poisonous substances leads to a sharp drop in 2. 517030-L-sub01-bw-Egorov Processed on: 8-2-2018. PDF page: 8.

(10) INTRODUCTION. survival upon a certain threshold. The exponential survival curve indicated that biological cells have a finite-size target volume, irradiation of which leads to cell death. It was established that the size of this target coincides with the size of the cell nucleus. Radiation damage is thus directly related to DNA damage. However, not all radiation damage can be explained by DNA strand rupture. In the nucleus DNA is wound around protein spools, the so-called histones, which give DNA its compact supercoiled form. Proteins are furthermore essential for all metabolic processes in the cell which is why their radiation damage can induce failure of cellular functions. It is therefore clear that understanding radiation action upon proteins and peptides is essential for a better understanding of biological radiation damage on the molecular level. Generally, biomolecular radiation damage can be either direct or indirect. In the case of direct damage, DNA or other relevant biomolecules are damaged directly due to the action of ionizing radiation. In the case of indirect damage, biomolecular damage is due to free radicals formed upon interaction of ionizing radiation with the chemical environment, e.g. water or other biomolecules. Both direct and indirect damage to DNA appear to be reduced by the presence of histones, which were found to protect cellular DNA [7, 8].. 1.2. ASTROBIOLOGY. Ionizing radiation induced damage is one of the limiting factors for survival of biomolecules in space, on the surface of exoplanets or on the early Earth. Another astrobiologically relevant topic is the prebiotic synthesis of complex molecules. Amino acids, the building blocks of peptides and proteins were found in high abundance on carbonaceous chondrites meteorites [9-11] and on cometary material [12]. This implies that the existence of simple peptides and proteins in space could be possible. Their potential degradation by radiation in space or on planets with very dilute atmospheres is important for understanding the initial steps for the origins of life on the early Earth and other planets. This thesis touches the question of protein and peptide survival and fragmentation pathways upon their interaction with soft X-rays and VUV photons respectively in the Chapter 3 and Chapter 4.. 1.3 X-RAY MICROSCOPY AND CRYSTALLOGRAPHY Research on small biological objects in vivo such as a living cell is an arduous task. For example, in conventional optical microscopy the performance is diffraction limited to resolutions of about half the wavelength. For better resolution a number 3. 517030-L-sub01-bw-Egorov Processed on: 8-2-2018. PDF page: 9.

(11) INTRODUCTION. Figure 1.1 Three-dimensional structure of myoglobine observed in 1957 using X-ray crystallography [16].. of beyond-the-diffraction-limit approaches have been introduced over the years but it is still most straightforward to reduce wavelength. Electron microscopy is based on the very short de Broglie wavelength of energetic electrons but requires the sample to be dehydrated, embedded in resin and sliced ultra-thin [13]. This makes the application of this method to large biological objects such as relatively thick biological cells very difficult. The other possibility for short wavelength studies is the use of X-rays [14]. X-rays were first applied for research on material structures in 1913 by W.L. Bragg and W.H. Bragg [15]. The first application of X-rays in the research on complex biological samples was crystallography of myoglobin which yielded the threedimensional structure of this small protein (see Figure 1.1) [16]. Typically, nowadays for these types of diffraction studies, hard X-rays (several 10 keV) from synchrotron sources are employed which is why in the following the technique will be denoted as hard X-ray crystallography. It is important to note, that high doses of X-rays are able to damage the samples, in particular if the crystals are not cooled [17]. Cooling of the sample crystals strongly reduces secondary damage, as it prevents free radical diffusion [18]. 4. 517030-L-sub01-bw-Egorov Processed on: 8-2-2018. PDF page: 10.

(12) INTRODUCTION. Figure 1.2 An image of the mother and daughter yeast cell pair. Picture a shows a 2D slice of the soft X-ray 3D tomogram. The grey scale corresponds to the absorption coefficient. Picture b shows the same pair of cells, but with the subcellular components colored according to their X-ray attenuation [27].. Despite its paramount importance for determination of protein structure, hard Xray crystallography cannot be applied to targets in vivo as the technique requires a periodic sample structure, i.e crystalline targets. For X-ray imaging of complex biological system that lack periodicity such as cells or cell organelles, soft X-ray transmission microscopy is a powerful technique. Biological samples under natural conditions imply the presence of water. It is thus beneficial to employ soft X-rays with energies in the water window [19, 20]. The water window refers to the energy range where water is transparent for soft X-rays, but carbon-containing molecules are not. This corresponds to soft X-rays with energies between the K-absorption edge of carbon at about 280 eV and the K-absorption edge of oxygen at about 530 eV. First significant progress in soft X-ray microscopy was made in the 1970s and 1980s by the group of Schmahl et al [21, 22] and Kirz et al [23, 24], who developed fullfield transmission soft X-ray microscopy and scanning transmission soft X-ray microscopy, respectively. Both of them used synchrotron radiation. Initially, the resolution of the method was 100-200 nm, and improved to 50 nm in the late 1980s. At present, resolutions around 10 nm are possible [25, 26]. The example of a soft Xray scanning tomography image shown in Figure 1.2 ([27]) has a resolution of 36 nm. It is notable that even though it is generally possible to investigate watercontaining samples, it is usually necessary to keep the sample at cryogenic temperatures in order to avoid secondary radiation damage. In gas-phase irradiation experiments only primary radiation damage is possible. This implies that soft X-ray gas-phase experiments could facilitate the assessment and the unraveling of primary and secondary damage occurring during soft X-ray microscopy of frozen biological samples. . Soft X-ray microscopy techniques are not limited to biological systems. They are also widely applied in research on solar cells, batteries and magnetic structures, works of art and archeology. Generally speaking, biological samples are most 5. 517030-L-sub01-bw-Egorov Processed on: 8-2-2018. PDF page: 11.

