title: Investigation of enzymatic behavior of benzonase/alkaline phosphatase in the digestion of oligonucleotides and DNA by ESI-LC/MS.
abstract: 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.
authors.affiliation: Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts 02138, USA.
authors.forename: Qing
authors.initials: Q
authors.lastname: Liao
authors.forename: Norman H L
authors.initials: NH
authors.lastname: Chiu
authors.forename: Chang
authors.initials: C
authors.lastname: Shen
authors.forename: Yi
authors.initials: Y
authors.lastname: Chen
authors.forename: Paul
authors.initials: P
authors.lastname: Vouros
mesh_terms: 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*
chemical_list: D009841:Oligonucleotides; D004247:DNA; D004720:Endonucleases; D000469:Alkaline Phosphatase