Mass Spectroscopy - Instrumental Analytics

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Questions and Answers

How does proton transfer occur during analyte ionization in MALDI?

Proton transfer occurs from photo-excited matrix molecules to the analyte, stabilizing it and encouraging cation attachment.

What is the significance of the matrix in MALDI-MS?

The matrix enhances the absorption of laser light, increases analyte intensity, and reduces fragmentation of the analyte ions.

Describe the crystallization process in the ionization principle of MALDI.

The solvent evaporates, leading to crystallization where analytes become part of the crystal structure.

What role does the electrostatic field play in the ionization process after analyte ionization?

The electrostatic field accelerates the ionized analytes towards the mass analyzer based on their charge. Signup and view all the answers

Define the purpose of TOF technology in mass spectrometry.

TOF technology measures the mass of the analyte by timing the flight duration from ionization to detector arrival. Signup and view all the answers

What is involved in calibrating the mass spectrometer with known reference molecules?

Calibration involves using reference molecules with known molecular weights to establish a relationship for mass determination. Signup and view all the answers

Explain how fragmentation is minimized in the MALDI process.

Fragmentation is minimized when proteins are embedded in a matrix that absorbs laser energy efficiently, preserving analyte integrity. Signup and view all the answers

How do ions with different m/z values behave during the acceleration phase in TOF?

Ions with different mass-to-charge ratios are accelerated to different velocities, allowing for their separation. Signup and view all the answers

What is the primary purpose of mass spectroscopy?

To determine the molecular mass of ions in vacuum. Signup and view all the answers

Name two ionization techniques used in mass spectrometry.

Matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI). Signup and view all the answers

What roles do the ion source, mass analyzer, and ion detector play in a mass spectrometer?

The ion source generates ions, the mass analyzer separates them based on mass-to-charge ratio, and the ion detector measures the abundance of the ions. Signup and view all the answers

Explain the role of the high potential difference in electrospray ionization.

It forces the spraying of charged droplets from the electrospray needle towards the counter electrode. Signup and view all the answers

Describe the first step in the MALDI process.

The formation of a 'Solid Solution' where the matrix is in excess, isolating analyte molecules from each other. Signup and view all the answers

How does matrix excitation occur in the MALDI process?

The laser beam is focused on the matrix-analyte solid solution, causing localized vibrational excitation and disintegration. Signup and view all the answers

What happens to the clusters ejected from the surface during the MALDI process?

Matrix molecules evaporate away, leaving the free analyte in the gas-phase. Signup and view all the answers

What is the function of the Faraday cup in a mass spectrometer?

It acts as an ion detector that measures the current produced by the incoming ions. Signup and view all the answers

Study Notes

Instrumental Analytics - Mass Spectroscopy

Focuses on determining the molecular mass of ions in a vacuum environment.
Analytical technology utilized for determining the molecular mass of ions.

Mass Spectroscopy - Components

Ion Source:
- Electrospray ionization (ESI)
- Matrix-assisted laser desorption/ionization (MALDI)
Mass Analyzer:
- Combination of electric and magnetic fields
- Quadrupole (high frequency)
- Time-of-flight (TOF) analyzer
Ion Detector:
- Faraday cup

Mass Spectroscopy - MALDI

Mechanism:
- Formation of a solid solution, crucial for analyte isolation.
- Matrix excitation by pulsed laser light, causing localized disintegration of the solid solution.
- Analyte ionization via photo-excited matrix molecules. It results in the generation of characteristic ions such as [M+X]+.
Advantages:
- Analyzes large biomolecules without destruction. Improves analysis of large biomolecules.
- Higher absorption and intensities of analyte molecules (proteins), leading to less fragmentation.

Mass Spectroscopy - ESI

Mechanism:
- Analyte solution flow undergoes high potential difference (2.5-4 kV) relative to a counter electrode.
- Charged droplets are repelled towards the source sampling cone.
- Solvent evaporation occurs as droplets traverse the space between the needle tip and the cone.
Advantages:
- Eases analysis of large biomolecules because it doesn't destroy them during ionization.

