Utility of accurate monoisotopic mass measurements to confidently identify lambda exonuclease generated single-stranded amplicons containing 7-deaza analogs by electrospray ionization FT-ICR mass spectrometry

被引:11
|
作者
Frahm, JL
Mason, CJ
Muddiman, DC
机构
[1] Virginia Commonwealth Univ, Dept Chem, Richmond, VA 23284 USA
[2] Mayo Proteom Res Ctr, WM Keck FT ICR Mass Spectrometry Lab, Rochester, MN 55905 USA
[3] Mayo Clin & Mayo Fdn, Coll Med, Dept Biochem & Mol Biol, Rochester, MN 55905 USA
基金
美国国家卫生研究院;
关键词
FT-ICR; dual-electrospray source; 7-deaza; mass measurement accuracy; average nucleotide;
D O I
10.1016/j.ijms.2004.02.004
中图分类号
O64 [物理化学(理论化学)、化学物理学]; O56 [分子物理学、原子物理学];
学科分类号
070203 ; 070304 ; 081704 ; 1406 ;
摘要
A 53-base pair region on the long arm of chromosome 22 was amplified using PCR with 7-deaza-modified deoxynucleotides. Increased amplification efficiency was achieved by doubling the concentration of the modified deoxynucleotide triphosphates. Incorporation of 7-deaza purines has been previously shown to selectively eliminate fragmentation pathways during gas-phase sequencing of nucleic acids by sustained off-resonance irradiation collision-induced dissociation (SORI-CID) and infrared multiphoton dissociation. However, 7-deaza analogs result in significant duplex stability precluding interrogation of the single-stranded species by tandem mass spectrometry. Herein, we demonstrate the use of lambda exonuclease to successfully overcome this problem and are able to obtain single-stranded PCR products containing 7-deaza adenine and guanine nucleobases. Mass accuracy was used as our metric to determine complete incorporation of 7-deaza residues in PCR products > 15 kDa; less than or equal to 3 ppm neutral monoisotopic mass measurement accuracies were routinely achieved. High mass measurement accuracy was obtained using a dual electrospray source and subsequently, using an isotopic fitting algorithm, the best fit between the theoretical and experimental isotopic distributions was determined using a chi-square value. Theoretical isotopic distributions were generated using an average nucleotide (averatide) chemical formula developed herein which was based on the relative frequencies of AT and GC base pairs in the human genome. Single-stranded PCR products were fragmented using SORI-CID and as expected, cleavage at the 7-deaza modified sites was not observed. Collectively, this integrated approach can facilitate top-down sequencing of PCR products by a variety of tandem mass spectrometry methods. (C) 2004 Elsevier B.V. All rights reserved.
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页码:79 / 87
页数:9
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