Steven C. Pomerantz

[44] Analysis of RNA hydrolyzates by liquid chromatography-mass spectrometry

Publisher Summary This chapter discusses analysis of Ribo Nucleic Acid (RNA) hydrolyzates by liquid chromatography-mass spectrometry. Reversed-phase high-performance liquid chromatography (HPLC) is an experimentally effective technique for the separation of nucleosides, particularly for the analysis of enzymatic digests of RNA and Deoxyribo-nucleic acid (DNA). Depending on the problem at hand, identification of nucleoside constituents can often be made on the basis of retention times and Ultraviotet (UV) absorbance characteristics. As is common, in general, when chromatographic methods are used for purposes of identification, as opposed to simply for separation, the reliability of the method suffers as the complexity of the mixture increases, or when components of unknown or unexpected identity are encountered. The development of directly combined HPLC-mass spectrometry [liquid chromatography (LC)/MS] based on the thermospray interface provides a method which can be effectively applied to the analysis of nucleosides in nucleic acid digests, and is a powerful extension of the capabilities of either technique alone. Procedures for nuclease digestion of RNA to ribonucleotides, followed by dephosphorylation using alkaline phosphatase, are followed which, in general, permit direct injection of the crude digest directly into the HPLC.

Determination of oligonucleotide composition from mass spectrometrically measured molecular weight

Extensive calculations for molecular mass versus subunit composition have been made for oligonucleotides from RNA and DNA to determine the extent to which base compositions might be derived from mass spectrometrically determined molecular weights. In the absence of compositional constraints (e.g., any numbers of A, U, G, C), measurement of molecular weight leads to only modest restrictions in allowable number of base compositions; however, if the compositional value for any one residue is known, such as from selective chemical modification or enzymatic cleavage, the number of allowable base compositions becomes unexpectedly low. For example, hydrolysis of RNA by ribonuclease T1 produces oligonucleotides for which G=1, for which all base compositions can be uniquely specified up to the 14-mer level, solely by measurement of mass to within ±0,01%. The effects of methylation, phosphorylation state of nucleotide termini, and knowledge of chain length on the determination of subunit composition are discussed.

Tandem Mass Spectrometry for Structure Assignments of Wye Nucleosides from Transfer RNA

The tricyclic wye nucleoside family of eight known members constitutes one of the most complex and interesting series of posttranscriptionally modified nucleosides in transfer RNA. The principal reaction paths represented in collision‐induced dissociation mass spectra of wye bases and their analogs have been studied in order to determine those structural features that can be readily established by mass spectrometry. The main routes of fragmentation are determined by the presence vs. absence of an amino acid side chain at C‐7 (1H‐imidazo[1,2‐a]purine nomenclature). The common methionine‐related side chain is cleaved at two points, providing a ready means of establishing the presence and net level of side chain modification. For those molecules without a side chain, the initial reaction steps are characteristically controlled by the presence vs. absence of methyl at N‐4, allowing determination of the methylation status of that site. In the latter case initial opening of the central (pyrimidine) ring, in analogy to the dissociation behavior of guanine, causes loss of identity of C‐6/C‐7 so that placement of a single methyl at either site is not possible. Subsequent complex reaction paths follow, which include loss of CO and sequential loss of two molecules of HCN. †In honor and celebration of the 70th birthday of Professor Leroy B. Townsend.

Applications of LC/MS and Tandem Mass Spectrometry to the Characterization of Nucleosides in Mixtures

Abstract Liquid chromatography-mass spectrometry (LC/MS) and tandem mass spectrometry (MS/MS) provide new approaches for structural studies of nucleosides, in the nanogram range, in mixtures. Examples are given of the use of LC/MS for rapid screening of synthesis reaction mixtures, and of MS/MS for the detection and characterization of nucleoside isomers in RNA hydrolysates.