32.17
MALDI-TOF Kütle Spektrometrisi
Kütle spektrometrisi, kütle/yük oranına, yani m/z’ye göre kimyasal ve biyolojik örneklerde bulunan geniş bir yelpazedeki bileşikleri tanımlayabilen ve…
MALDI-TOF mass spectrometry is an analytical technique for detecting and characterizing biological samples such as peptides, carbohydrates, and DNA.
MALDI-TOF contains an ionization source to generate gaseous ions and a mass analyzer for ion detection based on the ion’s time of flight.
First, the sample peptides are mixed with a matrix and loaded onto the target plate.
When struck by a laser, the sample desorbs, producing positively charged ions. That is why this technique is called, matrix-assisted laser desorption ionization.
Ions then move into the mass analyzer, where they are sorted, depending on the time required to travel through the length of the flight tube.
This time of flight is proportional to the square root of the mass-to-charge ratio. Among the ions with the same charge, smaller ions move faster.
The detector collects these ions and passes the information to a computerized system. It generates a peptide mass fingerprint to identify the unknown protein by comparing the peptide fragments with a database library.
View the full transcript and gain access to JoVE Core videos
Q1: What are the main components of a MALDI-TOF mass spectrometer?
MALDI-TOF mass spectrometers contain three essential components: an ionization source that generates gaseous ions from biological samples, a mass analyzer that sorts ions based on their time of flight through a drift region, and a detector that collects ions and sends data to a computerized system. The matrix-assisted laser desorption ionization source enables soft ionization of peptides, lipids, and carbohydrates without fragmenting them, maintaining sample integrity.
Q2: How does the matrix function in MALDI ionization?
The matrix consists of organic molecules mixed with the sample that absorb laser energy and mediate ionization. When the laser strikes the sample, matrix molecules energetically ablate from the surface, absorb the laser energy, and carry sample molecules into the gas phase. This process ionizes sample molecules to carry a single positive charge while protecting them from fragmentation, enabling intact analysis of biological molecules.
Q3: Why do smaller ions reach the detector first in MALDI-TOF?
Ion separation in MALDI-TOF depends on mass-to-charge ratio. Smaller ions move faster through the flight tube because their time of flight is proportional to the square root of the mass-to-charge ratio. Among ions with the same charge, those with lower mass travel more quickly, allowing the detector to collect them first and create a peptide mass fingerprint for protein identification.
Q4: What is a peptide mass fingerprint and how is it used?
A peptide mass fingerprint is a pattern generated by the computerized system after the detector collects ions from the mass analyzer. This fingerprint represents the masses of peptide fragments from an unknown protein. By comparing the peptide mass fingerprint against a database library, researchers can identify the unknown protein and characterize its composition and structure.
Q5: How does a reflectron improve MALDI-TOF resolution?
A reflectron is an optional component that reflects ions back through the flight tube, increasing the ion flight path. This extended flight time between ions of different mass-to-charge ratios allows better sample resolution, enabling ions of the same mass to reach the detector simultaneously. This improvement enhances the accuracy and precision of mass measurements in biological sample analysis.
Q6: What biological samples can MALDI-TOF analyze?
MALDI-TOF can analyze a wide variety of biological molecules including peptides, carbohydrates, lipids, and DNA. It can identify microorganisms from clinical samples, ascertain biomolecules in tissues, and analyze protein samples isolated by electrophoresis or chromatography. The soft ionization capability maintains sample integrity, making it ideal for characterizing intact biological entities without fragmentation.
Q7: How does MALDI-TOF compare to other mass spectrometry analyzers?
MALDI sources can be coupled with different analyzers beyond time-of-flight, including triple quadrupoles and Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometers. MALDI-FT-ICR instruments are known for exceptionally high mass resolution, where sample ions move through an ICR cell in a magnetic field while separating by mass-to-charge ratio, offering enhanced analytical capabilities for complex biological samples.