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22nd International Symposium on Plasma Chemistry July 5-10, 2015; Antwerp, Belgium Towards the molecular mechanism of biomolecules in water treated by atmospheric plasma jet in He/O 2 gas mixture D. Dewaele1, V. Layes3 , C.C.W. Verlackt4, E.C Neyts4, E. Maes2,5, G. Baggerman2,5, J. Benedikt3, F. Lemière1,2 and F. Sobott1,2 1 Biomolecular & Analytical Mass Spectrometry, Department of Chemistry, University of Antwerp, 2020 Antwerpen, Belgium 2 UA-VITO Center for Proteomics, University of Antwerp, 2020 Antwerpen, Belgium 3 Ruhr-University Bochum, Research Department Plasmas with complex interaction, 44780 Bochum, Germany 4 Research group Plasmant, Department of Chemistry, University of Antwerp, 2610 Antwerpen-Wilrijk, Belgium 5 Flemish Institute for technological Research (VITO), 2400 Mol, Belgium Abstract: To obtain a better knowledge of the molecular mechanisms underlying the plasma treatment of biological materials (e.g., cells, tissues) a model study was set up. Different model compounds were selected and treated with the atmospheric plasma jet device operated in He with an admixture of O 2 . Samples were further analysed by mass spectrometry and the global effect of the oxidation was determined. Keywords: oxidation, biomolecules, mass spectrometry, atmospheric plasma jet 1. Introduction The use of cold atmospheric plasma can be especially useful in medicine, e.g. for sterilization of medical equipment or the treatment of living cancer tissue [1,2]. Several earlier studies have already tested the effect of plasma treatment on viruses and bacteria [3,4]. This study intends to extend the knowledge of the molecular effect of this plasma treatment on different classes of biomolecules using mass spectrometry based approaches. A first class of biomolecules included in this study were the oligosaccharides. Oligosaccharides play an important biological role, for example anchored to integral membrane protein they take care of cell-cell contact or form as glycoproteins important structural compounds in cells. Next to the sugar compounds, different peptides (7-11 amino acids) and proteins of different sizes were examined. A recent study of the research group of Julia Bandow already addressed the effect of plasma treatment on a molecular basis and revealed the oxidation of the catalytic cysteine in glyceraldehyde 3-phosphate dehydrogenase by mass spectrometry [5]. The use of native mass spectrometry allowed us not only to see the global oxidation effect (extent of oxidation) on the intact proteins but also made it possible to study the structure e.g. possible unfolding [6]. casein, haemoglobin). All samples were dissolved in UPLC purified water and 50mM ammonium acetate was added to the protein samples. Samples were treated with a micro-scale atmospheric pressure plasma jet (µ-APPJ) that was operated in He with an admixture of O 2 (1400 sccm He, 8.4 sccm O 2, 220V r.s.m. ). The effect of the plasma treatment for the different molecules was followed over time (1, 3, 5, 7, 9, 12, 15 min). 400 µl of sample was placed in a glass vial for each time point and treated (fig.1). After treatment samples were collected and stored at -20°C. The effect of the plasma treatment was further studied by mass spectrometry using an ESI- Q-TOF- Synapt G2 HDMS T-wave ion mobility mass spectrometer (Waters). Samples were introduced in the mass spectrometer via nano-electrospray using in-house-made gold-coated borosilicate needles The different biomolecules were first analysed intact to detect the global effect (mass shift) i.e. total amount of oxidation events of the treatment. Samples were diluted to 5-20 µM in their original buffer, being water or 50 mM ammonium acetate. For the standard proteins additional analysis of the position of oxidations and possible conformational changes of the native fold due to oxidation were performed by LC-MSMS of a tryptic digest and ion mobility mass spectrometry (IM-MS). 2. Methods and Materials Different biomolecules were used as model compounds: i) carbohydrates (glucose, N-acetyl-glucosamine, cellulose, N,N,N’’-triacetylchitotriose), ii) peptides (bradykinin, HIV-Tat, Angiotensin 1-7), iii) proteins (ubiquitin, cytochrome C, human serum albumin, β- P-III-10-5 1 Peptides Three short peptides (7-11 amino acids) were plasma treated and oxidation of the peptides was followed in time (1, 4.5, 8min). Figure 3 shows the comparison of the plasma treatment on the angiotensin fragment 1-7 and bradykinin (fig.3). Unlike angiotensin, short plasma treatment (1 min) of bradykinin showed a large and relatively high intensity set of oxidation products. Prolonging the exposure time resulted in an even higher modification rate of the peptide. b) Fig. 1. Practical set-up for sample treatment 3. Results/ Discussion Carbohydrates a) In a first experiment, oxidation of glucose and glucosamine were followed in time. Analysis of the oxidation products showed an extensive oxidation of the sugars, where the major peak was identified as the oxidation of the aldehyde group to a carboxylic acid. Additional oxidation products were identified as the oxidation of the primary alcohols to aldehydes. Fig. 2. Plasma treatment of cellotriose. The control sample only showed the presence of the intact cellotriose ionized by sodium (527 Da). Exposure to the cold plasma source resulted in the loss of a monomeric oxidized glucose molecule (219 Da) and in the appearance of the unmodified and oxidized disaccharide. Next oligosaccharides, longer sugar polymers, were studied. Short plasma treatment resulted next to the oxidation of the trisaccharide, in the cleavage of the glycosidic bond. As seen in figure 2 plasma treatment of cellotriose showed the appearance of monomeric oxidized glucose molecules (219 Da) already after a short treatment time, and this signal increases with is enhanced by longer exposure. Similar results were observed for the N,N,N’’-triacetylchitotriose. 