Mass spectrometric methods for distinguishing structural isomers of glutathione conjugates of estrone and estradiol

Collisionally activated decompositions (CAD) of M+H]⁺ ions from two sets (estrone and estradiol) of three isomeric glutathione (GSH) conjugates were studied by using five tandem mass spectrometric methods: (1) low energy (LE) CAD in an ion trap, (2) LE CAD in a triple quadrupole, (3) electrospray ionization (ESI)-source CAD in a tandem four sector, (4) high energy (HE) CAD of both ESI-produced and fast-atom bombardment (FAB)-produced ions in a tandem four-sector mass spectrometer, and (5) metastable-ion decompositions of FAB-produced ions. Four types of fragment ions are produced. The first type, formed from cleavage of the peptide backbone, gives rise to modified b₂, modified y₂, y₂, and b₁ ions. These fragments are observed with all the methods and show that the catechol estrogen attachment is at the cysteine moiety of the GSH. Internal fragment ions are the second type, and they also support that the modification is at cysteine. The third type involves fragmentation of the C–S bond to give an ion containing the steroid bonded to the sulfur. The fourth type of fragment ion is similar to the third but involves oxidation of the steroid ring and reduction of the GSH moiety; it is the most isomer specific of the four. The isomer-specific ions are of relatively low abundance in the product-ion spectra taken on the triple quadrupole and ion trap, but their abundances can be improved by increasing the collision energy. ESI source-CAD and the HE-CAD spectra of the isomers are the most distinctive because abundant product ions of all four types are seen in a single spectrum.