LABILE HYDROGEN COUNTING IN BIOMOLECULES USING DEUTERATED REAGENTS IN DESORPTION CHEMICAL IONIZATION AND FAST-ATOM-BOMBARDMENT MASS-SPECTROMETRY

The number of mobile hydrogens in biomolecules can be determined by fast atom bombardment (FAB) and desorption chemical ionization (DCI) mass spectrometry in association with, respectively, condensed-phase and gas-phase H/D exchange reactions. From the difference of molecular ion masses recorded before and after D labeling, the number of mobile hydrogens could be easily determined on a variety of macrocyclic antibiotics, carrying up to 20 mobile hydrogens, as well as on some peptides and saccharides. In FAB experiments a new procedure has been developed which involves an in situ deuteration of both sample and matrix by repeated treatment with D2O directly on the FAB probe tip. This versatile procedure achieves high levels of deuterium substitution (94%-96%), avoids the risk of H/D back-exchange with atmospheric water, and allows the use of any water-soluble FAB matrix. In the DCI method the sample is instantly evaporated in the cold ion source filled with a rather high pressure of deuterated ammonia (ND3). Under such conditions, both molecular and fragment ions leaving the ion source have undergone an almost complete (90%-98%) H/D exchange process with ND3. The presence of D-labeled fragment ions in DCI mass spectra significantly helps the comprehension of molecular structures and fragmentation mechanisms. Experimental parameters influencing the extent of H/D exchange are discussed in detail.