Interpretation of the Mass Spectrum
A mass spectrum is generated by a Matrix-Assisted Laser Desorption/Ionization Time of Flight (MALDI-TOF) Mass Spectrometer. A mass spectrum is a useful tool to aid in verifying that a synthetic oligonucleotide has the correct molecular weight. It is about an order of magnitude more precise in the estimation of molecular weight than a gel electrophoretogram, even when the latter is properly calibrated. It does have limitations, however, some of which are the following:
- At present, desalted (but not otherwise purified) oligonucleotides with more than 50 nucleotides are difficult to analyze by MALDI-TOF mass spectrometry.
- T-rich sequences are very easily measured, G-rich sequences are very difficult to measure, and A- and C-rich sequences are of intermediate difficulty.
- Peak height is NOT proportional to the amount of material present. Short oligonucleotides are desorbed and ionized more readily than longer ones, so that the peak corresponding to a 12-mer, for example, may be larger than that for a 24-mer, even though the 12-mer represents only a small percentage of failure sequence in the synthesis of the 24-mer.
- Depurination is a feature of the MALDI process. Consequently, in addition to the peak of calculated mass, one often sees a peak of mass 115 to 151 mass units less than the major peak. Often a second peak of lower intensity and somewhat broader mass range, corresponding to the loss of two purine bases, is seen.
Some of the other features of MALDI-TOF molecular weight measurement are:
- Oligonucleotides are converted to the ammonium salt for MALDI mass spectrometry. Ammonia separates from the oligo during the desorption process, so the oligo flies as the free acid form of DNA, i.e., the phosphate group bears a proton to neutralize the charge. The mass contribution for each monomer is thus:A = 313.22
C = 289.18
G = 329.22
T = 304.21
- In calculating the mass of an oligo, remember that there are one fewer phosphates than nucleosides, and the value of 61.98 mass units must be subtracted.
- A peak is nearly always present at m/2z. Mass spectrometry does not measure mass, but rather the ratio of mass to charge. Ideally molecules bear a single charge (e-) and are measured as M-H+. But a large percentage of the molecules lose 2 electrons in the desorption process and are characterized as M-2H+. Remember that the abscissa of the spectrum is mass/charge, not simply mass.
- Sodium ions, if present, may partially replace the ammonium ion. Sodium ions desorbed with the DNA add 22 mass units per sodium ion. To the degree that sodium replaces ammonium, peaks are broadened, because multiple species can now be present (mono-, di-, tri-, etc. adduct).
- The error in mass measurement is typically around 0.2%. For reference, the mass of a typical 30-mer would be in the range of approximately 9,000 Daltons for which the expected error would be equivalent to the molecular weight of water, 18 Daltons. The presence of a single protecting group used during the synthesis (isobutyryl = 86, benzoyl = 121, or dimethoxytrityl = 302) can thus be readily detected.