Investigation of enzymatic behavior of benzonase/alkaline phosphatase in the digestion of oligonucleotides and DNA by ESI-LC/MS.
We have developed an ion-pairing HPLC-MS method that has sufficient separation power, selectivity, and sensitivity to investigate the enzymatic behavior of benzonase/alkaline phosphatase upon digestion of oligonucleotides and DNA. Mass spectrometry revealed that this enzyme pair can nonspecifically digest oligonucleotides and DNA into fragments ranging from 2 to 10 nucleotides, i.e., sizes suitable for routine mass spectrometric measurements. Trimers, tetramers, and pentamers are the most prominent digested products. This makes benzonase/alkaline phosphatase a promising choice for DNA and DNA adduct related studies that require a nonspecific enzyme. A computer software program developed in-house was critical in automating the processing of mass spectral data. The methodology described here provides a systematic approach for evaluating the behavior of DNA-cleaving enzymes by mass spectrometry.
Liao
Qing
Q
Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts 02138, USA.
Chiu
Norman H L
NH
Shen
Chang
C
Chen
Yi
Y
Vouros
Paul
P
D000469:Alkaline Phosphatase / Q000737:chemistry / Q000494:pharmacology*; D002853:Chromatography, Liquid / Q000379:methods*; D004247:DNA / Q000737:chemistry / Q000187:drug effects*; D004720:Endonucleases / Q000737:chemistry / Q000494:pharmacology*; D009841:Oligonucleotides / Q000032:analysis* / Q000737:chemistry; D012984:Software; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
D009841:Oligonucleotides; D004247:DNA; D004720:Endonucleases; D000469:Alkaline Phosphatase