How to Interpret MALDI Mass Spectra

The following description details how to interpret MALDI-ToF data for peptide mass fingerprinting. It is intended as an aid for customers with little knowledge of mass spectrometry, in order to facilitate data anaysis. Interpretation of MALDI-ToF data for different applications is beyond the scope of this document.

Ions are generated by Matrix-Assisted Laser Desorption/ionisation (MALDI), accelerated by a high electric potential, and separated by the time taken to reach a detector (Time-of-Flight (ToF)). The time-of-flight is directly proportional to the mass-to-charge ratio of an ion, and hence a mass spectrum is obtained.

An example of a mass spectrum is shown in Figure 1. Some features to note are:

1) that the x axis represents m/z - that is mass divided by charge. In MALDI-ToF, ions are nearly always singly charged [M+H]+ species (where M represents the molecule of interest; and H is hydrogen). To obtain an exact Molecular Weight, it is thus a simple matter of subtracting the mass of a single hydrogen (1.0079) from any mass shown on a spectrum.

2) the data generated by the IFR and JIC joint proteomics facility will fall in the m/z range of 800-4000 Da for Peptide Mass Fingerprinting.

3) although the peaks look like individual "sticks" when shown at full scale, they are actually groups of peaks. An expanded view of the peak at ~2465 is shown in Figure 2. The group of peaks shown in Figure 2 is called an isotope distribution. This is caused by naturally occuring Carbon-13 (13C has an abundance of approximately 1% of 12C). As a peptide of this size contains many carbon atoms (for this particular peptide 112 carbon atoms), then the contribution from 13C can be significant. The peak on the left is the 'monoisotopic' peak, and only contains 12C. The next contains one 13C (and thus has a mass of 1Da greater). The next has two 13C (and thus has a mass of 2Da greater than the monoisotopic peak) etc. etc.
All annotations on mass spectra supplied to customers will be for the monoisotopic peaks only.
So for the example in Figure 2, only the mass 2465.1990 would be given.

4) that the y axis represents absolute intensity - that is the number of ions of each species that reach the detector. However, abundance in the gas-phase is not usually representative of abundance in solution.

5) a number of masses have been deliberately excluded from the mass list supplied. Peaks resulting from autolysis of trypsin, from commonly occuring keratin peaks, and from sodium adduct peaks have been removed. This will aid in database searching.

All the peaks that have been annotated on the mass spectrum have been put into a mass list (usually in the form of an excel spreadsheet), and it is these that are used to search against a database (see section about database searching).