Maldi
 
Maldi-TOF MS
  
 
 
 
 
 

Background

The first use of mass spectrometry in the identification of proteins by either peptide mass fingerprint identification or peptide sequencing dates from just some years ago [3, 6, 9]. The reason for this late application of mass spectrometry in the analysis of proteins is the only recent development of soft ionizations methods for peptides and proteins. Ionization of (poly)peptides is required for mass spectrometry because sample molecules must be available as ionized gas. However, hard ionization methods used in mass spectrometry result in the fragmentation of (poly)peptides. In 1988 Karas and Hillenkamp [4] developed ‘Matrix Assisted Laser Desorption Ionization’ (MALDI) which is now used as a soft ionization method in time-of-flight mass spectrometry (TOF MS).

The combination of TOF MS with MALDI is ideal because a TOF mass-analyzer does not depend on continuous ion formation and offers a wide mass range in which high resolution (isotope level) mass measurements can be performed [1, 3, 4, 5, 6, 7, 9, 10].

MALDI in theory and practice

After protein digestion by a proteolytic enzyme (e.g. trypsine), peptides are mixed with low molecular weight compounds which have an absorption maximum at the wavelength of the laser (e.g. a-cyano-4-hydroxy cinnamic acid) [2, 12]. Such compound is referred to as matrix. Mixtures of peptides and matrix are then applied and dried as spots on a metal target plate. Different matrix substances and application methods are used depending on the nature of the compound to be analyzed and type of measurement used in MALDI-TOF MS. The sample plate is inserted into a vacuum lock and transferred into a vacuum chamber (ion source). MALDI takes place inside this ion source. A pulsed laser beam (337nm) is fired into the spot and matrix molecules absorb the applied energy. This results in desorption of matrix and co-crystallized analyte molecules into the gas phase which move away from the target at supersonic speed. At the same time, charged matrix molecules collide with analyte molecules and transfer their charges on the peptide molecules.

Figure 1. Schematic representation of Matrix-assisted laser desorption and ionization
(click to enlarge).

MALDI produces mainly singly-charged metastable ions, which are able to hit the detector prior to decomposition (lifetime > 10-5 s). If the TOF measurement is performed in the positive ion mode, a general mass shift of +1 Da is observed for all peptide ions. The mono-isotopic form of this ion type is denoted [M+H]+ due to the ionization process in which a proton is exchanged from matrix to analyte [1, 4, 10, 11].

After ‘MALDI’, ionized peptides are accelerated in an electric field with the help of a high voltage grid. All ions are accelerated to the same kinetic energy to ensure that all ions of identical mass move at the same speed. The accelerated ions are then introduced into the high vacuum flight tube (‘field free drift region’) and continue to fly until they reach the detector. Since light ions reach the detector earlier than heavy ions, recorded flight times are used to calculate ion masses in m/z (mass per charge). Resolution and mass accuracy is thus dependent on the time window wherein ions of the same mass reach the detector. This is for a large part determined by the start velocity distribution.

Figure 2. Schematic representation of MALDI-TOF mass spectroscopy
(click to enlarge).

Pulsed extraction and reflectron tubes are used to minimize and correct start velocity distribution, respectively. During pulsed extraction, the voltage of the acceleration grid is switched on with a time delay of a few hundred nanoseconds from the laser impulse. Once the acceleration grid voltage is applied, ions with a higher start velocity will be further away from the target than slower ions and receive a smaller portion of kinetic energy. For example, a slow ion that has traveled 1% of the distance obtains 99% of the potential, whereas a fast ion might have traveled 15% and will obtain only 85%. Pulsed extraction greatly reduces start velocity distribution and improves resolution in reflector mode [10]. Reflectron tubes are used to reverse the drift direction of the ions in an electric counter field (Figure 2). Ions of the same mass but higher start energy drift deeper into the reflector before being reflected and fly a longer distance before they reach the detector. In this way, they catch up with the slower moving ions at a certain point after the reflector. The detector is located at this focusing point.

Reflector tubes greatly enhance resolution up to the isotope level by correcting start velocity distribution and by prolonging the flight time. Although use of the reflector mode results in higher resolution, reflection reduces sensitivity and high molecular weight (poly)peptides can only be detected in the linear mode. [1, 10, 11].


Figure 3. Representative MALDI-TOF MS spectrum (click to enlarge).
The Figure shows the m/z spectrum of the alpha subunit of methyl coenzyme M reductase of the methane-forming archaebacterium Methanothermobacter thermautotrophicus. Mass spectral peaks are labeled with mono-isotopic mass-to-charge (m/z) values used for database searching.

MS then results in a number of peaks with recorded m/z values derived from the peptide fragments of the proteolytically cleaved protein under analysis. The peaks of each peptide include of [M+H]+ ion, which is usually the highest peak, as well as peaks that stem from the (C13-)isotope forms of the peptide. In addition, artifacts peaks will be present, generally of known m/z values, caused by impurities or by matrix adducts. All (relevant) peaks seen on the MALDITOF MS spectrum are manually selected as their mono-isotopic peptide peaks ([M+H]1+ ) and stored in a peak search list. The list is submitted to a computer search program (MASCOT), which compares experimentally obtained m/z values with theoretically calculated m/z values of tryptic peptides of proteins from a genomic open reading frame database.