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On the use of ESI‐QqTOF‐MS/MS for the comparative sequencing of nucleic acids
Author(s) -
Oberacher Herbert,
Pitterl Florian
Publication year - 2009
Publication title -
biopolymers
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.556
H-Index - 125
eISSN - 1097-0282
pISSN - 0006-3525
DOI - 10.1002/bip.21156
Subject(s) - chemistry , mass spectrum , oligonucleotide , ion , fragmentation (computing) , fragment (logic) , spectral line , tandem mass spectrometry , mass spectrometry , analytical chemistry (journal) , algorithm , computer science , chromatography , dna , physics , biochemistry , organic chemistry , astronomy , operating system
The usability of a quadrupole—quadrupole—time‐of‐flight (QqTOF) instrument for the tandem mass spectrometric sequencing of oligodeoxynuleotides was investigated. The sample set consisted of 21 synthetic oligodeoxynucleotides ranging in length from 5 to 42 nucleotides. The sequences were randomly selected. For the majority of tested oligonucleotides, two or three different charge states were selected as precursor ions. Each precursor ion was fragmented applying several different collision voltages. Overall 282 fragment ion mass spectra were acquired. Computer‐aided interpretation of fragment ion mass spectra was accomplished with a recently introduced compa rative s equencing algorithm (COMPAS). The applied version of COMPAS was specifically optimized for the interpretation of information‐rich spectra obtained on the QqTOF. Sequences of oligodeoxynucleotides as large as 26‐mers were correctly verified in >94% of cases (182 of 192 spectra acquired). Fragment ion mass spectra of larger oligonucleotides were not specific enough for sequencing. Because of the occurrence of extensive internal fragmentation causing low sequence coverage paired with a high probability of assigning fragment ions to wrong sequences, tandem mass spectra obtained from oligonucleotides consisting of 30 and more nucleotides could not be used for sequence verification neither manually nor with COMPAS. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 401–409, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com