(13) INTRODUCTION. vulnerable to ionizing radiation. Consequently, the issue of radiation damage is most relevant for soft X-ray microscopy of biological matter. The focus of this thesis is the interaction of energetic photons with peptides and proteins in the gasphase, rather than in the condensed phase. Gas-phase studies are inherently free of any secondary radiation damage. Therefore, interactions with gas-phase peptides and proteins are relevant for condensed phase techniques such as soft X-ray microscopy, as they are a perfect testbed for the determination of the sole primary radiation action induced by photoabsorption.. 1.4. PHOTON ABSORPTION EXPERIMENTS. Due to experimental challenges in the production of sufficiently dense gas phase targets of proteins and peptides, gas-phase protein and peptide photoabsorption spectroscopy experiments were preceded by experiments on thin films of amino acids and peptides. The first near-edge X-ray absorption fine structure spectroscopy (NEXAFS) study of amino acid and peptide thin films was performed in 1997 [28]. This study has shown that for small dipeptides such as Gly-Tyr, the total NEXAFS spectrum can be constructed as a superposition of the NEXAFS spectra of the constituting amino acids. In the following years this idea was further developed and C, N and O K-edge NEXAFS spectra of all common amino acids were measured [29]. It was shown in [30] that the superposition principle also holds for NEXAFS spectra of larger proteins. Accordingly, soft X-ray microscopy has been applied to thin films in order to map the position of proteins on the sample [31]. Initially, gas-phase photo-absorption experiments with energetic photons were only performed for relatively small systems such as amino acids and very small peptides [32-37]. VUV and soft X-ray spectroscopy of larger gas-phase peptides and proteins has been pioneered by Bari and coworkers who performed VUV absorption mass-spectrometry of leucine enkephalin [38] and by Milosavljevic et al. who performed photoionization studies on cytochrome C [39]. In the past 6 years the field has significantly evolved. However, one of the most important tasks to the research field remains the establishment of a tool kit to determine the structure of single, gas-phase proteins. A first logical step into that direction is the development of experimental procedures and techniques to distinguish between the folded, native protein conformation and its unfolded, denatured conformation. For example, the influence of protein folding on the evolution of the ionization potentials of BPTI, ubiquitin and cytochrome C with protonation state was studied by means of VUV absorption experiments in [40]. Experiments with soft X-rays have shown that it is also possible to observe a shift of the C 1s ionization potential transition upon protein conformational change [41]. 6. 517030-L-sub01-bw-Egorov Processed on: 8-2-2018. PDF page: 12.

(14) INTRODUCTION. Both findings hint at potential applications of soft X-ray and VUV absorption mass-spectroscopy for research on protein tertiary structure. In addition to dedicated structural studies, numerous other VUV absorption experiments were performed unveiling patches of information. Research of leucine enkephalin [42] and substance P [43] has allowed for a detailed insight into the correlation of fragmentation pathways with photon energy which can be linked to the initial excitation of a specific electron orbital. For example, the production of the leucine enkephalin b3+ fragment shows a maximum yield at 6.9 and 9.6 eV, which can be related to exciting the Δ2Δ3‫( כ‬NV1) transition and Δ1Δ3‫( כ‬NV2) transition, respectively. The specific nomenclature of the molecular break-up fragments of a protein or peptide is presented in section 1.5. Experiments studying the effect of the absorption of 6.4-16 eV photons by ubiquitin have shown that the kind of fragments generated after VUV photon absorption depends on the photon energy [44]. Most notably, in the case of dissociative photoionization backbone fragments are distributed evenly along the backbone, while for photodissociation at photon energies below the ionization potential of the protein fragmentation is dominated by “a” fragments. VUV experiments with leucine enkephalin complexes have shown that the addition of a few water molecules to a peptide dimer significantly increases its stability upon photoabsorption [45]. Systematic research on VUV photon ionization of [YGnF+H]+ peptides with n varying from 1 to 10 has provided insights both into the charge transfer mechanisms in peptides and into the molecular size effects on the fragmentation patterns [46]. The fragmentation spectra of the smaller peptides are totally dominated by immonium fragments stemming from the Y and F residues at the ends of the peptides. The larger species undergo significant backbone scission or non-dissociative ionization. This was explained by the fact that the G chain in the larger peptides becomes too long for efficient charge transport to the Y and F end terminal residues. First soft X-ray absorption experiments with leucine enkephalin [47] and cytochrome C [48] have shown strikingly different overall patterns of the resulting photoabsorption mass spectra. In the case of leucine enkephalin, the mass spectrum is dominated by small fragments, while for cytochrome C nondissociative ionization and small neutral loss channels dominate. The reason for such a difference is investigated in detail in Chapter 3. Last, but not least multiple EUV photon ionization of ubiquitin experiments have revealed that this relatively large molecular system behaves like an ensemble of small peptides [49]. This implies that ionization triggers an ultrafast localized 7. 517030-L-sub01-bw-Egorov Processed on: 8-2-2018. PDF page: 13.

(15) INTRODUCTION. Figure 1.3 Peptide bond between two amino acids. The drawing was made in PyMOL.. response by the peptide that absorbed the photon before the deposited energy is redistributed over the system. All in all it can be clearly seen that the emerging field of gas-phase peptides photoabsorption studies has significantly matured over the last years. For one, the role of the size of the proteins in the handling of ultrafast energy deposition by photon absorption is being unraveled [46-48]. The transition from a small molecule-like behavior to a large molecule-like behavior in VUV and soft X-ray photoabsorption experiments is analyzed in detail in Chapter 3 and 4. Another important issue researched during the last years is the application of synchrotron radiation to protein conformation studies [40, 41]. In Chapter 5 we will investigate the dependency of the molecular response to soft X-ray photons for different peptide conformations.. 1.5. PEPTIDES AND PROTEINS. A protein consists of one oȱ ȱ ȱ ȱ ΅-amino acid residues, connected by peptide bonds (Figure 1.3). Chains longer than 40 amino acid residues are usually referred to as proteins, while shorter ones are called peptides. In this thesis both proteins (insulin (porcine, 51 residues), ubiquitin (bovine, 76 residues), cytochrome C (equine, 104 residues)) and various peptides (leucine enkephalin (5 residues), YG5F (7 residues), YG10F (12 residues), angiotensin I (10 residues), gramicidin A (15 8. 517030-L-sub01-bw-Egorov Processed on: 8-2-2018. PDF page: 14.

(16) INTRODUCTION. Figure 1.4 The 4 levels of protein structure [51].. residues), melittin (26 residues), and the collagen fragment PK26-P (26 residues)) are investigated. Proteins have four levels of structure as it can be seen in Figure 1.4 . Each protein has a primary structure that corresponds to the sequence of the amino acid residues forming the polypeptide. Biological functionality of polypeptides originates from their secondary and tertiary structure. Secondary structure refers 9. 517030-L-sub01-bw-Egorov Processed on: 8-2-2018. PDF page: 15.

