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Oligonucleotide sequence and composition determined by matrix-assisted laser desorption/ionization

C M Bentzley1, M V Johnston, B S Larsen

  • 1Department of Chemistry and Biochemistry, University of Delaware, Newark 19716, USA.

Analytical Chemistry
|July 1, 1996
PubMed
Summary
This summary is machine-generated.

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Matrix-assisted laser desorption/ionization with time-of-flight mass spectrometry (MALDI-TOF) accurately determined oligonucleotide molecular weights. This method, combined with enzymatic digestion, successfully identified individual nucleotides within short DNA strands.

Area of Science:

  • Biochemistry
  • Analytical Chemistry
  • Molecular Biology

Background:

  • Accurate molecular weight determination is crucial for oligonucleotide characterization.
  • Traditional sequencing methods can be time-consuming and require specific reagents.

Purpose of the Study:

  • To evaluate matrix-assisted laser desorption/ionization with time-of-flight mass spectrometry (MALDI-TOF) for oligonucleotide molecular weight determination.
  • To assess the feasibility of using MALDI-TOF in conjunction with enzymatic digestion for nucleotide sequencing.

Main Methods:

  • Oligonucleotides (12-60 bases) were analyzed using MALDI-TOF.
  • Enzymatic digestion with calf spleen phosphodiesterase was employed to generate nucleotide fragments.
  • MALDI-TOF mass spectra of digest mixtures were analyzed to identify molecular ions and mass differences.

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Main Results:

  • MALDI-TOF provided molecular weight measurements with mass accuracy better than 0.1%.
  • Enzymatic digestion produced a characteristic oligonucleotide ladder in the MALDI-TOF spectra.
  • Mass differences between peaks in the spectra successfully identified individual nucleotides in 12- and 24-base strands.

Conclusions:

  • MALDI-TOF is a highly accurate method for determining oligonucleotide molecular weights.
  • Combining MALDI-TOF with enzymatic digestion offers a viable approach for nucleotide sequencing of short oligonucleotides.
  • The resolution and mass accuracy of MALDI-TOF are sufficient for unambiguous nucleotide identification.