MALDI-MS

Early attempts utilized pulsed laser light for ionization.
Initial spectra showed low intensities and fragmentation.
Advances led to matrix use, embedding proteins in small organic molecules, enhancing analysis.
This technique produces higher intensities of analyte molecules (proteins) with significantly reduced fragmentation.

Ionization Principle (MALDI)

Analyte mixes with a large excess of matrix (1000-10000 fold molar excess).
Solvent evaporates, causing crystallization where analytes are part of the crystal.
Surface of the crystal is excited with short wavelength laser light for ionization.
Only intact proteins result when laser energy is correctly-tuned.
An electrostatic field (100-1000V/mm) next to the sample accelerates ions to the mass analyzer based on polarity.

TOF Technology

Analyte mass is determined via electronic measurement from ionization to detection.
Electrostatic field accelerates ions beyond keV energies.
Analyte separation in a field-free region based on mass-to-charge ratio (m/z) produces different velocities.
Known flight distance and acceleration voltage, enable calculation of m/z ratio post-measurement.

MALDI TOF Resolution

Modern MALDI enables isotope composition resolution (<1 dalton) in peptides and proteins.
Limitations of MALDI concerning energy distribution and ion desorption.
Reflector design helps with starting condition issues for more reliable results.

Ion Detection

Secondary electron amplifier uses several dynodes (high potential ~10kV) and opposite charges of ions.
Analogous signal generates the spectrum.

Matrix Preparation

Matrix principles aim to isolate the analyte in crystals and allow laser absorption for suitable analysis.
Matrix solution mixing with sample solution, often achieved in methanol or acetonitrile for dissolving matrix molecules (100mM concentration).

Sample Preparation

Protein purity is critical for analysis.
High salt concentrations, detergents, and hydrophobic protein are problematic.
Separation and purification techniques include reversed phase chromatography (with volatile solvents).

MALDI Spectra

Can produce both positive and negative spectra (deprotonated molecules for negative).
Increased molecular weight results in more multiple charged monomers (M+2H+, M+3H+).
Single charged species usually have highest concentrations.

Isotope Influence

Natural elements contain mixtures of isotopes.
Isotope distributions exist for all naturally occurring molecules.
Software calculates isotope distributions without manual user intervention.
Software usually normalizes peaks on the most intense ones.

ESI with Quad

ESI involves atmospheric pressure ionization, followed by vacuum application.
Ionization chamber connected to a mass analyzer by a micro-aperture.
Nitrogen flow prevents neutral particles from entering the analysis portion.
Capillary solution and electric field are needed for ionization.

Quadrupole Analyzer

First described in 1953, it consists of four metal electrodes producing an alternating AC/DC field.
Ions of the appropriate m/z ratio follow stable oscillating trajectories. Other ions collide with metal 'stabs' stopping them.

Proteomics

Sample preparation involves cell or tissue lysis, protein extraction, and protein digestion (using trypsin or chymotrypsin).
Proteins are analyzed via chromatography coupled with MS.
Analytes are isolated, detected, and measured based on mass-to-charge (m/z) ratio and retention time.

AK Characterization

Covers primary structure analysis (e.g., using ESI-MS, LC-MS, CE-SDS); secondary structure analysis (circular dichroism, FT-IR) and higher-order structure analysis (e.g., HDX-MS, XRD).
Functional assessment includes equilibrium dissociation constants, ligand binding assays, competition ELISAs, and cell-based assays.

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Description

This quiz explores the principles and components of mass spectroscopy, focusing on ion sources, mass analyzers, and detectors. It highlights techniques such as electrospray ionization and matrix-assisted laser desorption/ionization, essential for molecular mass determination. Ideal for students of analytical chemistry and instrumentation.