RNA fragmentation studied in a matrix-assisted laser desorption/ionisation tandem quadrupole/orthogonal time-of-flight mass spectrometer.
We have studied the fragmentation behaviour of short, singly protonated oligoribonucleotides on a MALDI Qq-TOF instrument with the aim of using this instrumental set-up to characterise modifications of RNA molecules. Individual ion species from enzymatically generated mixtures were isolated in one quadrupole and subjected to collision-induced dissociation in a second quadrupole followed by separation of the resulting product ions in an orthogonal time-of-flight mass analyser. Complex spectra were generally observed with nearly all types of cleavages along the phosphodiester backbone and of the N-glycosidic bonds (and combinations of these) occurring, albeit at different relative intensities. The most labile part of the backbone was found to be the 5'-P-O bond, resulting in c- and y-ions. Loss of neutral cytosine and guanine occurred equally often, whereas neutral loss of adenosine was less prevalent. Loss of uracil, either neutral or charged species, was not observed. Because the fragmentation pattern observed here is significantly different from what has been reported for singly protonated oligodeoxyribonucleotides, we suggest that the 2'-substituent in the sugar plays a central role in the fragmentation mechanisms of nucleic acids. Finally, we used the acquired knowledge about oligoribonucleotide fragmentation to characterise an in vivo methylated oligoribonucleotide by tandem mass spectrometry.
Kirpekar
F
F
Department of Biochemistry and Molecular Biology, Odense University SDU, Campusvej 55, DK-5230 Odense M, Denmark. f.Kir@BMB.sdu.dk
Krogh
T N
TN
D001483:Base Sequence; D001411:Geobacillus stearothermophilus / Q000235:genetics / Q000378:metabolism; D009843:Oligoribonucleotides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011522:Protons; D012313:RNA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012323:RNA Processing, Post-Transcriptional; D019642:RNA, Archaeal / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012329:RNA, Bacterial / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012341:RNA, Ribosomal, 5S / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D016945:Sulfolobus acidocaldarius / Q000235:genetics / Q000378:metabolism
D009843:Oligoribonucleotides; D011522:Protons; D019642:RNA, Archaeal; D012329:RNA, Bacterial; D012341:RNA, Ribosomal, 5S; D012313:RNA