17th International Mass Spectrometry Conference :: Prague, 2006
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|Presentation date:||Thu, Aug 31, 2006|
|Presentation time:||14:30 – 16:00|
Dmitri Zagorevski1, Michael F. Aldersley1, James P. Ferris11 Rensselaer Polytechnic Institute, Troy, NY, United States
Correspondence address: Dmitri Zagorevski, Rensselaer Polytechnic Institute, Chemistry and Chemical Biology, 209C Cogswell Lab., 110 8th St., Troy, NY, 12180 United States.
Keywords: MALDI; Oligonucleotides; Reaction Monitoring; Sample Preparation.
Novel aspect: A procedure allowing direct MALDI analysis of oligonucleotides from montmorillonite is developed. Large even-numbered guanosine clusters are detected and characterized by mass spectrometry.
The generation of RNA oligomers from activated nucleic acids on a montmorillonite catalyst is one of the hypotheses of the origin of life on the Earth.1 The analysis of the reaction products involves their extraction from the catalyst followed by chromatographic separation. Chromatography does not provide a "clean" separation of complex mixtures and the detection of large oligomers that are formed in very small quantities is difficult. Another concern is whether oligomers extracted from the mineral represent all the products formed in the reaction. We are reporting that MALDI-TOF mass spectrometry can be successfully used for the analysis of complex mixtures of oligonucleotides synthesized on montmorillonite directly from the catalyst without their separation or sample clean up.
Activated nucleotides (ImpX, X = A, U, G or C) in NaCl and MOBS buffer were subjected to catalytic oligomerization on montmorillonite. Reaction mixtures remaining on the catalyst were mixed with a matrix (6-aza-2-thiothymine or 2,4,6-trihydroxyacetophenone) and dibasic ammonium citrate and subjected to MALDI-TOF analyses. Unlike unpurified solvent extracts, these samples showed signals due to RNA oligomers. Compounds having up to 40 nucleic acids were detected in linear negative ion mode. The characteristic feature of the mass spectra was an enhanced sensitivity to high-molecular weight oligomers. Reflectron mode MALDI mass spectra identified the formation of (pX)n, cyclic-(pX)n and (pX)n-1X. The size of RNA oligomers detected directly from the montmorillonite was approximately the same as that observed for the desalted mineral extracts confirming that the extraction procedure was indeed removing the longest oligomers.
High tolerance of montmorillonite to salts and buffers was used for analysis of RNA samples with high concentrations of salts and buffers. The procedure involves mixing of the sample with montmorillonite, matrix and the ammonium salt followed by MALDI analysis. Homoionic Li-montmorillonite showed distinctive advantages over natural and Na- and K-enriched minerals. The size of oligomers detected from Li-montmorillonites was comparable with that for desalted samples.
Homoionic montmorillonites can be used for H/alkali metal-exchange. The number of metal atoms incorporated into the ion is related to the number of acidic H-atoms in RNA and DNA molecules. We found that the maximum number of alkali metals in negatively charged ions of RNA related to the number of phosphoric groups as (PO3)n=(Li, Na, K)m + 1. For DNA molecules, a different equation was found: (PO3)n=(Li, Na, K)m + 2.
The analysis of oligonucleotides from ImpG demonstrated the domination of even numbered oligomers above n=6. A cyclic octamer, [c-(pG)8-2H+K]-, dominated the mass spectra. MS/MS experiments on even-numbered species were consistent with the formation of cage-type structures.
1. J. P. Ferris, Elements 1, 145 (2005).