2 Fig. 3. Plasma treatment of angiotensin (A) and bradykinin (B). Angiotensin shows stepwise mass shifts of 16 Da, which implies the addition of an extra oxygen atom. For bradykinin mass shift 14 and 16 Da are observed, implying a more complex behaviour. This implies the formation both hydroxyl and carbonyl groups into the peptide. The observed difference in behaviour could be explained by the nature and reactivity of the amino acids. An option to evaluate and get a better understanding of this findings could be provided by reactive molecular dynamics simulations. P-III-10-5 c) Proteins Analysis of the intact plasma treated proteins revealed the occurrence of oxidations. In first instance the mass spectra acquired under native conditions showed no unfolding of the treated proteins. Ion mobility adds an additional dimension to the data, which allows for separation depending shape. Performing additional IMMS experiments confirmed that no major conformational changes could be observed due to modification of the amino acids. Figure 4 shows the native mass spectra of the plasma treated ubiquitin after 1 and 5 minutes. Plasma treatment for 1 minute resulted in a mixture of unmodified ubiquitin and modified/oxidized ubiquitin molecules. Prolonging the exposure to 5 minutes resulted in the total disappearance of unmodified ubiquitin. Furthermore it suggests that the mass spectra show the presence of ubiquitin once and twice oxidized. The presence of oxidation on proteins could be explained by (1) the structure/accessibility for the reactive oxygen species, (2) the nature and reactivity of the amino acids or (3) a combination of these two. or of in vitro oxidation using Fe(II)/ H 2 O 2 [7] electrochemical oxidation on ubiquitin [8] had similar findings, but identified the methionine at position 1 as the major oxidation peak. Whether this is also true for plasma treated proteins in water environment is still questionable and needs further evaluation. Table 1: Possible oxidation positions in ubiquitin determined by LC-MSMS analysis. Light grey marked oxidations were also observed in the control sample Amino acid Modification (Da) (position) Met (1) Oxidation (+16) Sulfone (+32) Asp (21) Hydroxylation (+16) Phe (45) Dihydroxylation (+32) Lys (48) Dihydroxylation (+32) Aminoadipic semialdehyde (-1) Tyr (59) Hydroxylation (+16) His (68) Oxidation (+16) The further elucidation of the precise effect of the plasma treatment (i.e. oxidations) off the different model proteins will contribute to the better understanding of the molecular effect of the cold plasma treatment on proteins. Fig. 4. Native mass spectra of the [M+5H]5+ of control ubiquitin and plasma treated ubiquitin respectively, 1 and 5 minutes. 2 mass shifts of 16 Da were observed, which implied the addition of 2 oxygen atoms. To pinpoint the modified amino acids and therefore the exact location of the oxidation, a tryptic digestion on the protein samples was performed. The three peptide digestion mixtures were analysed by LC-MSMS (LTQOrbitrap Velos, Thermo). The preliminary data analysis was performed with the PEAKS 6.0 software package, which enabled the identification of the different posttranslational modification under which for example oxidations. The posttranslational analysis of the tryptic digest revealed the presence of 6 possible oxidation positions (see table 1). This data on the ubiquitin peptide mixtures suggest that the intact protein mass shift reflects a mixture of ubiquitin oxidation sites. The position of these particular amino acids also suggest that the reactive oxygen species mainly have an effect on the surface of the protein. Earlier papers that studied for example the effect P-III-10-5 4. Conclusion In summary, we studied the molecular mechanisms underlying the cold plasma treatment on different biomolecules. First results point to the chain breaking of the longer oligosaccharides, which could prove to have an important biological relevance. A different reactivity of the oxidation could be identified in the different peptides and proteins, which ranged from slow and few oxidation products to fast and extensive oxidation mechanisms, depending on the nature of the surface exposed amino acids and the secondary structure 5. References [1] J.-W. Lackmann, J.E. Bandow, Appl. Microbiol. Biotechnol. 98 (2014) 6205. [2] M.G. Kong, G. Kroesen, G. Morfill, T. Nosenko, T. Shimizu, J. van Dijk, J.L. Zimmermann, New J. Phys. 11 (2009) 115012. [3] H. Yasuda, T. Miura, H. Kurita, K. Takashima, A. Mizuno, Plasma Process. Polym. 7 (2010) 301. [4] V. Raballand, J. Benedikt, J. Wunderlich, a von Keudell, J. Phys. D. Appl. Phys. 41 (2008) 115207. [5] J.-W. Lackmann, S. Schneider, E. Edengeiser, F. Jarzina, S. Brinckmann, E. Steinborn, M. Havenith, J. Benedikt, J.E. Bandow, J. R. Soc. Interface 10 (2013) 20130591. [6] A. Konijnenberg, A. Butterer, F. Sobott, Biochim. Biophys. Acta 1834 (2013) 1239. [7] S. Huilin, Liqing, D.E.K. and R.A.S. Robinson, J.Phys.Chem. B 117 (2013) 164. 3 [8] 4 C. Mcclintock, V. Kertesz, R.L. Hettich, 80 (2008) 3304. P-III-10-5