10.2 Mass spectrometry of plasma-treated samples

Mass spectrometry is a powerful tool in plasma medicine, analyzing molecular changes in treated samples. It ionizes molecules, measures their mass-to-charge ratio, and provides crucial information about structure, composition, and abundance of plasma-altered biological molecules.

Various ionization techniques, mass analyzers, and detectors enable precise analysis of plasma-treated samples. This method helps researchers understand plasma-induced modifications, identify biomarkers, study drug metabolism, and characterize protein changes, advancing our knowledge of plasma-cell interactions and therapeutic effects.

Principles of mass spectrometry

• Analyzes molecules by ionizing them and measuring their mass-to-charge ratio
• Provides crucial information about molecular structure, composition, and abundance in plasma medicine research
• Enables identification and quantification of complex biological molecules altered by plasma treatment

Ionization techniques

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• Electron ionization (EI) bombards molecules with high-energy electrons to create positive ions
• Electrospray ionization (ESI) nebulizes liquid samples into charged droplets
• Matrix-assisted laser desorption/ionization (MALDI) uses laser energy absorbed by a matrix to ionize molecules
• Atmospheric pressure chemical ionization (APCI) ionizes vaporized samples through gas-phase ion-molecule reactions

Mass analyzers

• Quadrupole mass analyzer uses oscillating electric fields to separate ions based on their m/z ratio
• Time-of-flight (TOF) analyzer measures the time taken for ions to travel a fixed distance
• Ion trap analyzer captures ions in a three-dimensional electric field
• Fourier transform ion cyclotron resonance (FT-ICR) analyzer provides ultra-high mass resolution
• Orbitrap analyzer traps ions in an electrostatic field and measures their oscillation frequency

Detectors in mass spectrometry

• Electron multiplier amplifies the ion signal by generating secondary electrons
• Faraday cup collects ions directly and measures the resulting current
• Microchannel plate detector consists of multiple electron multipliers for improved sensitivity
• Array detector simultaneously detects multiple m/z values for increased speed and sensitivity

Plasma treatment of samples

• Modifies surface properties and chemical composition of biological samples
• Generates reactive species that interact with sample molecules, creating new compounds
• Enhances ionization efficiency and improves mass spectrometry analysis in plasma medicine

Types of plasma sources

• Dielectric barrier discharge (DBD) plasma generates non-equilibrium plasma at atmospheric pressure
• Atmospheric pressure plasma jet (APPJ) produces a stream of reactive species for localized treatment
• Microwave-induced plasma creates high-density plasma for efficient sample treatment
• Inductively coupled plasma (ICP) generates high-temperature plasma for elemental analysis

Plasma-sample interactions

• Reactive oxygen species (ROS) oxidize organic molecules in the sample
• Reactive nitrogen species (RNS) induce nitration and nitrosylation of biomolecules
• UV radiation from plasma causes photochemical reactions and bond cleavage
• Charged particles in plasma modify surface charge distribution of samples

Chemical modifications by plasma

• Oxidation of lipids and proteins leads to formation of carbonyl and hydroxyl groups
• Nitration of aromatic amino acids (tyrosine) alters protein structure and function
• Crosslinking of polymers increases molecular weight and changes physical properties
• Fragmentation of large molecules produces smaller, more easily ionized species

Sample preparation methods

• Crucial step in mass spectrometry analysis of plasma-treated samples
• Affects ionization efficiency, spectral quality, and overall analytical performance
• Tailored to specific sample types and target analytes in plasma medicine research

Solid vs liquid samples

• Solid samples require dissolution, extraction, or direct ionization techniques (MALDI)
• Liquid samples can be directly analyzed using electrospray ionization or undergo further preparation
• Biological tissues often require homogenization and extraction before analysis
• Cell cultures may need lysis and protein precipitation steps

