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Questions and Answers
What are the essential properties that make certain matrices suitable for MALDI in mass spectrometry?
What are the essential properties that make certain matrices suitable for MALDI in mass spectrometry?
Low molecular weight, quick crystallization with analyte, good aqueous solubility, often acidic, and strong light absorption in the UV range.
Why is it challenging to predict the most effective matrix for a specific analyte in MALDI?
Why is it challenging to predict the most effective matrix for a specific analyte in MALDI?
The interaction between the polarity of the analyte and the matrix plays a significant role, and sometimes combinations of matrices yield better results.
Describe the ionization mechanism that occurs during MALDI.
Describe the ionization mechanism that occurs during MALDI.
The matrix absorbs laser light, leading to the desorption of ionized matrix molecules into a plume, where proton and charge transfers ionize the analyte.
What is a significant advantage of using MALDI for the mass analysis of biomolecules?
What is a significant advantage of using MALDI for the mass analysis of biomolecules?
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How does MALDI imaging contribute to biomedical research, specifically in cancer surgery?
How does MALDI imaging contribute to biomedical research, specifically in cancer surgery?
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What type of charge do MALDI typically generate during the analysis of biomolecules?
What type of charge do MALDI typically generate during the analysis of biomolecules?
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In the context of matrix selection for MALDI, what role does matrix acidity play?
In the context of matrix selection for MALDI, what role does matrix acidity play?
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List two major applications of MALDI in the field of biomolecule analysis.
List two major applications of MALDI in the field of biomolecule analysis.
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What distinguishes the MALDI process from Electrospray Ionization (ESI)?
What distinguishes the MALDI process from Electrospray Ionization (ESI)?
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Why is MALDI considered a 'soft ionization' technique?
Why is MALDI considered a 'soft ionization' technique?
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What is the primary advantage of using MALDI in biological analysis?
What is the primary advantage of using MALDI in biological analysis?
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How does Electrospray Ionization (ESI) facilitate the analysis of non-volatile molecules?
How does Electrospray Ionization (ESI) facilitate the analysis of non-volatile molecules?
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Describe the key functional principle of MALDI imaging in discovering new antibiotics.
Describe the key functional principle of MALDI imaging in discovering new antibiotics.
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What role did Koichi Tanaka play in the development of soft laser desorption techniques?
What role did Koichi Tanaka play in the development of soft laser desorption techniques?
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Explain the significance of the 2002 Nobel Prize in Chemistry related to ionization techniques.
Explain the significance of the 2002 Nobel Prize in Chemistry related to ionization techniques.
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What are the two ionization methods commonly used in biomolecule analysis to keep molecular ions intact?
What are the two ionization methods commonly used in biomolecule analysis to keep molecular ions intact?
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What are the characteristics of the charged droplets generated by ESI?
What are the characteristics of the charged droplets generated by ESI?
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What is the connection between MALDI and the concept of chemical warfare among bacteria?
What is the connection between MALDI and the concept of chemical warfare among bacteria?
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Explain the significance of using a solid matrix in the MALDI process.
Explain the significance of using a solid matrix in the MALDI process.
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How does the soft ionization of ESI benefit the analysis of complex biological samples?
How does the soft ionization of ESI benefit the analysis of complex biological samples?
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How does the choice of solvent influence the crystallization process in MALDI sample preparation?
How does the choice of solvent influence the crystallization process in MALDI sample preparation?
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What is a common approach for sample clean-up before performing MALDI?
What is a common approach for sample clean-up before performing MALDI?
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Describe how MALDI imaging techniques contribute to the field of biomolecule analysis.
Describe how MALDI imaging techniques contribute to the field of biomolecule analysis.
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What is the primary outcome of using 'soft' ionization techniques like MALDI?
What is the primary outcome of using 'soft' ionization techniques like MALDI?
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What challenge does sample reproducibility present in MALDI, and how might it be addressed?
What challenge does sample reproducibility present in MALDI, and how might it be addressed?
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In what way is MALDI described in terms of ion production, and what are its sensitivity levels?
In what way is MALDI described in terms of ion production, and what are its sensitivity levels?
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Study Notes
Biological Applications of Mass Spectrometry (MS)
- Mass spectrometry (MS) is a powerful tool for studying various molecules, including biological molecules, environmental pollutants, inorganic complexes, toxins, drugs, etc.
