Matrix-Assisted Laser Desorption/Ionization (MALDI) mass spectometry

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Matrix-Assisted Laser Desorption/Ionization (MALDI) mass spectometry

For researchers who anticipate running many samples, there is an open access program in which users run their own samples.

What it can do

  • measure intact mass of proteins, peptides and other molecules
  • isotopic resolution of molecules up to mass of 3500
  • mass range of 700 to 70,000
  • smaller molecules can be measured in some cases-matrix interferes below 600
  • non-peptide molecules may not ionize well
  • positive ions (protonated) used most often; negative ions (deprotonated molecule) used for negatively charged molecules
  • not good for mixtures
  • salt can be removed with ZipTip or diluted for some samples
  • some detergents can be tolerated in concentrated samples-need special procedure
  • accuracy of 0.1%. For detecting small modifications to proteins, mass determination by ESI may be necessary


Sample requirements

  • 20 µM or more solution
  • minimum 1 µL for direct spotting on plate, 5 µL if desalting needed
  • sample must be miscible with acidic solution of 80% methanol
  • molecule must ionize
  • limited ability to analyze molecules <700
  • need to know solvent composition
  • detergent may prevent analysis
  • unused sample can be returned

Principle

  • A sample is mixed with an aromatic carboxylic acid matrix; most common are 4-cyanohydroxycinnamic acid and sinapinic acid.
  • The sample-matrix mixture is dried on a plate, then loaded into the mass spectrometer which is evacuated to a high vacuum.
  • MALDI ionizationA laser illuminates the sample and the matrix transfers energy to the sample molecule which moves into the vapor phase.
  • The energy also ionizes the molecule giving it a charge (positive ions most commonly used).
  • An electric field accelerates the ion down a tube about 1 meter long.
  • The kinetic energy of the ion when it reaches the detector can be calculated using Newtonian physics with the equation: energy= ½ mass *(velocity)2. The energy of the ion can also be calculated from: charge * voltage, where the voltage of about 20,000 is known from instrument settings, and charge is an integral number, most often 1.
  • From measurement of the time taken for the ion to move from the plate on which it was loaded to the detector, the mass can be calculated.
  • Although the instrument measures the time for an ion to move from where it goes into the vapor phase to the detector, normally results are shown as mass/charge. The mass/charge can be calculated from the quantities listed above, but normally a set of standards is used to calibrate the instrument.
  • delayed extraction;a delay between the laser pulse to move molecules into vapor phase and application of the accelerating voltage.
  • reflector mode in which an electrostatic lens reflects ion, focusing them, and thus increasing the resolution between different molecules. Useful up to about 6000.

Data

The instrument reports a mass to charge ratio (m/z). In MALDI, most molecules have a single charge, so the m/z measurement is equal to the mass of the ionized molecule.
Most often, positive ions are measured so that the m/z measurement is for the original molecule plus a hydrogen ion. The commonly used Protein Prospector tool (http://prospector.ucsf.edu/) calculates m/z for positively charged peptides.
Sometimes we measure negatively charged ions which are formed by removing a proton from the sample molecule, and thus have a m/z about 1 unit less than the original molecule.

For peptides up to about 4000, molecules differing in size by 1 mass unit can be distinguished. Thus for a peptide, multiple species show because of the different isotopes of oxygen (O-18) and nitrogen (N-15) in particular. For peptides up to mass of 2000, the most abundant isotopic form of a peptide is one containing only the most common isotopic forms of the elements i.e. all O-16, all N-14). Above 2000, molecules containing one or more heavy isotopes are more prevalent.

Above about 4000, the instrument does not have enough resolution to distinguish molecules differing by a mass of one so we use the more sensitive linear mode i.e. we do not use the electrostatic reflector, and report average masses, which Protein Prospector and ExPASy (http://ca.expasy.org/tools/pi_tool.html) tools can calculate.

For proteins, expected accuracy is 0.2% or less. A problem with estimating masses of large molecules is having a good standard. Above 44,000, there is no readily available high purity proteins available. Although BSA is supplied as a standard, even one manufacturer warns that it is not a reliable standard, because it may bind other molecules which increase its mass.

Molecules differ in how readily they ionize. Thus a MALDI measurement of a mixture of peptides is not a good way to determine relative amounts of peptide or purity of a sample because one molecule may ionize much more readily than another, particularly in mixtures, and give a stronger signal even though there is less of it than another molecule.

We can perform more accurate measurements with the ESI instrument, but the sample requirements are more stringent, there is a longer wait time to perform measurements and the cost is higher.

Acidic peptides can bind sodium and potassium ions, which come from glass vessels as well as buffers. Acidic peptides may show only masses 22 and 38 mass units higher than expected because the peptides bind a sodium or potassium ion. In some cases, this can be confirmed by measuring the mass of negative ions, which is usually less sensitive.