Electrospray quadrupole mass spectrometry analysis of model oligonucleotides and polymerase chain reaction products: Determination of base substitutions, nucleotide additions/deletions, and chemical modifications

ESI FTICR mass spectrometry is the only technique currently used for accura te molecular weight analysis of PCR products above 100 bp in size. This is important in demonstrating the potential for MS in making major contributio ns in the molecular biology and genomics areas, In the near future, it is m ore likely that less expensive, wore user friendly MS techniques will be us ed for high-throughput analyses (including MALDI TOF and ESI quadrupole), T here have been numerous reports on the use of MALDI TOF, The current report is to the first to evaluate the use of ESI-quadrupole analysis of PCR prod ucts, Synthetic oligonucleotides (30 and 89 mers) and polymerase chain reac tion products of varying molecular weight (62, 88, 89, and 114 bp) were ana lyzed by ESI using a quadrupole MS. The mass accuracy for nucleic acids in the 30-62 bp range was shown to allow determination of nucleotide substitut ions and additions/deletions. For higher molecular weight PCR products (88- 114 bp), the mass accuracy of ESI-MS distinguishes single or multiple nucle otide insertions/deletions. In addition, ESI quadrupole MS allows determina tion of molecular weight of both strands of higher molecular weight ds PCR products anti can distinguish nucleotide modifications (e.g., with biotin), In conclusion, it is demonstrated that ESI-MS occupies an intermediate pos ition (as compared to MALDI TOF and ESI FTICR) with regard to mass accuracy and resolution in analysis of nucleic acids.