Download Towards the molecular mechanism of biomolecules in water treated by atmospheric plasma jet in He/O2 gas mixture

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-
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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.
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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
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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
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