Protein Facility

Matrix Assisted Laser Desorption/Ionization Time-of-Flight (MALDI-TOF) is a mass analysis technique that was pioneered by Professor Franz Hillenkamp and Dr. Michael Karas of the University of Munster in Germany. The facility has an Perseptive Biosystems (Applied Biosystems) Voyager DE-PRO. MALDI offers a quick and easy method of mass analysis using a minimal amount of sample. The principles involved in the MALDI mass analysis can be seen in this figure.

From Finnigan MAT

Principles of MALDI

• The sample is dispersed in a large excess of matrix material which will strongly absorb the incident light.
  • The matrix contains chromophore for the laser light and since the matrix is in a large molar excess it will absorb essentially all of the laser radiation
  • The matrix isolates sample molecules in a chemical environment which enhances the probability of ionization without fragmentation
• Short pulses of laser light focused on to the sample spot cause the sample and matrix to volatilize
• The ions formed are accelerated by a high voltage supply and then allowed to drift down a flight tube where they separate according to mass
• Arrival at the end of the flight tube is detected and recorded by a high speed recording device From Finnigan MAT

   

The time-of-flight of the ion is converted to mass using the following principles:

• An accelerating potential (V) will give an ion of charge z an energy of zV. This can be equated to the kinetic energy of motion and the mass (m) and the velocity (v) of the ion
  zV = 1/2mv²
• Since velocity is length (L) divided by time (t) then
  m/Z = [2Vt²]/L²
• V and L cannot be measured with sufficient accuracy but the equation can be rewritten
  m/Z = B(t-A)²
  where A and B are calibration constants that can be determined by calibrating to a known m/Z

Mass of an ion is determined by the following method:

• 1. Measure time-of-flight (t) of the ion
• 2. External or internal calibration is used to determine the constants A and B so the time-of-flight can be converted to mass
  m/Z = B(t-A)²
• 3. Store B/V so changes in the 20 kV voltage supply does not effect calibration
  It is assumed that all ions have the same kinetic energy.

Samples are loaded onto metal plates for analysis on the instrument. A sample concentration of 1 mg/mL is ideal and usually from one to ten picomoles of sample is required for analysis. This is spotted onto the sample position on the metal strip and then 0.5 uL of matrix (usually 10 mg/mL) is applied to the sample position as well. There are many different matrices that can be used for MALDI-TOF. Some of the most common include Sinapinnic Acid (SA) for protein samples, alpha-Cyano-4-hydroxycinnamic acid (ACH) for peptide samples, and a 9:1 mixture of 2,5-Dihydroxybenzoic acid and 2-hydroxy-5-methoxybenzoic acid (sDHB) for carbohydrate and sometimes protein samples. DNA can also be analyzed using MALDI-TOF by employing different matrices. New matrix solutions are now in development which will yield greater sensitivity and resolution.

MALDI technology has many applications in the biochemical field. It can be used to easily monitor and optimize enzymatic digests, characterize proteins, or can be used for quality control for peptide synthesis. MALDI has also been used as a method of N-terminal and C-terminal protein/peptide sequencing. There are also applications in the rapid conformation of post translational modifications and the quantitation of drugs and chelators conjugated to monoclonal antibodies.

• MALDI Sample Preparation
  MALDI samples should be free of SDS, and should not be radioactive. It is best to remove buffer salts and detergents (e.g. by dialysis) prior to analysis and to dissolve the sample in a suitable solvent (e.g. 0.1% TFA/water) which will not degrade the spectrum. If there is too much salt in a sample, the salt signal intensity is so large that it effectively suppresses out the sample signal, giving no sample spectrum. In cases where it is not possible to remove these contaminants the sample should be in a higher concentration. It may then be possible to dilute the sample to the point where the contaminants will have little effect on the spectrum.
  
  Levels of buffers and detergents which exceed the following limits will probably cause noticeable degradation of the spectrum:

  Phosphate buffer	>50mM
  Ammonium bicarbonate	>30mM
  Tris buffer	>100mM
  Guanidine	>1M
  Detergents(e.g. Triton-X)	>0.1%
  SDS	>0.01%
  Alkali metal salts	>1M
  Glycerol	>1%
  Sodium Azide	>1mM

  
  
  
• Common Matrices used in MALDI-TOF
• Resolution and Mass Accuracy on the Perseptive Biosystems Voyager DE-PRO
• MALDI Mass Spectrometry Submission Form (PDF)
• MALDI Peptide Spectrum Interpretation, Nick Wedd
• Protein Prospector-Proteomics tools for mining sequence databases in conjunction with Mass Spectrometry experiments