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Determine the precence of, and identify, species (proteins, peptides, etc.) in a sample.

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Materials Needed

  • MALDI-TOF Mass Spectrometer
  • Sample Plates
  • Appropriate matrix solutions
  • Reasonably pure analyte


Protein Isolation:

  • Commonly use of 1- or 2-D gel electrophoresis
  • Any method that will net a pure protein or peptide assay

Sample Preparation:

  • MALDI samples are analyzed in solid state, prepared by suspending the analyte in a liquid matrix. The analyte and matrix are then spotted onto a sample plate and dried. The matrix solution dries and forms into crystals which have analyte suspended inside.
  • Things to take into account for sample prep:
    • concentration of matrix and analyte
    • hydrophobicity
    • hydrophilicity
    • solubilities
    • ionic strengths
  • Be warned - precipitation of analyte or sample can occur if an incorrect choice is made.

Sample Analysis:

  • Laser will hit the sample a vaporize the crystals releasing some of the analyte along with it.
  • Sample then enters a vacuum and accelerated by a electric field down the 'flight tube' to the detector.
  • The detector measure the time-of-flight (TOF) for a ion to reach it and relates that to the mass/charge (m/z) ration.
    • TOF is the mass component
    • The charge of the analyte is calculated by the detector upon contact
  • Sequencing can be done in terms of peptides or amino acids
    • peptides(MS)
      • protein fractionation by chemical agents will yield characterizable peptides
      • peptides will be compared to databases entry and used to identify your protein
    • amino acids(MS/MS)
      • chemical degradation of either the N- or C- terminus.
      • standards used to generate calibration curve
      • amino acid sequence determined by order of occurrence in spectrum


John J. Lennon

Kings College



User notes

Matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is generally regarded as the workhorse of proteomics due to its easy of use in identification of biomolecules.

Heating sample to make it dry faster can cause problems with protein incorporation into the growing crystals leading to a bad sample. Once dried the sample is very stable and can be stored for a ccouple days if stored properly.

Rinsing of crystal may help increase the quality of the spectra. Depending on the matrix, using μLs of ddH2O will do the job.


  • linear vs. reflectron
  • linear is quicker and easier
  • reflectron improves mass resolution
    • A second electric field is generate to reflect the ions. The greater the size of the ion the farther it penitrates into the field before being repulsed (momentum: ρmv2).
    • allows for compensation of kinetic energy difference in analyte with the same m/z

Size Limitations of MALDI-TOF MS

  • Lower bounds: ~500 Daltons
    • by doing MS/MS this can be lowered to allow analysis of amino acids
      • Electrospray is more exact than MALDI for aa analysis
  • Upper bound: >300 KiloDaltons
    • not clearly defined at this point


[1] [2] See Help:References for how to manage references in EcoliWiki.

  1. Blackstock, WP & Weir, MP (1999) Proteomics: quantitative and physical mapping of cellular proteins. Trends Biotechnol. 17 121-7 PubMed EcoliWiki page
  2. Egelhofer, V et al. (2002) Protein identification by MALDI-TOF-MS peptide mapping: a new strategy. Anal. Chem. 74 1760-71 PubMed EcoliWiki page