- MS excels at elucidating the structure of biomolecules like proteins and nucleic acids, due to their complex structures and large size as polymers.
- Recent advances in MS instrumentation are driven by the need for enhanced biomolecular analysis methods.
- MS is vital for studying fundamental biological processes such as protein folding, enzyme function, biosynthesis, and protein-protein interactions.
Biology Primer - The Central Dogma
- There is a correlation between DNA (gene) sequence and protein amino acid sequence.
- DNA undergoes transcription to produce mRNA.
- mRNA then undergoes translation to synthesize proteins.
- Nucleic acids are polymers composed of nucleotides.
- Proteins are polymers composed of amino acids.
Biology Primer - Protein Function
- Proteins perform diverse functions, including:
- Catalysis: Enzymes
- Structural: Keratin
- Transport: Hemoglobin
- Trans-membrane transport: Na+/K+ ATPases
- Toxins: Venom, ricin
- Contractile function: Actin, myosin
- Hormones: Insulin
- Storage: Proteins in seeds and eggs
- Defensive: Antibodies
Biology Primer - Protein Structure
- Proteins have four levels of structure:
- Primary: Linear sequence of amino acids.
- Secondary: Certain amino acid sequences form hydrogen bonds to create helices and sheets.
- Tertiary: Secondary structures fold into 3D shapes.
- Quaternary: Multiple proteins interact to form a larger structure.
- Mass spectrometry (MS) can be used to characterize all protein structures.
Studying Protein Structure with MS
- MS is useful in studying protein dynamics and structures, compositions, and quantities through methods like MS-based proteomics
- MS techniques can be applied to investigate protein folding, protein complex assembly, and other protein dynamics.
- Techniques include:
- In-vivo transients
- Oligomer topology
- MS of intact complexes
- Cross-linking MS
- Affinity-tag MS
- MS-based proteomics -Oxidative foot-printing MS -H/D-exchange MS -Electron-mediated foot-printing MS -Limited-proteolysis MS
Why are proteins good models for learning about MS?
- Proteins have extensive structural and functional diversity.
- They have inspired the development of various mass spectral analyses.
- The common ionization modes (ESI and MALDI) are routinely used to study proteins and biological systems.
- They are excellent model systems for understanding the different ways mass spectrometry can study and discriminate molecular structures.
What do mass spectrometers do?
- Mass spectrometry (MS) is a suite of analytical techniques that ionize chemical species, transfer them to the gas phase, and then sort them based on their mass-to-charge ratio (m/z).
- MS is a gas phase technique.
- Molecules to be analyzed must be ionized to interact with electric fields used in the mass spectrum.
- An ion's potential energy in an electric field is proportional to its charge and the field's potential difference. Potential energy converts to kinetic energy during acceleration.
Basic Components of All Mass Specs
- MS instruments have several basic components:
- Sample Introduction (via HPLC, GC, direct infusion)
- Ion source (to generate ions)
- Generate ions (often soft ionization) such as ESI or MALDI
- Mass analyzer (to separate ions based on m/z)
- TOF, Quadrupole, IT, FT-ICR, Orbitrap
- Components separating or manipulating ions
- Detector (to detect ions)
- Photo multipliers
- Data System (to process and analyze data)
The Mass Spectrum
- The output of a mass spectrometry (MS) experiment is a mass spectrum.
- It is a plot of relative intensity (y-axis) versus m/z (x-axis).
- Mass Spectrum is essentially a histogram of ions detected
- The resolving power of the mass analyzer influences the appearance of the histogram.
- This information on a compound or mixture of compounds depends on the ion generation, analysis, and manipulation techniques used in gas phase process.
Examples of Mass Spectra
- Mass spectra display the relative abundance of ions corresponding to different m/z values.
- EI, MALDI, and ESI provide different types of mass spectra for various applications.
Important Concepts - Monoisotopic Mass
- The monoisotopic mass is the mass of a compound calculated using the most abundant isotopes of each atom.
- To calculate monoisotopic mass, determine most abundant isotopes for common elements (1H, 12C, 14N, 160, 32S, 31p.) then input the values into the equation provided
Important Concepts - Average Mass
- Average mass (molecular weight) is the mass calculated using atomic masses from the periodic table and weighted naturally.
- Average mass is a weighted average of the atomic masses.