(17) INTRODUCTION. Figure 1.5 Peptide and protein fragmentation notation.. to the formation of alpha-helices, beta-pleated sheets and more. These helices and sheets are held together by means of intramolecular hydrogen bonds. The secondary structures fold into the tertiary one. This is caused by a large variety of properties of residue sidechains and the resulting ionic interactions, hydrogen bonds, van der Waals dispersion forces, sulphur bridges, hydrophobic and hydrophylic interactions. Complexes of two or more proteins are referred to as quaternary structures. Protein functionality implies interactions with other components of the living cell. This ability can be defined, for example, by the protein mobility in cell membranes, its ability to change its structure to allow interactions with other components, etc.. Tertiary and secondary structure of polypeptide define these characteristics and we can only fully understand how proteins work if we understand all these levels of polypeptides organization. One of the promising methods to investigate polypeptide three-dimensional structure in the gas phase could be circular dichroism NEXAFS, which might be sensitive to the polypeptide secondary structure. Circular dichroism is the difference in molecular photoabsorption cross section between left- and right- circularly polarized photons of the same energy and it is routinely applied to quantify for instance helicity contents in liquid phase proteins using visible or UV light. In this thesis we will use the common nomenclature for peptide fragmentation shown in Figure 1.5. Depending on the number and localization of charges after backbone scission, charged N-terminal fragments: a-,b-,c- or C-terminal fragments: :x-,y-,z- or both can be formed [50]. Other possible fragmentation channels are combinations of double bond scissions from both, N-terminal and C-terminal sides, leading to formation of so-called internal fragments. A particular class of internal fragments are the immonium ions, containing a single side chain and formed by a combination of a-type and y-type scissions. Further in the text they will be noted using their amino acid letter code.. 10. 517030-L-sub01-bw-Egorov Processed on: 8-2-2018. PDF page: 16.

(18) INTRODUCTION. 1.6. THESIS OUTLINE. This thesis is organized as following: x In chapter 2 I will highlight the major features of the home-built tandem mass spectrometer “PAULTJE”, as most experiments performed in the course of this thesis were performed with this apparatus. I will focus on technical improvements to the setup, implemented during this thesis. I will furthermore describe the Nanocluster Trap setup, used for a number of experiments presented in chapter 5. Last but not least, I will give a brief description of the 3rd generation synchrotron facility BESSY II and the relevant beamlines. x Chapter 3 is devoted to the protein size dependency of molecular survival upon soft X-ray photoionization at the C K-edge. Smaller peptides were found to fragment upon photoabsorption, while larger systems tend to photoionize non-dissociatively. x Chapter 4 investigates the size dependence from non-dissociative ionization to massive fragmentation in VUV photoionization of protonated peptides. The smaller amount of deposited energy as compared to soft X-ray absorption results in qualitatively and quantitatively different fragmentation patterns and a lower mass at which the transition between the different regimes occurs. x Chapter 5 presents a soft X-ray spectroscopy study on protonated melittin. The influence of protonation state on non-dissociative single and double ionization will be discussed and directly related to the secondary structure. The influence of protonation on backbone scission will be discussed in detail.. 11. 517030-L-sub01-bw-Egorov Processed on: 8-2-2018. PDF page: 17.

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(20) BIBLIOGRAPHY. [21] G. Schmahl and D. Rudolph, Optik, 29, 577 (1969). [22] B. Niemann, D. Rudolph and G. Schmahl, Appl.Opt., 15, 1883 (1976). [23] H. Rarback, D. Shu, S. Feng, H. Ade, J. Kirz, I. Mcnulty, D. Kern, T. Chang, Y. Vladimirsky, N. Iskander, D. Attwood, K. Mcquaid and S. Rothman, Rev.Sci.Instrum., 59, 52 (1988). [24] J. Kirz, H. Ade, E. Anderson, D. Attwood, C. Buckley, S. Hellman, M. Howells, C. Jacobsen, D. Kern, S. Lindaas, I. Mcnulty, M. Oversluizen, H. Rarback, M. Rivers, S. Rothman, D. Sayre and D. Shu, Phys.Scripta, T31, 12 (1990). [25] J. Vila-Comamala, K. Jefimovs, J. Raabe, T. Pilvi, R.H. Fink, M. Senoner, A. Maassdorf, M. Ritala and C. David, Ultramicroscopy, 109, 1360 (2009). [26] W. Chao, J. Kim, S. Rekawa, P. Fischer and E.H. Anderson, Optics Express, 17, 17669 (2009). [27] D.Y. Parkinson, G. McDermott, L.D. Etkin, M.A. Le Gros and C.A. Larabell, J.Struct.Biol., 162, 380 (2008). [28] J. Boese, A. Osanna, C. Jacobsen and J. Kirz, Journal of Electron Spectroscopy and Related Phenomena, 85, 9 (1997). [29] Y. Zubavichus, A. Shaporenko, M. Grunze and M. Zharnikov, Journal of Physical Chemistry A, 109, 6998 (2005). [30] Y. Zubavichus, A. Shaporenko, M. Grunze and M. Zharnikov, J Phys Chem B, 112, 4478 (2008). [31] J. Stewart-Ornstein, A.P. Hitchcock, D. Hernandez Cruz, P. Henklein, J. Overhage, K. Hilpert, J.D. Hale and R.E.W. Hancock, J Phys Chem B, 111, 7691 (2007). [32] O. Plekan, V. Feyer, R. Richter, M. Coreno, M. de Simone, K.C. Prince and V. Carravetta, Journal of Physical Chemistry A, 111, 10998 (2007). [33] K. Wilson, M. Jimenez-Cruz, C. Nicolas, L. Belau, S. Leone and M. Ahmed, Journal of Physical Chemistry A, 110, 2106 (2006). [34] V. Feyer, O. Plekan, R. Richter, M. Coreno, K.C. Prince and V. Carravetta, Journal of Physical Chemistry A, 112, 7806 (2008). [35] O. Plekan, V. Feyer, R. Richter, M. Coreno and K.C. Prince, Mol.Phys., 106, 1143 (2008). [36] F. Talbot, T. Tabarin, R. Antoine, M. Broyer and P. Dugourd, J.Chem.Phys., 122 (2005). [37] M. Thompson, W. Cui and J. Reilly, Angew.Chem.-Int.Edit., 43, 4791 (2004). 13. 517030-L-sub01-bw-Egorov Processed on: 8-2-2018. PDF page: 19.