Extraction techniques

• Liquid-liquid extraction separates analytes based on their solubility in immiscible solvents
• Solid-phase extraction (SPE) uses adsorbent materials to selectively retain and elute analytes
• Supercritical fluid extraction employs supercritical CO2 for efficient extraction of non-polar compounds
• Microwave-assisted extraction accelerates extraction process using microwave energy

Sample concentration

• Evaporation under nitrogen stream concentrates samples by removing volatile solvents
• Lyophilization (freeze-drying) removes water from samples while preserving heat-sensitive compounds
• Solid-phase microextraction (SPME) concentrates analytes on a fiber coating
• Molecular imprinted polymers (MIPs) selectively extract and concentrate specific target molecules

Mass spectrometry analysis

• Provides detailed molecular information about plasma-treated samples
• Enables identification and quantification of chemical changes induced by plasma treatment
• Crucial for understanding the effects of plasma on biological systems in plasma medicine

Qualitative vs quantitative analysis

• Qualitative analysis identifies compounds based on their mass spectra and fragmentation patterns
• Quantitative analysis determines the concentration of specific analytes using calibration curves
• Relative quantification compares abundance of analytes between different samples or conditions
• Absolute quantification requires internal standards with known concentrations

Spectral interpretation

• Mass spectrum displays ion intensity vs mass-to-charge ratio (m/z)
• Molecular ion peak (M+) represents the intact molecule and provides molecular weight information
• Fragment ions result from molecule fragmentation and provide structural information
• Isotope patterns help confirm elemental composition and identify halogenated compounds

Data processing techniques

• Peak detection algorithms identify and measure ion signals in mass spectra
• Deconvolution separates overlapping peaks and resolves complex spectra
• Background subtraction removes chemical noise and improves signal-to-noise ratio
• Normalization adjusts for variations in sample amount or instrument response

Applications in plasma medicine

• Mass spectrometry analyzes molecular changes induced by plasma treatment in biological systems
• Provides insights into mechanisms of plasma-cell interactions and therapeutic effects
• Supports development of targeted plasma treatments for various medical applications

Biomarker identification

• Discovers molecular signatures associated with plasma-induced cellular responses
• Identifies oxidative stress markers (8-OHdG) in plasma-treated tissues
• Detects changes in lipid profiles following plasma exposure
• Analyzes post-translational modifications of proteins induced by plasma treatment

Drug metabolism studies

• Investigates plasma-induced changes in drug metabolism and pharmacokinetics
• Identifies new metabolites formed through plasma-mediated drug modifications
• Quantifies changes in drug concentrations following plasma treatment
• Analyzes plasma-induced alterations in drug-protein binding

Protein modifications

• Characterizes oxidative modifications of proteins (carbonylation) caused by plasma treatment
• Identifies nitration and nitrosylation of amino acid residues in proteins
• Analyzes plasma-induced protein crosslinking and aggregation
• Detects changes in protein phosphorylation patterns following plasma exposure

Challenges and limitations

• Mass spectrometry analysis of plasma-treated samples faces several technical and analytical challenges
• Addressing these limitations improves accuracy and reliability of results in plasma medicine research
• Ongoing developments in instrumentation and methodology aim to overcome these obstacles

Matrix effects

• Co-eluting matrix components suppress or enhance ionization of target analytes
• Biological matrices (plasma, tissue extracts) introduce complex interferences
• Internal standards and matrix-matched calibration help compensate for matrix effects
• Advanced sample preparation techniques (immunoaffinity extraction) reduce matrix interference

Ion suppression

• Occurs when matrix components compete with analytes for ionization
• Reduces sensitivity and affects quantitative accuracy of mass spectrometry analysis
• Dilution of samples or use of alternative ionization techniques can mitigate ion suppression
• Chromatographic separation before mass spectrometry analysis reduces co-elution of interfering compounds

Sample degradation

• Plasma-induced modifications can continue during sample storage and preparation
• Oxidation and hydrolysis of unstable compounds lead to loss of analytes
• Antioxidants and stabilizers added to samples prevent further degradation
• Rapid sample processing and analysis minimize degradation-related artifacts

