GSH and NAc are complementary to covalent protein binding studies because
trapping studies are easily implemented and amenable to high throughput
screening. Mass spectra and/or NMR spectra of the trapped adducts often
provide valuable information of bioactivation pathways to guide further
structural modifications. In this section, the use of these approaches will be
reviewed, and generic protocols will be described along with typical examples.
Future advances in this field will be discussed briefly at the end of this section.
14.1 GLUTATHIONE,N-ACETYLCYSTEINE, AND POTASSIUM
CYANIDE AS TRAPPING AGENTS
14.1.1 Introduction
It is known that most chemically reactive intermediates are electrophiles that
can react with cellular proteins to form covalently bound drug–protein
adducts. In order to provide guidance for chemical modification of lead
compounds, it is desirable to characterize structures of reactive intermediates
for understanding of bioactivation mechanism. However, reactive intermedi-
ates are generally unstable in biological systems, and readily react with
proteins. On the contrary, reactive intermediates can also react with some
trapping agentsin vitroto form corresponding adducts. The most commonly
used agents for trapping reactive intermediates are glutathione,N-acetylcys-
teine, and cyanide ion. These trapping agents can have nucleophilic addition to
reactive intermediates to form corresponding adducts. Glutathione is an
endogenous tripeptide consisting ofL-g-glutamyl-L-cysteinyl-glycine (Glu-Cys-
Gly) with a molecular weight of 307 Da. It is the most commonly used trapping
agent in qualitative analysis of GSH-reactive intermediate adducts, because the
resulting adducts may be conveniently detected via constant neutral loss
scanning of m/z 129 at positive ion mode with triple quadrupole mass
spectrometry (Tang and Miller, 2005). The sensitivity and selectivity of analysis
of GSH adducts may be increased using an exact neutral loss detection ofm/z
129.0426 at positive ion mode via sequential low and high energy MS
acquisition in a hybrid quadrupole time-of-flight mass spectrometer (Castro-
Oerez et al., 2005) or precursor scanning ofm/z272 at negative ion mode in
triple quadrupole mass spectrometer (Dieckhaus et al., 2005). Glutathione
ethyl ester (GSH–EE) may also be used as a trapping agent. The use of GSH–
EE improved the detection capacities for reactive intermediates (Soglia et al.,
2004), because an approximately 10-fold increase in sensitivity in mass
spectrometry analysis was observed for adducts containing the GSH–EE
moiety compared to GSH. In most cases, mass spectra of GSH adducts offer
less structural information than anticipated because most fragment ions of
GSH adducts are derived from the GSH moiety. Alternatively, NAc, a thiol
molecule with a molecular weight of 163 Da, may be used as a trapping agent.
Because the NAc moiety has less fragments than the GSH moiety, the MS–MS
450 PROTOCOLS FOR ASSESSMENT