(21) BIBLIOGRAPHY. [38] S. Bari, O. Gonzalez-Magaña, G. Reitsma, J. Werner, S. Schippers, R. Hoekstra and T. Schlathölter, J.Chem.Phys., 134 (2011). [39] A.R. Milosavljevic, C. Nicolas, J. Lemaire, C. Dehon, R. Thissen, J. Bizau, M. Refregiers, L. Nahon and A. Giuliani, Phys.Chem.Chem.Phys., 13, 15432 (2011). [40] A. Giuliani, A.R. Milosavljevic, K. Hinsen, F. Canon, C. Nicolas, M. Refregiers and L. Nahon, Angewandte Chemie-International Edition, 51, 9552 (2012). [41] A.R. Milosavljevic, C. Nicolas, M.L.J. Rankovic, F. Canon, C. Miron and A. Giuliani, Journal of Physical Chemistry Letters, 6, 3132 (2015). [42] M.L. Rankovic, F. Canon, L. Nahon, A. Giuliani and A.R. Milosavljevic, J.Chem.Phys., 143 (2015). [43] F. Canon, A.R. Milosavljevic, L. Nahon and A. Giuliani, Physical Chemistry Chemical Physics, 17, 25725 (2015). [44] F. Canon, A.R. Milosavljevic, G. van der Rest, M. Refregiers, L. Nahon, P. Sarni-Manchado, V. Cheynier and A. Giuliani, Angewandte Chemie-International Edition, 52, 8377 (2013). [45] A.R. Milosavljevic, V.Z. Cerovski, F. Canon, L. Nahon and A. Giuliani, Angewandte Chemie-International Edition, 52, 7286 (2013). [46] O. Gonzalez-Magaña, G. Reitsma, S. Bari, R. Hoekstra and T. Schlathölter, Physical Chemistry Chemical Physics, 14, 4351 (2012). [47] O. Gonzalez-Magaña, G. Reitsma, M. Tiemens, L. Boschman, R. Hoekstra and T. Schlathölter, Journal of Physical Chemistry A, 116, 10745 (2012). [48] A.R. Milosavljevic, F. Canon, C. Nicolas, C. Miron, L. Nahon and A. Giuliani, J.Phys.Chem.Lett., 3, 1191 (2012). [49] T. Schlathölter, G. Reitsma, D. Egorov, O. Gonzalez-Magaña, S. Bari, L. Boschman, E. Bodewits, K. Schnorr, G. Schmid, C.D. Schroeter, R. Moshammer and R. Hoekstra, Angewandte Chemie-International Edition, 55, 10741 (2016). [50] P. Roepstorff and J. Fohlman, Biomed.Mass Spectrom., 11, 601 (1984). [51] OpenStax College, "Proteins", (2013).. 14. 517030-L-sub01-bw-Egorov Processed on: 8-2-2018. PDF page: 20.

(22) EXPERIMENT. Chapter 2 Experiment Most experiments presented in this thesis were performed with the tandem mass spectrometer PAULTJE. For a few experiments the nanocluster trap mass spectrometer [1] installed at the BESSY II synchrotron was used. PAULTJE was described in detail previously in the PhD theses of Sadia Bari [2] and Olmo Gonzalez-Magaña [3]. I will focus on the changes and improvements of the setup made during my PhD project which mainly concern the improvement of the biomolecular ion beam current. A high current which defines the target density is a stringent requirement for being able to perform experiments on gas phase molecules. Key aspects of a high-fluence source are variable frequency and amplitude RF supplies for ion funnel and octupole, allowing for maximum control over ion transmission, as well as large diameter inlet capillaries. In order to understand the dependency of ion transmission on RF-amplitude, frequency and pressure, SIMION 8.0.4 simulations of the ion transmission through the ion funnel and octupole were performed and compared with experimental data. Based on the performance tests, the entire design of the existing ion funnel chamber was improved. The photoabsorption experiments were performed at VUV and soft X-rays beamlines of the 3rd generation synchrotron BESSY II. Therefore, also a short description of the BESSY II facility and in particular the beamlines used by us is given.. 15. 517030-L-sub01-bw-Egorov Processed on: 8-2-2018. PDF page: 21.

(23) EXPERIMENT. 2.1. PAULTJE. The PAULTJE apparatus is a mobile tandem mass-spectrometry setup able to perform a wide range of experiments with gas-phase molecular ions. Examples are soft X-ray and VUV photoabsorption studies on peptides and proteins [4-7], nucleotides [8] and polyaromatic hydrocarbons (PAH) [9, 10], keV ion collision experiments [11, 12], femtosecond laser experiments [13, 14] and hydrogenation and abstraction reactions involving PAHs [15, 16]. The interaction of target molecular ions with synchrotron radiation, keV ion beams or atomic hydrogen takes place in the heart of the setup – the 3D RF Paul Trap, for which’s invention, Wolfgang Paul was awarded the Nobel Prize [17]. In order to gently transfer biomolecular ions into the gas-phase electrospray ionization (ESI), another Nobel Prize invention [18], is employed. Molecular ions which are produced by the electrospray ionization source are generated at atmospheric pressure. However, typical pressures in the Paul trap are as low as 10-6-10-7 mbar in order to prevent photon or keV ion interactions with residual gas in the trap. The transfer of ions from atmospheric pressure to high vacuum is a sophisticated multistage process, whose efficiency defines the transmission of ions from the source to the trap. The process starts with the ions being transferred from the atmospheric pressure region through a heated capillary (T=350 K) into a RF ion funnel (3-7x10-1 mbar) where the ions are phase space compressed. Subsequently, the ions enter a RF-octupole (3-5x10-4 mbar), in which they are accumulated for hundreds of ms up to several seconds by controlling the bias voltage on the exit diaphragm of the octupole. The ions are extracted from the octupole by lowering the exit diaphragm bias voltage for a duration of about 50 ms. The ions then enter the RF quadrupole mass-filter, which is used to mass-select the molecule with the m/z of interest. Eventually, the ions enter the Paul trap, where they are subjected to a 50 ms pulse of helium buffer gas which facilitates trapping by collisionally cooling of excess ion kinetic energy. The cooled and trapped ions are then exposed to the photon or keV ion beam for a well-defined period, before a second helium buffer gas pulse is injected in order to cool down the kinetic energy of fragments formed in dissociation processes of the precursor molecular ions. Finally the ions are extracted into the time of flight analyzer (p=5x10-8-2x10-7 mbar) and detected by a microchannel plate detector. Due to the limited ion capacity of the Paul trap (104-105), a large number of cycles, typically 500-2000, is required in order to obtain a good signal-to-noise ratio.. 16. 517030-L-sub01-bw-Egorov Processed on: 8-2-2018. PDF page: 22.

(24) EXPERIMENT. Figure 2.1 Sketch of the experimental setup. The length of the time of flight (TOF) system between the 3D RF trap and the MCP detector at the end of the TOF tube is not to scale.. 17. 517030-L-sub01-bw-Egorov. PDF page: 23. Processed on: 8-2-2018.

(25) EXPERIMENT. Figure 2.2 Sketch of the Nanocluster trap interfaced with the UE52-PGM beamline at BESSY II [1]. For our experiment the cluster source is replaced by our ESI+funnel combination.. 2.2. NANOCLUSTER TRAP SETUP. Most experiments presented in chapter 5 of this thesis were performed with the Nanocluster trap operated by the research group of T. Lau [1]. The Nanocluster trap, see Figure 2.2, is a stationary tandem mass-spectrometry setup permanently interfaced to the BESSY II UE52_PGM beamline. The apparatus is equipped with a superconducting solenoid magnet for magnetization of metal clusters and is mostly used for soft X-ray magnetic circular dichroism experiments [19, 20]. For our experiments we interfaced our ESI and RF ion funnel with the Nanocluster Trap. Due to the much larger ion capacity of the linear RF trap, it can accommodate mass selected ion currents of 100 pA or more. Typical ion currents of conventional combined ESI and RF funnel devices are of the order of 5 pA. The improvements made to our system to fully exploit the trapping capacity of the Nanocluster trap will be highlighted in the following subsection. Ions transferred into the gas-phase are guided by the RF hexapole ion guide (Figure 2.2, 1. ion guide) into the RF mass filter, where ions with the required m/z can be selected. Afterwards mass-selected ions are focused by electrostatic lenses and deflected by a static quadrupole bender into the liquid helium cooled linear RF-quadrupole ion trap. In contrast to the PAULTJE setup, in the Nanocluster trap, helium buffer gas is present during the entire operation cycle. Ions can be cooled to liquid helium temperature (4K, this corresponds to final internal temperatures of the ions between 10K and 15K due to RF heating in the trap). Ions fill the trap up to the space charge limit and are then subjected to polarized soft X-ray radiation with typical photon fluxes of 1012 18. 517030-L-sub01-bw-Egorov Processed on: 8-2-2018. PDF page: 24.

