Analysis of biological and synthetic ribonucleic acids by liquid chromatography-mass spectrometry using monolithic capillary columns.
Ion-pair reversed-phase high-performance liquid chromatography (IP-RP-HPLC) has been evaluated as a method for the fractionation and desalting of ribonucleic acids prior to their characterization by electrospray ionization mass spectrometry. Monolithic, poly(styrene-divinylbenzene)-based capillary columns allowed the rapid and highly efficient fractionation of both synthetic and biological ribonucleic acids. The common problem of gas-phase cation adduction that is particularly prevalent in the mass spectrometric analysis of ribonucleic acids was tackled through a combination of chromatographic purification and the addition of ethylenediaminetetraacetic acid to the sample at a concentration of 25 mmol/L shortly before on-line analysis. For RNA molecules ranging in size from 10 to 120 nucleotides, the mass accuracies were typically better than 0.02%, which allowed the characterization and identification of failure sequences and byproducts with high confidence. Following injection of a 500 nL sample onto a 60 x 0.2 mm column, the limit of detection for a 120-nucleotide ribosomal RNA transcript from Escherichia coli was in the 50-80 fmol range. The method was applied to the analysis of synthetic oligoribonucleotides, transfer RNAs, and ribosomal RNA. Finally, sequence information was derived for low picomole amounts of a 32-mer RNA upon chromatographic purification and tandem mass spectrometric investigation in an ion trap mass spectrometer. Complete series of fragment ions of the c- and y-types could be assigned in the tandem mass spectrum. In conclusion, IP-RP-HPLC using monolithic capillary columns represents a very useful tool for the structural investigation and quantitative determination of RNAs of synthetic and biological origin.
Hölzl
Georg
G
Institute of Analytical Chemistry and Radiochemistry, Leopold-Franzens-University, Innrain 52a, and Institue of Legal Medicine, Innsbruck Medical University, Müllerstrasse 44, A-6020 Innsbruck, Austria.
Oberacher
Herbert
H
Pitsch
Stefan
S
Stutz
Alfred
A
Huber
Christian G
CG
D002614:Chelating Agents / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation / Q000379:methods*; D009843:Oligoribonucleotides / Q000032:analysis*; D011786:Quality Control; D012313:RNA / Q000032:analysis*; D012329:RNA, Bacterial / Q000032:analysis; D012335:RNA, Ribosomal / Q000032:analysis; D012343:RNA, Transfer / Q000032:analysis; D012346:RNA, Transfer, Amino Acyl / Q000138:chemical synthesis; D021241:Spectrometry, Mass, Electrospray Ionization / Q000295:instrumentation / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000295:instrumentation / Q000379:methods
D002614:Chelating Agents; D009843:Oligoribonucleotides; D012329:RNA, Bacterial; D012335:RNA, Ribosomal; D012346:RNA, Transfer, Amino Acyl; D012313:RNA; D012343:RNA, Transfer