Factors determining the performance of triple quadrupole, quadrupole ion trap and sector field mass spectrometer in electrospray ionization mass spectrometry. 2. Suitability for de novo sequencing.
The sequence coverage by fragment ions resulting from collision-induced dissociation in a triple stage quadrupole (TSQ) and a quadrupole ion trap (QIT) mass spectrometer of 10-20-mer oligonucleotides was investigated. While (a-B) and w ion series were the most abundant on both instruments, additional ion series of sequence relevance were preferably formed in the TSQ. Thus, a total number of 83 fragment ions were used to deduce the complete sequence of a 10-mer oligonucleotide of mixed sequence from a tandem mass spectrum recorded on the TSQ. The complete sequence was also encoded in the 28 fragments that were obtained from the QIT under comparable fragmentation conditions. Spectrum complexity increased considerably at the cost of signal-to-noise ratio upon fragmentation of a 20-mer oligonucleotide in the TSQ, whereas spectrum interpretation with longer oligonucleotides was significantly more straightforward in spectra recorded on the QIT. The extent of fragmentation had to be optimized by appropriate setting of collision energy and choice of precursor ion charge state in order to obtain full sequence coverage by fragments for de novo sequencing. Moreover, full sequence information was also dependent on base sequence because of the low tendency of backbone cleavage at thymidines. Tandem mass spectrometry on the QIT yielded redundant information that was successfully utilized to deduce the complete sequence of 20-mer oligonucleotides with high confidence.
Premstaller
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A
Institute of Analytical Chemistry and Radiochemistry, Leopold-Franzens-University, Innrain 52a, 6020 Innsbruck, Austria.
Huber
C G
CG
D001483:Base Sequence; D007202:Indicators and Reagents; D013058:Mass Spectrometry / Q000295:instrumentation / Q000379:methods*; D009838:Oligodeoxyribonucleotides / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000295:instrumentation / Q000379:methods*
D007202:Indicators and Reagents; D009838:Oligodeoxyribonucleotides