(26) EXPERIMENT. Figure 2.3 Timing scheme of the Nanocluster Trap experiment [1].. photons/s yielding about 105 daughter ions per second. Afterwards the ions are extracted from the ion trap into the reflectron time-of-flight mass spectrometer. The timing scheme for extraction is depicted in Figure 2.3. A fraction of the ions contained in the trap is extracted into the TOF spectrometer with every voltage pulse to the ion trap exit aperture. After a delay, tuned for detection of specific daughter ions, the first acceleration stage of the TOF spectrometer is pulsed and the daughter ion’s m/z and intensity is detected. At the end of the cycle, the obtained mass spectra are averaged and the ion trap is emptied by switching off the RF voltage. Subsequently, a new photon energy is set and a new measurement cycle starts. ȱȱȱȱȱȱȱȱȱȱȦ̇ǅŗŝŖŖǯȱ ȱȱȱȱ reach the MCP detector which detects the signal induced by 103-104 ions. About 110 % of the extracted ions is detected due to the efficiency of the detector. In addition to that, the extraction efficiency is not the same for ions with different m/z which affects the direct comparison of mass spectra.. 19. 517030-L-sub01-bw-Egorov Processed on: 8-2-2018. PDF page: 25.

(27) EXPERIMENT. Figure 2.4 Sketch of the ion funnel with capillary and octupole ion guide.. 2.3 OPTIMIZATION OF FLUENCE ESI SOURCE 2.3.1. THE. HIGH-. Ion funnel basics. The invention of electrospray ionization has provided a powerful tool for transfer of biomolecular ions from the liquid phase into the gas phase. Initially, the most common method for transfer from atmospheric pressure into vacuum was the application of a skimmer. However the ion transmission in this case is usually only around 0.01% [21]. Initially, our group was using a skimmer for the ion transfer, which was subsequently replaced by an ion funnel allowing for more efficient ion transmission. One of my first tasks was the optimization of the ion funnel performance. The key problem in the ion transfer is that according to the Liouville theorem it is not possible to slow down and focus ions at the same time, due to phase space volume conservation. However, the necessary ion transmission cannot be achieved without the slowing down and compression of the ion beam. The phase space can 20. 517030-L-sub01-bw-Egorov Processed on: 8-2-2018. PDF page: 26.

(28) EXPERIMENT Table 2.1 Tabulation overview of the standard settings of the ESI, ion funnel (IF) and octupole (8-pole), see Figure 2.4 , for the ion transmission experiments. Vpp indicates the peak-to-peak voltages of a specific RF field.. ESI (kV) Capillary (V) Elect1 (V) 4.53. 100. 8-pole bias (V). 8-pole RF (kHz). 8. 700. 110. Elect26 (V) Diaph (V) IF RF (kHz) IF RF Vpp (V) 13. 11. 8-pole RF 8-pole exit 8-pole Vpp (V) (V) pulse (V) 190. 5. 50. 500. 240. Einzel lens (V). IF pressure (mbar). 7.7. 0.3. be compressed by means of collisional damping, which takes place in low vacuum RF devices such as the ion funnel. The funnel was first implemented in 1997 by [22] in order to optimize the transmission efficiency. Subsequently, a large number of ion funnel designs was developed, while key features of the ion funnel remained the same. Typical funnels consist of a stack of rings, whose internal diameters gradually decrease towards the funnel exit, see Figure 2.4. An RF field is applied to the ring electrodes in a way that the phase difference between adjacent electrodes is 180 degrees. As in a conventional ring electrode trap [23] ions are confined radially. At the same time ions are pushed forward by the application of a continuous DC gradient between the first and the last ring. Due to collisional damping the ions are eventually focused near the exit of the ion funnel and transferred to the octupole. The funnel we are using is sketched in Figure 2.4 together with the entrance capillary and the octupole. The funnel is based on the design described in [24].. 2.3.2. Ion inlet system improvement. One of the most obvious possible improvements of the initial stage of the ion guiding system is the increase of the capillary diameter. An increase of the capillary diameter from 0.508 to 0.762 mm (standard commercial capillary inner diameters) increases the capillary cross section by more than a factor of two and as a consequence the biomolecular ion flow into the funnel increases by a similar factor. The implementation of such a large diameter capillary however has implications on the required pumping speed. We use an IGX6/100l Edwards root pump with a booster. It has a pumping speed of 600 m3/h, and allows for a minimum pressure in the ion funnel chamber of 0.3 mbar.. 2.3.3. Ion funnel and octupole ion transmission experiments. In order to quantify and optimize the high fluence ESI source it was necessary to perform ion transmission experiments. We investigated the transmission 21. 517030-L-sub01-bw-Egorov Processed on: 8-2-2018. PDF page: 27.

(29) EXPERIMENT. Figure 2.5 Ion transmission as a function of the ion funnel’s RF frequency for two pressures inside the ion funnel of p=0.3 mbar and p=0.7 mbar. System settings are listed in the Table 2.1.. dependency on ion funnel and octupole settings by using a beam of [leucine enkephalin+H]+, a model peptide for mass-spectrometry with m=555.6 Da. In conventional mass spectrometric systems, ion funnels are operated with RF voltage generators with fixed frequency. We use a much more versatile approach, where sine signals of variable frequency and amplitude are generated by a function generator and amplified by RF power amplifiers. Full control over frequency and amplitude allows for much better optimization of the ESI output. We have systematically investigated the influence of the diaphragm voltage, capillary voltage, first electrode voltage, octupole RF voltage and frequency, ion funnel RF amplitude and frequency. The basic experimental settings used in the optimization are summarized in Table 2.1. Throughout the following sections, these settings are used, unless stated otherwise. Additionally, we have varied the pressure in the ion funnel chamber from 0.3 to 0.7 mbar by modifying the pumping speed to investigate the pressure effect on ion transmission. On the respective graphs, ion transmission is normalized to the maximal ion transmission in the measurement series and labeled as relative transmission. It was established during the experiments that the ion transmission depends strongly on the ion funnel. 22. 517030-L-sub01-bw-Egorov Processed on: 8-2-2018. PDF page: 28.

