Photon and electron action spectroscopy of trapped biomolecular ions - from isolated to nanosolvated species

Abstract

In an effort to understand the vast complexity of the underlying processes within a cell at a molecular level, the first step lies in revealing the fundamental physical and chemical properties, as well as the structure, of biopolymers (proteins and DNA). With the development of modern experimental techniques it has become possible to study these large molecules under well-defined conditions in the gas phase, by closely inspecting their interactions with energetic photons and electrons. In this Thesis, we present the experimental setups for the action spectroscopy of peptides, proteins and nucleotides, as well as the hydrated complexes (hydrated nucleotides), in the gas phase. We present the details and the operation of the two experimental setups based on coupling the linear quadrupole ion trap with: (1) a VUV or a soft X-ray synchrotron beamline and (2) a focusing electron gun. In the case (1), the existing experimental setup consisting of a commercial quadrupole ion trap mass spectrometer (LTQ XL from Thermo Scientific), equipped with an electrospray ion source, was coupled to the VUV beamline DESIRS and the soft X-ray beamline PLEIADES at the synchrotron SOLEIL (France). The setups were used to study the photo-induced ionization/fragmentation of trapped biopolymers and nanosolvated species. The results obtained with this setups include VUV action spectroscopy of protonated Leucine-Enkephalin peptide (both a monomer and a dimer) and a nanosolvated nucleotide Adenosine monophosphate (AMP), in (5-15) eV photon energy range. The inner-shell action spectroscopy in the soft X-ray energy range (around C and N K-edge), was performed for multiply charged precursor of Ubiquitin protein. The photo-dissociation and photo-fragmentation ion yields for several fragment ions from all above mentioned macromolecules were extracted and the obtained spectral features were discussed considering relevant photon-induced processes. In the case (2), new experimental setup was developed by coupling the same LTQ XL ion trap with a focusing electron gun, in order to perform an electron activation tandem mass spectrometry, as well as an electron-impact action spectroscopy of trapped biopolymer ions. The ion optic simulations using SIMION program were performed in order to investigate the propagation of the electron beam in the RF+DC ion trap. Tests measurements for electron-induced fragmentation of Substance P, Melittin and Ubiquitin are presented for the impact energy of 300 eV. Finally, we present the electron-impact inner-shell action spectroscopy of the multiply charged Ubiquitin protein, in the vicinity of C K-edge energies of (280-300) eV. The electron-impact results are compared with the soft X-ray photon-impact action spectroscopy results obtained for the same target.

U nastojanju da se razume ogromna složenost procesa u okviru ćelije na molekulskom nivou, prvi korak je otkrivanje fundamentalnih fizičko-hemijskih osobina, kao i strukture biopolimera (proteina i DNK). Razvojem savremenih eksperimentalnih tehnika omogućeno je proučavanje velikih biološki relevantnih molekula pod jasno definisanim uslovima u gasnoj fazi, izučavanjem njihovih interakcija sa fotonima i elektronima velikih energija. U ovom radu su predstavljene eksperimentalne postavke za akcionu spektroskopiju peptida, proteina, nukleotida, kao i nanosolvatisanih kompleksa (hidratisani nukleotidi) u gasnoj fazi. Prikazani su detalji i princip rada dve eksperimentalne postavke zasnovane na povezivanju linearne kvadrupolne jonske zamke sa: (1) sinhrotronskim fotonskim mlazom (VUV i meki X-zraci) i (2) fokusirajućim elektronskim topom. U slucaju (1), postojeća eksperimentalna aparatura koja sadrži linearnu kvadrupolnu jonsku zamku u okviru komercijalnog masenog spektrometra (LTQ XL od firme Thermo Scientific) povezana je sa VUV mlaznom linijom DESIRS i mlaznom linijom za meke X-zrake PLEIADES na sinhrotronu SOLEIL (Francuska). Aparatura je upotrebljena za izučavanje foto-indukovanih procesa jonizacije i fragmentacije zarobljenih jona biopolimera i nanosolvatisanih čestica. Rezultati dobijeni na ovoj aparaturi uključuju VUV akcionu spektroskopiju protonisanog leucin-enkefalin peptida (monomer i dimer), kao i nanosolvatisanog nukleotida adenosin monofosfata (AMP), u opsegu energija fotona od (5-15) eV. Akciona spektroskopija unutrašnje ljuske koristeći meke X-zrake (u oblasti C i N K-ljuske), urađena je za višestruko naelektrisani prekursor proteina ubikuitin. Za sve gore pomenute makromolekule, izučavani su absorpcioni spektri za neke od dobijenih jonskih produkata, pri čemu su analizirane uočene spektralne karakteristike dobijene u pomenutim fotonski indukovanim procesima. U slucaju (2) razvijen je novi eksperimentalni sistem zasnovan na povezivanju iste LTQ XL jonske zamke sa elektronskim topim sa mlazom elektrona srednjih energija, koja omogućava tandem masenu spektrometriju i elektronski indukovanu akcionu spektroskopiju zarobljenih jona biopolimera. Korišćenjem programa SIMION, urađene su simulacije sa ciljem ispitivanja transmisije elektrona kroz jonsku zamku sa RF+DC potencijalima. Inicijalni testovi aparature uradjeni su fragmentacijom peptida supstance P i melitin, kao i ubikuitin proteina pri energijama elektrona u oblasti oko 300 eV. Na kraju, prikazani su rezultati elektronski-indukovane akcione spektroskopije višestruko naelektrisaniog jona ubikuitin proteina, u oblasti energija oko C K-ljuske (280-300) eV. Ovi rezultati su upoređeni sa rezultatima dobijenim za istu metu pri fotonskiindukovanim (X-zraci) procesima iz iste oblasti energija.