Simplified sample preparation for the analysis of oligonucleotides by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry.
Matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOFMS) for the analyses of oligonucleotides has generally been carried out using negative ionisation conditions, usually following ammonium ion-exchange chromatography and the addition of ammonium buffers to the MALDI matrix. The molecular ion region is complex, due to the varying degrees of ammoniation of the phosphate backbone of the oligonucleotide. This gives rise to an overall decrease in sensitivity compared with similar size peptides and can cause ambiguity of assignment of the relative molecular mass of the sample. This study describes the use of H(+) ion exchange resin in situ as the means of removing alkali metal ions from the phosphate backbone of the oligonucleotide. An increase in resolution, sensitivity and identification of the molecular species is reported, with little or no difference in sensitivity observed between positive or negative ionisation spectra. This method is now used for routine screening of synthetic oligonucleotides with a gain in sensitivity of 1-2 orders of magnitude compared with previous methods, and mass assignment errors of +/-0.1% are routinely recorded for externally calibrated data.
Langley
G J
GJ
Department of Chemistry, University of Southampton, Highfield, Southampton SO17 1BJ, UK.
Herniman
J M
JM
Davies
N L
NL
Brown
T
T
D007475:Ion Exchange Resins; D009841:Oligonucleotides / Q000032:analysis*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000379:methods; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods
D007475:Ion Exchange Resins; D009841:Oligonucleotides