(30) EXPERIMENT. Figure 2.6. The dependency of ion transmission on the voltage on the exit diaphragm of the funnel for funnel pressures of p=0.3 mbar and p=0.7 mbar. System settings are listed in the Table 2.1.. diaphragm voltage, pressure, RF frequency and peak-to-peak voltage (see Figure 2.5 - Figure 2.7). The influence of the voltages on the first electrode and capillary appears less significant (see Figure 2.8). Figure 2.5 shows the dependency of ion transmission on the ion funnel’s RF peakto-peak voltage and frequency for two pressures. It can be clearly seen that the ion transmission dramatically varies with the amplifier frequency. The ion transmission as a function of RF frequency exhibits a broad peak with a maximum ȱǅŚśŖȱ £ȱȱƽŖǯŝȱǰȱȱǅŝŖŖȱ £ȱȱƽŖǯřȱǯȱȱ¡ȱȱȱ transmission notably shifts to lower frequencies with increasing pressure. Figure 2.6 shows the ion transmission as a function of the voltage on the exit diaphragm of the funnel for gas pressures inside the funnel of p=0.3 mbar and p=0.7 mbar. For both pressures the transmission dependency on the diaphragm voltage maximizes over a range of voltages, i.e 9-13 V. The distribution is somewhat broader for the lower pressure of p=0.3 mbar. This can be explained by the higher energy of the ions in the end of the ion funnel. This happens due to less collisional damping events at a lower pressure. 23. 517030-L-sub01-bw-Egorov Processed on: 8-2-2018. PDF page: 29.

(31) EXPERIMENT. Figure 2.7 Relative ion transmission through the ion funnel as a function of the RF peak-to-peak voltage (Vpp) for various frequencies. System settings are listed in the Table 2.1.. 24. 517030-L-sub01-bw-Egorov Processed on: 8-2-2018. PDF page: 30.

(32) EXPERIMENT. Vcap = 120 V Vcap = 60 V Vcap = 0 V. Relative transmission. 1.0. 0.8. 0.6. 0.4. 0.2. 0.0. 0. 50. 100. 150. 200. 250. Electrode 1 (V). 0. 50. 100. 150. 200. Capillary (V). Figure 2.8 Left panel: relative ion transmission as a function of the voltage on electrode 1 for 3 different capillary voltages (Vcap). Right panel: relative ion transmission as a function of capillary voltage for a fixed electrode 1 voltage of 110 V. System settings are listed in the Table 2.1.. Figure 2.7 depicts the ion transmission as a function of the RF peak-to-peak voltage for 6 different frequencies. It can be clearly seen that the working range is extremely narrow for low frequencies (100, 200 kHz) and tends to broaden and shift to higher voltages with increasing frequency. Figure 2.8 shows the ion transmission as a function of capillary voltage and electrode 1 voltage. The measurements indicate that the operating region for these parameters is very broad. The lower relevance of the electrode1 and capillary voltage to the ion transmission can be explained by the notable gas flow near the exit of the capillary which is additionally pushing ions towards the exit of the ion funnel, thus making the values of the voltages near the entrance area less important for the ion transmission. Figure 2.9 shows the ion transmission as a function of octupole RF frequency. It can be clearly seen that similar to the case of the ion funnel’s RF frequency, the operation range shifts to higher peak-to-peak voltages when the frequency increases. However, there is no observable operation range broadening as found for the ion funnel. Having mapped the ion transmission as a function of the relevant parameters, it is now possible to choose optimum settings for ESI operation. It is also clear that using a wide band RF power amplifier is key to optimizing the transmission, as the transmission is very sensitive to experimental settings, as can be seen from Figure 2.5. 25. 517030-L-sub01-bw-Egorov Processed on: 8-2-2018. PDF page: 31.

(33) EXPERIMENT. 1.0. f=500 kHz. f=700 kHz. f=900 kHz. f=1200 kHz. 0.8. Relative transmission. 0.6. 0.4. 0.2 1.0. 0.8. 0.6. 0.4. 0.2. 0. 50. 100. 150. 200. 250. 50. 100. 150. 200. 250. Octupole RF Vpp (V) Figure 2.9 Ion transmission as a function of octupole RF peak-to-peak amplitude for various frequencies. System settings are listed in Table 2.1.. 2.3.4. RF ion funnel SIMION simulation. In order to develop an ion funnel design for maximum ion transmission and to define precision limits for the maximum allowed misalignment between e.g. funnel and octupole it was necessary to model ion trajectories through the ion funnel and the octupole. To this end, the ion optics simulation package SIMION 8.0.4 was used [25]. Initially, SIMION calculates the electric field at every point of a userdefined potential array by solving the Laplace equation. Subsequently, ion trajectories can be calculated in a three dimensional space using a 4th-order RungeKutta method. Collisions of the electrosprayed ions with residual gas molecules in the ion funnel chamber were taken into account by the application of a hard sphere 26. 517030-L-sub01-bw-Egorov Processed on: 8-2-2018. PDF page: 32.

(34) EXPERIMENT. Figure 2.10 Ion funnel simulation result sample for [leucine-enkephalin+H]+. Simulation settings are based on the Table 2.1. Starting ion energy is 1-3 eV.. collision model. This model is based on the following approximations: x. Ion collisions are treated as collisions between two hard spheres. Energy transfer occurs solely via these collisions.. x. The collisions are elastic.. x. Background gas is assumed to be neutral.. x. The background gas velocity follows a Maxwell-Boltzmann distribution.. x. The mean velocity of the background gas may be non-zero.. x. Kinetically cooling and heating collisions are considered, i.e, both positive and negative energy transfer from the buffer gas to the ions is possible.. x. Background gas as a whole is unaffected by ion collisions: no heating, ionization or fragmentation processes are taken into account.. Knowing the approximate current of ions from experiments it was possible to calculate the ion repulsion effect on ion trajectories. The method employed only takes into account the Coulomb repulsion between ions. However, it assumes that the ion beam propagates evenly in the ion funnel, while in reality some biomolecular ions can be trapped between ion funnel electrodes for a time longer than average ion flight time, leading to an extra space-charge. The ion trajectories 27. 517030-L-sub01-bw-Egorov Processed on: 8-2-2018. PDF page: 33.

