New techniques for microscale structural studies by mass spectrometry

A chiral GC/MS separation and identification of TFA derivatives of monosaccharides and methanolysis products of carbohydrates and glycoconjugates were developed using a Chirasil-Val column. Both ? and ? anomers were formed in the preparation of TFA derivatives. The ratio of ? anomers to ? anomers is characteristic for each monosaccharide which can provide additional information in compound identification. The developed methods have been successfully used to the identification of sugar moieties in modified queuosine nucleosides and modified DNA isolated from the Rhizobium bacteriophage DNA. A new HPLC separation method which is suitable for thermospray ionization was developed for the separation of mononucleotides. Thermospray LC/MS was shown to be a powerful tool in the structural elucidation of nucleotides and nucleosides. The major ions observed in the thermospray spectra of nucleotides were identified as MH+, NH2+, BH2+ and ions related to the sugar moieties, and their NH4+ cluster ions. Hydrolysis and thermolysis may play a major role in the thermospray ionization of nucleotides and nucleosides, although the ammonia chemical ionization mechanism may also be important. No spectrum difference has been observed for 5'-, 3'-, and 2'-mono-mucleotides; however, the studies of spectra cytidine 3',5'-cyclic monophosphate and cytidine 2',3'-cyclic monophosphate indicate that isomer differentiation by thermospray ionization may be possible. Combined HPLC-thermospray mass spectrometry of mono- and disaccharides, 1-0-methylglycosides and 0-permethyl mono- through tetrasaccharides have been studies to assess the potential role of thermospray ionization for microscale structural studies of saccharides and glycoconjugates and for high sensitivity detection of liquid chromatographic effluents. Using HCO2NH4 as eluant for reversed-phase HPLC, abundant MNH4+ ions are formed from monosaccharides and mono- and permethylated saccharides, and are suitable for monitoring subnanogram constituents in HPC effluents. Detection of 100 pg 1-0methylhexopyranosides with signal/noise>10 is demonstrated. Hydrolysis and thermolysis appear to be the predominant mechanisms in the thermospray ionization in intact mono-, di-, tri-, and tetrasaccharides; therefore consecutive losses of H20 and ions corresponding to hydrolysis products are the major fragment ions. For permethylated carbohydrates, a mechanism analogous to ammonia CI may play the major role. Positional isomers of methylmonosaccharides are chromatographically separable and spectroscopically distinguishable; which may imply the potential of thermospray LC/MS in the structural elucidation of carbohydrates and glycoconjugates at the microscale.