MALDI-TOF

Question 1

Discuss how proteins are identified by Peptide Mass Fingerprinting (PMF) using Matrix Assisted Laser Desorption Ionization Time-of-Flight (MALDI-TOF) mass spectrometry.

Answer 1

1. General on MALDI-TOF and PMF
2. MALDI (ionisation source)
3. TOF (mass spectrometer)
4. PMF (protein identification method)

Question 2

1. genetal on maldi-tof and pmf

Answer 2

• (PMF) is a technique used to identify proteins by matching their constituent fragment masses (peptide masses) to the theoretical peptide masses generated from a protein or DNA database

Question 3

2. MALDI (ionisation source)

Answer 3

1. general info
   - MALDI works very well with thermolabile, non-volatile organic compounds (peptides)
   - commonly used in the analysis of proteins, glycoproteins, peptides, olidonucleotides and oligosaccharides
2. ions (positive ions, in a vacuum)
   - to detect and quantitate individual molecules they need to be converted to ions in order for them to be manipulated by external magnetic and electric fields
   - manipulation of ions is carried out under vacuum as this ensures that all other molecules have been pumped out and wont interfere with the detection and quantification of the sample

• ionisation of the sample is carried out by its bombardment with a laser light usually a pulsed nitrogen laser at 337 nm wavelength
• the laser generates photons (energiseed beams coming from the laser), which energise the peptides, when the peptides become energised they enter the mass spectrometer
• before laser bombardment the sample is mixed with an energy absorbing matrix which is responsible for converting the laser energy into excitation energy
• 2 types of EAMs; sinapinic acid for proteins and alpha-Cyano-4-hydroxycinnamic acid for peptides
• EAM molecules used at ratio 1000 matrix: 1 peptide

3. analyte and matrix spotting
   - the sample mixed with the appropriate matrix is placed onto a MALDI plate and is bombarded with a laser
   - absorption of UV radiation by chromophoric matrix causing evaporation and ionization of the matrix
   - when the solvent dries a thin crust containing the matrix and peptide is left behind
   - the laser hits spot with embedded peptides and matrix absorbs this energy (photons) allowing the electrons in the matrix to go to a higher energy state. higher energy state causes molecules to vibrate which as they do they desorb off the plate bringing the analyte with them
4. calibrating maldi plate
   - external calibration: Pep4 (mixture of 4 peptides with known mass introduced externally)
   - internal calibration:
   1. trypsin autodigestion peaks: creates autolysin peaks as it digest protein of interest and itself as it is a protein
   2. keratin peaks: comes off skin and is impossible to keep out of samples, digested by Trypsin
   3. spiking with an internal standard: after digesting protein of interest, add an external peptide at a known concentration and calibrate

Question 4

3. TOF (mass spectrometer)

Answer 4

• time of flight mass spectrometer measures the mass-dependant time it takes ions of various masses to move from the ion source to the detector
• for this to work successfuly the starting time must be well defined
• ions are formed in pulses, small ions reach the detector before large ones and the detector measures the time it takes
• 2 modes; linear (1m) and reflectron (3m), reflectron flight tube longer therefore allows peptides with similar masses to be seperated out
• mass spectrum is generated according to measured mass values of generated peptides and their abundance (intesity of signals)
• the abundance of an ion is measured by the current produced by electrons jumping to neutralise their charge when they reach the detector

Question 5

4. Peptide Mass Fingerprinting

Answer 5

• (PMF) is a technique used to identify proteins by matching their constituent fragment masses (peptide masses) to the theoretical peptide masses generated from a protein or DNA database
• first step, intact, unknown protein is cleaved with a proteolytic enzyme e.g trypsin to generate peptides
• as every protein has a unique set of peptides and therefore a unique set of peptide masses, identification can be accomplished by matching the observed peptide masses to the theoretical masses derived from a sequence database
• as the peptides are generated from protein resolved by 1D/2D gel elelctrophoresis, there is only a small number of peptides (all peptides coming from singular protein)
• peptide mass fingerprints can be searched for on a software called MASCOT and a confidence score can be calculated. anything above 40 is a high quality hit and indicates that its a match

FACTORS AFFECTING PERFORMANCE OF PMF
- PMF cannot deal with mixtures of more than 2 proteins
- specificity of the digestion: trypsin - need to generate tryptic peptides as opposed to spontaneous ones, for database search need to specify the enzyme used
- the mass assigned to peptides needs to be accurate so it will match peptide mass list in database