Sequencing regular and labeled oligonucleotides using enzymatic digestion and ionspray mass spectrometry.
A method using a combination of enzymatic digestion and ionspray mass spectrometry (MS) was developed for sequencing oligodeoxynucleotides (ODNs) containing more than 20 bases. Phosphodiesterase (PDE) digestion of ODNs produced truncated ODNs whose molecular weights (MWs) were determined by ionspray MS. It was demonstrated that reconstruction of MW spectra over a large MW range produced easy-to-read sequence ladders similar to those obtained using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Sample and enzyme cleanup, digestion control, and MW reconstruction were found to be crucial factors. For regular ODNs, both 5'- and 3'-PDE digestions are needed for complete sequencing. Late in the time course of PDE digestions, 5'-nucleoside monophosphates were found to produce artifactual peaks in the reconstructed MW spectra, and a table correlating base compositions and MS ions was compiled to handle such situations. For labeled ODNs, it is necessary to use collision-induced dissociation-tandem mass spectrometry (CID-MS/MS) for complete sequence determination. Sequencing of regular 22-mer and labeled 18-mer ODNs was demonstrated in this work.
Wu
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Diagnostics Division, Abbott Laboratories, 100 Abbott Park Road, Abbott Park, Illinois 60064, USA. Huaiqin.Wu@add.ssw.abbott.com
Chan
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Aboleneen
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D000818:Animals; D001483:Base Sequence; D002417:Cattle; D006868:Hydrolysis; D013058:Mass Spectrometry / Q000379:methods*; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides / Q000737:chemistry*; D010727:Phosphoric Diester Hydrolases / Q000737:chemistry*
D009841:Oligonucleotides; D010727:Phosphoric Diester Hydrolases