(35) EXPERIMENT. Relative transmission. 1.0 0.8 0.6 0.4 0.2 0.0 200. 400. 600. 800 1000 1200 1400. Ion funnel RF frequency (kHz). 4. 6. 8. 10. 12. 14. 16. 18. 20. Diaphragm voltage (V). Figure 2.11 Comparison of measured and simulated ion transmission: 1) left panel: Ion funnel frequency scan 2) right panel: diaphragm scan. White squares are the simulation results, black squares are the experiment results. The parameter settings are given in Table 2.1.. Figure 2.12 ȱȱȱȱȱǻΗǼȱ ȱȱȱȱȱȱȱȱȱ exit diaphragm. SIMION simulations were performed for gap distances of 1.5 mm, 2.5 mm and 5 mm.. obtained by means of the simulation method just described can be seen on Figure 2.10. The area in the vicinity of the capillary exit is known to be a region where supersonic expansion of the incoming biomolecular ion beam occurs [26]. 28. 517030-L-sub01-bw-Egorov Processed on: 8-2-2018. PDF page: 34.

(36) EXPERIMENT. However, the model applied is unable to simulate such gas flow dynamics which may play a significant role in the area near the exit of the capillary. This results in considerable uncertainties in the ion trajectories near their natural starting points adjacent to the capillary exit. Therefore, simulations for different capillary voltages are inconsistent with ion transmission measurements as a function of capillary voltage and not presented here. Because of these uncertainties and the aforementioned ones concerning charge repulsion effects, it was necessary to verify the reliability of the simulation. We started out by performing ion transmission (see Figure 2.11) tests as a function of the RF frequency and the voltage on the diaphragm. Similar trends are found when comparing the ion transmission efficiency experiments performed with the real setup in the region of the maximal transmission (ion funnel RF frequency=400-800 kHz, diaphragm voltage=8-14 V) to the simulations. Simulations with the diaphragm voltages above 14 V indicate severe ion trapping in the area between the last electrode and diaphragm, hampering ion transport. On the simulation side this results in very long computational times and therefore further simulations with diaphragm voltages above 14 V were not conducted. One of the most important design features of the ion funnel is the distance between the last electrode and diaphragm, the shorter it is the more efficient the transmission is between the ion funnel and octupole. It cannot be made infinitesimally small, so it is important to define the value below which a further decrease will not result in appreciable improved ion transmission. In order to establish this, simulations were run for gaps of 5 mm, 2.5 mm and 1.5 mm, see Figure 2.12. We obtained the following transmission values for different distances: 5 mm: 57%, 2.5 mm: 71%, 1.5 mm: 73%. It can be clearly seen that the transmission increase between 2.5 mm and 1.5 mm is not too large. For larger gaps the ion current decreases strongly. Therefore it was decided to take a distance of 2 mm. The aforementioned improvements in the ion funnel and capillary inlet system have resulted in a high-fluence electrospray source able to produce more than 2 nA of non-mass selected current and more than 150 pA of mass-selected current, what has made the experiments with the Nanocluster Trap feasible. Additionally, we performed a test to determine the maximal allowed misalignment of octupole and quadrupole. Allowing for an acceptable decrease in ion transmission due to misalignment of 10 %, misalignments up to 0.2 mm are acceptable.. 29. 517030-L-sub01-bw-Egorov Processed on: 8-2-2018. PDF page: 35.

(37) EXPERIMENT. Figure 2.13 Schematic representation of the operating principle of a microchannel plate (MCP) detector.. 2.4. MICROCHANNEL PLATE SENSITIVITY. A Microchannel plate (MCP) detector is a device commonly used in various fields of science and technology and capable of detection of incoming ions, electrons, neutrons and photons. Its operation is based on electron multiplication. A MCP consists of a regular array of tubes (microchannels) with a diameter of 2-50 μm (12 μm for our case). Each of the channels is an electronic multiplier (see Figure 2.13). The channels are inclined with respect to the surface normal (typically 5°-10°), therefore particles entering the channel are guaranteed to hit its wall. This triggers a cascade of electrons propagating through the tube. The amplification of a single MCP is around 104. Stacking two or more MCPs will yield an amplification of 108 or more. An issue playing a significant role in the detection of molecules with high m/z is the MCP sensitivity. Earlier, using C60 ions the dependency of a MCP detector sensitivity versus ion speed was measured [27]. It was shown that the detector efficiency can be approximated with the following equation: ܲ=. 1 + tanh ቀ. ‫ ݒ‬െ 28500 ቁ 11000 2. where v is the ion’s speed in m/s. From Figure 2.14 it can be clearly seen that for large molecules, P does not depend on the charge state of the molecule. The detection efficiency being independent of the molecule’s charge state was also ୰ା established in experiments of C଺଴ with gold surfaces [28, 29]. For light ions such as Ar ୯ା [30] electron emission and thus detection efficiency strongly depends on the charge state. This can be explained by the fact that the number of electrons emitted in an ion collision with a surface depends on the kinetic energy of the projectile 30. 517030-L-sub01-bw-Egorov Processed on: 8-2-2018. PDF page: 36.

(38) EXPERIMENT. Figure 2.14 MCP sensitivity with speed of singly, doubly, triply and quadruply charged C60 ions, taken from [27].. and on its potential energy, i.e. the projectile charge state. However, for larger molecules the potential energy is redistributed into vibrational excitations, which leads to fragmentation instead of electron emission. Kinetic-energy related electron emission dominates for the case of big molecules. All further experiments concern large biomolecular ions, so the MCP sensitivity is assumed to be independent of the charge state of the ion and to be described by the expression given above.. 2.5. BESSY II SYNCHROTRON FACILITY. All experiments mentioned in this thesis were performed at the third-generation synchrotron facility HZB-BESSY II (Berlin, Germany). A linear accelerator injects electrons into the storage ring up to a maximal operational value of the current of 300 mA at a kinectic energy of 1.7 GeV. Passing through periodic magnetic structures, the so called undulators, electrons are forced to oscillate and emit radiation with an energy distribution defined by the undulator periodicity and gap. The photon energy can be selected and controlled by the application of a monochromator behind the undulator. In total 11 undulators and more than 50 31. 517030-L-sub01-bw-Egorov Processed on: 8-2-2018. PDF page: 37.

(39) EXPERIMENT. Figure 2.15 PAULTJE interfaced with a BESSY II beamline at the HZB (Berlin, Germany). working stations operate at BESSY II. The Paultje setup interfaced with a beamline can be seen on Figure 2.15 Soft X-ray experiments were performed on the U49/2-PGM1 beamline [31] which produces photons with energy of 85-1600 eV with a flux of 5x1012 - 1.5x1013 photons/s. Undulator U49-2 is a periodic structure of magnets which consists of 84 periods each 49.4 mm long with a minimal gap of 15.64 mm. The monochromator is a plane grating monochromator (PGM). Usually, there is a significant photon flux drop around the carbon K-edge, due to photoabsorption by carbon-containing compounds on gratings. This has led to a significant irradiation time required to achieve a significant photoabsorption for YG10F and leucine– enkephalin: 1-3 seconds per cycle. Photoabsorption scales with the molecular size, so for the larger molecules we studied the irradiation time was less than 1 second per cycle. VUV experiments were performed using the U125-2 10m-NIM beamline [32] which consists of a quasi-periodic undulator and a normal incidence monochromator. This system provides the user with 6-40 eV photons with a flux of 5x1011-5x1012 32. 517030-L-sub01-bw-Egorov Processed on: 8-2-2018. PDF page: 38.