Identifying masses on a mass spectrum
- Monoisotopic mass: In asymmetric isotope distributions, the lowest m/z peak represents the monoisotopic mass
- Average mass: Weighted average of the isotope peaks
- Most abundant mass: The peak of greatest intensity
Important MS Concepts - Resolving Power
- Resolving power (RP) is a measure of the mass analyzer’s ability to distinguish between peaks of similar m/z.
- It is given by:
RP = m/Δm
where
- m equals the m/z of the ion
- Δm equals the full width at half maximum (FWHM),
What Resolving Power do you need?
- A resolving power approximating twice the molecule's molecular weight is typically necessary for isotopic resolution.
Resolving Power of Modern Mass Analyzers
- Different mass analyzers have different resolving powers and costs.
Important MS Concepts - Mass Accuracy
- Mass accuracy describes the agreement between the measured mass and the true value.
- It is commonly expressed using parts per million (ppm).
Mass Accuracies of Typical Mass Analyzers
- Different analyzers have different mass accuracies.
To summarize
- Monoisotopic mass: Calculated from most abundant isotopic masses of each atom.
- Average mass: Derived from weighted average of all isotopes of each element in periodic table.
- For small molecular weight compounds (under 10,000 Da), monoisotopic mass is often visible in mass spectrum from mass spectrometers with good resolving powers.
Basic Components of All Mass Spectrometers
- Mass spectrometers typically comprise:
- Inlet
- Ion source
- Mass analyzer
- Detector
- Data system
- High vacuum system
Why is Vacuum Needed?
- In mass spectrometry, ions must travel a specific distance before hitting a detector, preventing collisions with air molecules in the gas phase.
- The mean free path (λ) is the average distance a gas-phase ion travels before colliding with an air molecule.
- Without adequate vacuum, insufficient ion travel time to detector is achievable
Ion Sources for Biomolecules
- Soft ionization techniques (MALDI and ESI) keep molecular ions intact.
Matrix Assisted Laser Desorption/Ionization (MALDI)
- Laser vaporizes sample matrix, desorbing analyte ions.
- Typically used with protein or peptide analysis and soft ionization.
Sample Preparation
- Sample preparation methods for MALDI vary and impact final results.
- Using correct solvents/matrices/equipment.
Sample Clean-Up is critical for MALDI
- Removal of interfering molecules or contaminations (e.g., salts) is essential for accurate MALDI analysis.
MALDI Matrices
- Various matrices are used, each with unique characteristics like low molecular weight, polarity, and absorption at certain UV wavelengths.
Matrix Selection
- Matrix selection depends on the analyte characteristics for optimal analysis
- Polarity & acidity properties of matrices are useful for understanding protonation effects
Ionization Mechanism
- Matrix absorbs laser light in MALDI
- Analyte goes along with the plume
- Proton and charge transfers within the plume lead to analyte ionization
MALDI is well-suited for biomolecules
- MALDI is ideally suited for large biomolecule analysis (proteins > 300,000 Da.
- Provides excellent speed, affordability, sensitivity to analyze biomolecules with high precision.
MALDI Imaging
- MALDI imaging allows spatial mapping of molecules within complex samples, enabling visualization of disease versus healthy cells/tissues.
Chemical Warfare between bacteria
- MALDI imaging is useful in identifying chemical warfare used by bacteria to kill other microbes.
2002 Nobel Prize in Chemistry
- Koichi Tanaka's work showed soft laser desorption technique to ionize proteins without destruction and was foundational in MALDI methodology.
Electrospray Ionization (ESI)
- ESI transfers ions from solution to the gas phase at atmospheric pressure.
- ESI is widely used in LC-MS experiments and analysis of large or non-volatile molecules (e.g., proteins)
ESI generates charged droplets
- ESI generates charged droplets by transferring analytes from solution to the gas phase at atmospheric pressure.
- Charged droplets shrink and explode resulting in ions
Important Solvent Characteristics for ESI
- Organic solvents (MeOH or acetonitrile): Lower the surface tension for easier droplet formation.
- Low concentrations of volatile electrolytes (e.g., formic acid, acetic acid): Facilitate charge separation, creating smaller initial droplets for quicker desolvation.
Ionization Mechanism
- Solvent evaporation in ESI leads to droplet shrinkage, until electrostatic repulsion exceeds surface tension
- This causes droplet fission, resulting in tinier droplets and loss of small percentages of mass and charge.
