Mass Spectrometry

Mass Spectrometry

It is an interpretive technique wherein the molecular mass of the sample is provided in the

form of their mass to charge ratio via ionizing the molecules into charged molecules with the help of an ion source and depiction of molecular mass is done by mass analyzers.

Mass Spectrometry has four instrumental stages:

  • Introduction of the sample.
  • Ionization of the sample via ion source
  • Analysis of generated ion molecules via mass analyzer.
  • Detection

There are various types but today we are discussing here “MALDI-TOF Mass Spectrometry”.


MALDI TOF Mass Spectrometry where “MALDI” stands for Matrix-assisted laser desorption which is the “Ion source” utilized for ionizing the sample molecule and “TOF” stands for “Time of Flight” utilized as a mass analyzer.

The term “MALDI” was given by Franz Hillenkamp, Michael Karas in the year 1985.


 It is considered a “soft ionization” technique that involves a laser beam that strikes the analytes embedded in a matrix fixed on a solid surface known as a metal target. After the irradiation with the ion source, the molecules get ionized and vaporized accompanied by the matrix and lead to the analyzer.

The ion source utilized in MALDI-TOF is ESI which is Electrospray ionization utilized for the samples that cannot be vaporized and ionized in a single step like carbohydrates, protein, etc. The best part found is that at high temperatures, vaporization of the sample gets much faster and easier without decomposing the sample.

There are various ion analyzers, here in combination with MALDI the TOF is the most employed one, which stands for “Time of Flight”. TOF measures the time taken by the ions to reach the detector when applied by the equal electric potential. As the electric potential given is equal for all the ions, ultimately here their mass will act as the deciding factor, that which ion will reach faster to the detector. The smaller the ion, the faster the velocity and it will reach first to the detector and vice versa. Hence, with the help of the TOF analyzer, it becomes easier to get the data precise in no time.

The detector utilized is “PMT” called Photomultiplier tube which is analyzed by the impact of ions on the receiving tube which is in mathematical terms is that the number of electrons is directly proportional to the number of ions impacts and generates the signal to be analyzed.

The whole process leads to the destruction of the sample and the sample cannot be recovered.

 Flow chart of mass spectrometry

Selection of Matrix

  • The matrix plays a vital role as it acts as solid support for the sample and the right choice of the matrix makes the whole process a success. The selection is based on the polarity of the sample or a hit and trial method is approached. Eg: 3-Hydroxypicolinic acid, 3-Aminopicolinic acid, 2,5-Dihydroxybenzoic acid are utilized for DNA, oligosaccharides, and proteins.

Sample preparation

  1. The sample to be analyzed needs to be of concentration 0.1mg/ml in a solvent.
  2. Then the matrix according to the sample analyzed is dissolved to get a saturated solution of concentration 10mg/ml.
  3. Both the solutions, that is sample solution and the matrix solution is mixed together.
  4. Then the MALDI spectra range is adjusted according to the expected molecular mass or the m/z ratio which can range from 1000: 1 to 100,000: 1.
  5. The mixture is then dotted onto the metal target plate for further analysis.
  6. The mixture was allowed to dry which led to the formation of a solid deposit of sample planted into the matrix.
  7. Then the target plate is charged into the MALDI-TOF instrument and analyzed.

Sample Preparation

Why is MALDI-TOF is a “Soft ionization technique”?

It is considered a “soft ionization technique” as the ions have low internal energy and this led to the minimal to null fragmentation of the ions providing the accurate molecular mass. This advantage has taken the technique to great heights. 


  • Whole Mass Determination: The determination of intact mass plays a vital role in protein characterization. The generation of molecular weight will help to determine the intact structure of the protein that’s why it is considered the most suitable ionization technique for fragile components like proteins, DNA, peptides, oligosaccharides, polymers, etc. which can be further utilized for various purposes.
  • Peptide Mass fingerprinting: As MALDI-TOF has great accuracy, high resolution, and sensitivity that why is highly utilized in proteomics and is employed to identify proteins from simple mixtures by the method called peptide mass fingerprinting in which the peptides are produced via digesting proteins of interest with the help of specific enzymes and then analyzing the masses of these peptides with the service of MALDI TOF and further comparison can be done with theoretical procured masses of the peptide with the practical received ones.
  • Oligonucleotides analysis: As the curiosity is increasing, exploration of various areas is also increasing like the development of various molecular biological techniques and antisense nucleic acid drug technologies which is leading us to develop and analyze the complete oligonucleotide sequence primers, probes, and anti-sense drugs. The analysis of these fragile components requires a sensitive and accurate instrument that is fulfilled with the help of MALD-TOF Mass Spectrometry.
  • MALDI-TOF in virology: As known conventional methods like cell culture and immunological methods, PCR, and blot techniques are employed to identify viruses. Though they are considered as the gold standard for the identification purpose but are long-delayed therefore, there was a need for a fast technique for fulfilling the purpose. Hence many researchers employed the MALDI-TOF MS to identify viruses and received astonishing results depicting the method as more reliable, fast, and sensitive than the existing methods. Even in the year 2012, Piao and co-workers mingled the PCR technique with MALDI-TOF MS and received impressive results by detecting eight distinct viruses related to enteric infections in humans and named this technique as PCR-Mass assay.
  • In a similar way, the technique is utilized in the identification of microorganisms (Bacteriology), Detection of antibiotic resistance in bacteria, Identification of protein complexes.


  • Common solvents utilized for the sample preparation can be purified water, Acetonitrile, Ethanol, etc.
  • Trifluoroacetic acid is incorporated with the matrix and the sample to generate charged ions (M+H) ions.
  • Matrix utilized basically has low molecular weight consisting of chromophores.
  • Lasers used for energizing the samples to become protonated ions are UV lasers, Infrared lasers.
  • The mixed dried solution of the matrix and the sample that is recrystallized matrix is known as matrix cocrystal.
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