Advanced techniques

• Cutting-edge mass spectrometry methods enhance analysis of plasma-treated samples
• Provide higher sensitivity, selectivity, and structural information
• Enable comprehensive characterization of complex biological systems in plasma medicine

Tandem mass spectrometry

• MS/MS involves multiple stages of mass analysis for improved structural elucidation
• Collision-induced dissociation (CID) fragments precursor ions for detailed structural information
• Precursor ion scanning identifies compounds with specific structural features
• Neutral loss scanning detects molecules that lose a specific neutral fragment

Imaging mass spectrometry

• Maps spatial distribution of molecules in tissue sections or cell cultures
• MALDI imaging analyzes molecular changes across plasma-treated tissue surfaces
• Secondary ion mass spectrometry (SIMS) provides high spatial resolution for surface analysis
• Desorption electrospray ionization (DESI) enables ambient imaging of biological samples

High-resolution mass spectrometry

• Orbitrap and FT-ICR analyzers provide ultra-high mass resolution and accuracy
• Enables accurate mass measurements for elemental composition determination
• Resolves complex mixtures and identifies closely related compounds
• Improves confidence in compound identification and reduces false positives

Data analysis and interpretation

• Processes and extracts meaningful information from mass spectrometry data of plasma-treated samples
• Integrates multiple data sources to gain comprehensive insights into plasma-induced changes
• Crucial for translating mass spectrometry results into clinically relevant findings in plasma medicine

Statistical methods

• Principal component analysis (PCA) identifies patterns and groupings in complex datasets
• Partial least squares discriminant analysis (PLS-DA) classifies samples based on spectral features
• ANOVA and t-tests determine statistical significance of observed changes
• Multiple testing correction (Bonferroni, FDR) controls for false positives in large-scale analyses

Bioinformatics tools

• Pathway analysis software (KEGG, Reactome) maps identified molecules to biological pathways
• Protein interaction databases (STRING) reveal functional relationships between modified proteins
• Gene ontology (GO) enrichment analysis identifies overrepresented biological processes
• Machine learning algorithms predict functional consequences of plasma-induced modifications

Database searching

• Spectral libraries (NIST, MassBank) aid in compound identification by spectral matching
• Protein databases (UniProt, NCBI) support identification of modified peptides and proteins
• Metabolite databases (HMDB, METLIN) facilitate annotation of small molecules
• In-house databases of plasma-specific modifications improve identification accuracy

Emerging trends

• Cutting-edge developments in mass spectrometry push boundaries of plasma medicine research
• Enhance speed, sensitivity, and applicability of mass spectrometry in clinical settings
• Enable new insights into plasma-biological interactions and therapeutic mechanisms

Real-time analysis

• Rapid evaporative ionization mass spectrometry (REIMS) enables real-time tissue analysis during plasma treatment
• Ambient ionization techniques (DART, DESI) allow direct analysis of samples without preparation
• Ion mobility spectrometry-mass spectrometry (IMS-MS) provides rapid separation and analysis of complex mixtures
• Online monitoring of plasma-induced changes using microfluidic devices coupled to mass spectrometers

Miniaturization of instruments

• Portable mass spectrometers enable on-site analysis of plasma-treated samples
• Microfluidic paper-based analytical devices (μPADs) coupled to miniature mass spectrometers
• Handheld mass spectrometers for point-of-care diagnostics in plasma medicine applications
• Lab-on-a-chip devices integrate sample preparation and mass spectrometry analysis

Coupling with other techniques

• Hyphenated techniques combine mass spectrometry with complementary analytical methods
• LC-MS/MS improves separation and identification of complex plasma-treated samples
• Ion mobility-mass spectrometry (IM-MS) enhances separation of isomers and conformers
• Mass cytometry (CyTOF) enables high-dimensional analysis of cellular responses to plasma treatment