Ionization Mechanisms
- Large molecules (e.g., folded proteins): Ionizes via charge residue model (CRM).
- Small molecules (e.g., peptides, unfolded proteins): Ionize via ion-ejection model (IEM).
CRM Model for Folded Proteins
- Folded proteins in ESI-MS experiments tend to produce ions near the Rayleigh charge, which is the size of the water droplet.
ESI produces multiply charged ions
- ESI significantly improves the detection of large molecules (over 10⁶ Da) using standard mass spectrometers by generating multiply charged ions.
How does charge state affect MS spectrum?
- Charge state affects the spacing between isotope peaks.
- Spacing is inversely proportional to the charge state, making it easier to identify charge states & MW by analysis
Time-of-Flight (TOF)
- TOF mass spectrometers measure the time ions take to travel from the ion source to the detector.
- High resolution and suitability to measure large biomolecules often used in conjunction with ESI.
Time-of-Flight (TOF) Mass Analyzers
- Ions with higher velocity will reach the detector faster.
- TOF instruments often involve two separate instruments or parts:
- Ionization region
- Mass analyzer.
- High resolution, good sensitivity, and useful over wide mass range, making it good with large biomolecule assemblies over 100,000 Da
TOFs have Unlimited Mass Range
- High resolving power
- Wide mass range
- Commonly used with large biomolecules and biomolecular assemblies.
Increasing TOF resolution
- Techniques like delayed extraction increase ionization efficiency, allowing ions to equilibrate before reaching the accelerating region.
- This uniform arrival at the detector increases resolution because ions of similar m/z are more likely to reach the detector at the same time.
Increasing TOF resolution - Reflectron
- Reflectron mass spectrometers use a series of electrodes with increasing potential that causes ions with higher velocity to penetrate deeper, then slow and deflect back towards the detector.
- This helps resolve ions with similar m/z that had differing initial velocities.
Improvement of resolution
- Continuous extraction, linear mode, and reflector modes are techniques used to improve mass spectrum resolution in MALDI-TOF MS, particularly when analyzing multiple isotopes
Fourier Transform Mass Spectrometers (FTMS)
- FTMS provide high sensitivity, speed, and resolution, making them ideal for complex samples with many overlapping ions
Basic Principles of FTMS
- A constant external magnetic field traps ions in circular orbits between electrically charged plates. All have same translational kinetic energy, but differences in m/z affect rotation speed
Ion Cyclotron Resonance
- Trapped ions absorb energy when excitation field matches the cyclotron frequency. Ions orbit in circle to cause a periodic response as they passes plates. This periodic response becomes a free induction decay signal in mass spectrum
- Trapped ions having different m/z ratio will have different cyclotron frequency and results in more distinguishable peaks.
Measurement of ICR Signal (FTMS)
- Trapped ions absorb RF energy, increasing motion.
- Ions with different ω (cyclotron frequencies) are unaffected.
- Measuring the time-domain response gives frequency-domain data which converts to useful mass data
Coherent Motion of Ion Packet is critical
- The coherent motion of ion packets (remaining clustered as traveling in circular orbit) aids in generating a periodic response when passing electrodes.
Measurement of ICR Signal (II)
- The magnitude of oscillating current depends on the number of ions in the packet.
- The frequency is directly related to m/z.
Fourier Transform
- Fourier transform converts the time-domain signal to a frequency-domain signal.
- In FTMS, this is used to convert the signal(frequency data) into useful mass data, making it easier to identify individual ions.
Converting FID to Mass Spectrum
- FT is able to extract m/z info from free induction decay data.
- The longer the observation period, the more detailed the spectrum, as well as higher resolution
Rest of Course
- Tandem MS: Fragmentation techniques for analyzing protein/peptide sequences, and modifications.
- Hydrogen/Deuterium exchange MS: Studying H/D exchange to track dynamics in protein structure using peptide backbones.
- Native MS: Ionization and transfer of proteins into the gas phase in a folded state for characterization of 3º and 4º structures.
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Description
This quiz explores the essential properties and mechanisms of the Matrix-Assisted Laser Desorption/Ionization (MALDI) technique, particularly in the context of biomolecule analysis. It examines the challenges in matrix selection, advantages, and applications of MALDI in biomedical research, as well as comparisons with Electrospray Ionization (ESI). Test your knowledge on how MALDI is revolutionizing mass spectrometry.