(40) EXPERIMENT. photons/s. The undulator consists of 32 periods each 125 mm long with a minimal gap of 15.7 mm. The 10m-NIM monochromator is an off-Rowland Circle normal incidence monochromator (NIM) and contains a spherical grating with a 10 m focus. The exit slit width can be varied from 0 to 2000 μm. The larger width of the ¡ȱȱȱȱȱ̇ȱȱȱ¢ȱȱȱȱȱ¡ǯȱ ȱȱ ȱ ȱ ̇ȱ ȱ ȱ ȱ ȱ ǰȱ ¢ȱ ȱ ȱ ȱ ȱ for low (14 eV) and high (35 eV) photon energies photoflux is generally low. In addition to that, photoabsorption by peptides at these energies is low [5]. For this reason the slit width was kept to its maximum value of 2 mms in order to obtain the maximal photoflux. Another important issue to be mentioned here is contamination by higher harmonics photons. Higher order contamination of the photon beam is partly suppressed by the dislocations in the periodic pattern of the undulator making it quasiperiodic. The quasiperiodic structure of the undulator leads to slight red shift of the harmonics what results in their efficient filtering out by the monochromator. Experiments with the Nanocluster Trap were performed at the U52_PGM beamline for polarized soft X-ray radiation of 85-1600 eV energy and typical photon fluxes of 1012 photon/s [33]. The elliptical undulator U52 can produce polarized soft X-rays which can be either polarized linearly (any orientation) or circularly. It consists of 77 periods each 52 mm long and has a minimal gap of 16 mm.. 33. 517030-L-sub01-bw-Egorov Processed on: 8-2-2018. PDF page: 39.

(41) BIBLIOGRAPHY. BIBLIOGRAPHY [1] K. Hirsch, J.T. Lau, P. Klar, A. Langenberg, J. Probst, J. Rittmann, M. Vogel, V. Zamudio-Bayer, T. Moeller and B. von Issendorff, Journal of Physics B-Atomic Molecular and Optical Physics, 42, 154029 (2009). [2] S. Bari, "The influence of peptide structure on fragmentation pathways", PhD Thesis, University of Groningen (2010). [3] O. Gonzalez-Magaña, "Ionization-induced fragmentation dynamics of isolated complex molecules", PhD Thesis, University of Groningen (2013). [4] D. Egorov, L. Schwob, M. Lalande, R. Hoekstra and T. Schlathölter, Physical Chemistry Chemical Physics, 18, 26213 (2016). [5] S. Bari, O. Gonzalez-Magaña, G. Reitsma, J. Werner, S. Schippers, R. Hoekstra and T. Schlathölter, J.Chem.Phys., 134, 024314 (2011). [6] O. Gonzalez-Magaña, G. Reitsma, S. Bari, R. Hoekstra and T. Schlathölter, Phys.Chem.Chem.Phys., 14, 4351 (2012). [7] O. Gonzalez-Magaña, G. Reitsma, M. Tiemens, L. Boschman, R. Hoekstra and T. Schlathölter, J Phys Chem A, 116, 10745 (2012). [8] O. Gonzalez-Magaña, M. Tiemens, G. Reitsma, L. Boschman, M. Door, S. Bari, P.O. Lahaie, J.R. Wagner, M.A. Huels, R. Hoekstra and T. Schlathölter, Physical Review A, 87, 032702 (2013). [9] G. Reitsma, L. Boschman, M.J. Deuzeman, O. Gonzalez-Magaña, S. Hoekstra, S. Cazaux, R. Hoekstra and T. Schlathölter, Phys.Rev.Lett., 113, 053002 (2014). [10] G. Reitsma, L. Boschman, M.J. Deuzeman, S. Hoekstra, R. Hoekstra and T. Schlathölter, J.Chem.Phys., 142, 024308 (2015). [11] S. Martin, C. Ortega, L. Chen, R. Bredy, A. Vernier, P. Dugourd, R. Antoine, J. Bernard, G. Reitsma, O. Gonzalez-Magaña, R. Hoekstra and T. Schlathölter, Physical Review A, 89, 012707 (2014). [12] S. Bari, R. Hoekstra and T. Schlathölter, Physical Chemistry Chemical Physics, 12, 3376 (2010). [13] G. Reitsma, O. Gonzalez-Magaña, O. Versolato, M. Door, R. Hoekstra, E. Suraud, B. Fischer, N. Camus, M. Kremer, R. Moshammer and T. Schlathölter, International Journal of Mass Spectrometry, 365, 365 (2014). [14] T. Schlathölter, G. Reitsma, D. Egorov, O. Gonzalez-Magaña, S. Bari, L. Boschman, E. Bodewits, K. Schnorr, G. Schmid, C.D. Schroeter, R. Moshammer and R. Hoekstra, Angewandte Chemie-International Edition, 55, 10741 (2016). 34. 517030-L-sub01-bw-Egorov Processed on: 8-2-2018. PDF page: 40.

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(44) NEAR-EDGE X-RAY ABSORPTION MASS-SPECTROMETRY OF GAS-PHASE PROTEINS. Near-edge x-ray absorption massspectrometry of gas-phase proteins: the influence of protein size Multiply protonated peptides and proteins in the gas phase can respond to near edge X-ray absorption in three different ways: (i) non dissociative ionization and ionization accompanied by loss of small neutrals, both known to dominate for proteins with masses in the 10 kDa range. (ii) Formation of immonium ions, dominating for peptides in the 1 kDa range. (iii) Backbone scission leading to sequence ions which is typically weaker and has mainly been observed for peptides in the 1 kDa range. We have studied carbon 1s photoexcitation and photoionization for a series of peptides and proteins with masses covering the range from 0.5 kDa to more than 10 kDa. The gas phase protonated molecules were trapped in a radiofrequency ion trap and exposed to synchrotron radiation. Time of flight mass spectrometry was employed for investigation of the photoionization and photofragmentation processes. A smooth transition from the photofragmentation regime to the non-dissociative photoionization regime is observed. Mass spectra are most complex in the few kDa regime, where non-dissociative ionization, backbone scission and immonium ion formation coexist. The observed correlation between protein size and fragmentation, i.e. radiation damage, is of relevance for soft X-ray microscopy.. Published: Near-edge x-ray absorption mass-spectrometry of gas-phase proteins: the influence of protein size D. Egorov, L. Schwob, M. Lalande, R. Hoekstra; T. Schlathölter PCCP,18,26213(2016) 37. 517030-L-sub01-bw-Egorov Processed on: 8-2-2018. PDF page: 43.

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