Figure 2 -. Analysis of intact protein-inhibitor adducts.

a) In intact protein MS, protein analytes are directly analyzed without digestion. Each protein molecule sequesters protons during electrospray ionization, yielding multiple peaks according to their mass-to-charge ratio (m/z). A set of simultaneous equations with two unknowns can be set up to calculate the mass of the protein. In modern applications, this deconvolution is done algorithmically. b) Intact protein MS provides stoichiometry of covalent protein-inhibitor complex formation. By comparing the calculated mass difference across DMSO control to inhibitor-treated samples, the stoichiometry of binding can be calculated. c) In Bashore et al., analysis of an unexpected -34Da peak upon USP7 treatment with a cyanopyrrolidine inhibitor revealed the inhibition mechanism as beta-elimination of the enzyme-inhibitor adduct and subsequent destruction of the catalytic cysteine thiol. d) As protein mass increases, higher resolution is required to identify adjacent charge states. Top row shows simulated spectra for ideal homogeneous proteins of 25, 75, and 120kDa. Bottom row shows overlay of simulated spectra for protein and protein bound to 500Da inhibitor. e) In disulfide tethering, low molecular weight fragments bearing thiol groups are incubated with the protein of interest in a reducing environment. Under reduction-catalyzed disulfide exchange, only fragments with inherent affinity for the target at a nearby pocket remain bound to the protein of interest.