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An uclear receptor, peroxisome proliferator-activated receptor g (PPARg ), is al igand-dependent transcription factor involved in glucose homeostasis anda dipocyted ifferentiation. PPARg is the molecular target of various natural ands ynthetic molecules, includinga nti-diabetic agents such as rosiglitazone. Amide hydrogen/deuterium-exchange (H/D-Ex), coupled with proteolysis and mass spectrometry,w as applied to study the dynamics of theP PAR g ligand binding domain (LBD) with or withoutmolecules that modulate PPARg activity.The H/D-Ex patterns of ligand-free PPARg LBD show that the ligand binding pocket of LBD is significantly more dynamic than ther est of the LBD. Presumably,t he bindingp ocketi si ntrinsically disordered in order to accommodated ifferentl igands. The presence of twof ull agonists (rosiglitazone and GW1929), ap artial agonist (nTZDpa), and ac ovalenta ntagonist (GW9662), changedt he dynamics/conformation of PPARg LBD ands lowedt he H/D exchanger ate in variousr egionso ft he protein.T he full agonists slowed theH /D exchange more globally andtoagreater extent than the partial agonist or the antagonist, indicating thatthe full agonist stabilizes the PPARg LBDmorethan thepartial agonist or the antagonist. Oneinteresting observation is that thetwo fullagonists significantly stabilized helix12whilethe partial agonist andthe antagonist did not perturb the H/D exchange of this region. Ther esults showedt hatt he change in proteind ynamics induced by ligandb inding may be an important factor fort he activation of genes and that H/D-Ex is au sefulm ethodf or analyzingt he biological activity of drug leads.

Statistical analysis of data from 39 proteins (13 766 amino acid residues) digested with immobilized porcine pepsin under conditions compatible with hydrogen/deuterium (H/D) exchange (<1 degrees C, <30 s) was performed to examine pepsin cleavage specificity. The cleavage of pepsin was most influenced by the amino acid residue at position P1. Phe and Leu are favored residues each with a cleavage probability greater than 40%. His, Lys, Arg, or Pro residues prohibit cleavage when found at the P1 position. Pro also cannot be at position P2 (cleavage probability <0.3%). Occupation of the P3 position by His, Lys, or Arg, or occupation of the P2' position by Pro, also leads to very little cleavage (cleavage probability <1.7%). The average cleavage probability over the entire data set was 13.6%, which is slightly lower than the value previously obtained by Powers et al. (14.8%). This is due, in part, to the larger protein sizes used in the current study. While the specificity of pepsin was similar to that previously observed, higher selectivity was observed in the present study due to less experimental variation in the conditions used to generate our database.

The epitope of horse cytochrome c against monoclonal antibody E8 was determined using amide hydrogen/deuterium (H/D) exchange combined with immobilized antibody, on-line pepsin proteolysis, liquid chromatography (LC), and mass spectrometry (MS). The results were generally in good agreement with contact residues identified by an X-ray co-crystal structure of the E8-cytochrome c complex and results obtained by H/D exchange with nuclear magnetic resonance (NMR) spectrometry. The H/D exchange reaction of cytochrome c was carried out in the presence or absence of immobilized E8 antibody. Regions that gained less deuterium in the presence of the antibody than in its absence are defined as the epitope by the H/D exchange MS method. Control experiments were carefully designed to help identify the epitope with high confidence.

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