Shotgun sequencing small oligonucleotides by nozzle-skimmer dissociation and electrospray ionization mass spectrometry

Eur. J. Mass Spectrom. 11, 221–229 (2005)
DOI: 10.1255/ejms.736

Shotgun sequencing small oligonucleotides by nozzle-skimmer dissociation and electrospray ionization mass spectrometry

Zhaojing Meng and Patrick A. Limbach^*
Rieveschl Laboratories for Mass Spectrometry, Department of Chemistry, PO Box 210172, University of Cincinnati, Cincinnati, OH 45221, USA. E-mail Pat.Limbach@uc.edu

ABSTRACT:

Nozzle-skimmer dissociation in combination with de novo sequencing was investigated as an approach for increasing the throughput of oligonucleotide analysis attainable by electrospray ionization mass spectrometry. An experimental method allowing for the sequential generation of precursor and fragment ion data during direct infusion of sample was developed. These data can then be used with readily available de novo sequencing software to characterize small oligonucleotides. When this approach was applied to mixtures of oligonucleotides, it was found that de novo sequencing becomes limited due to spectral congestion and overlapping oligonucleotide m/z dissociation product values. Self-packed C₁₈ microspray emitters were investigated as a means of reducing spectral complexity. It was found that such emitters allow for the analysis of oligonucleotide mixtures with minimal component overlap, and these emitters provide additional benefits of pre- concentrating and desalting the sample. These developments can provide a route for the more rapid characterization of ribonucleic acid endonuclease digestion mixtures.

Keywords: RNA, DNA, MS/MS, oligodeoxynucleotide, sequence analysis, de novo sequencing, ESI-MS

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