Figure 8.

Caffeic acid phenethyl ester (CAPE) forms an adduct with Cys133 in MD2. Recombinant mouse MD2 (1 μg) was incubated with CAPE (20 μM) for 1 h at 37°C prior to in-solution digestion and micro liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. MS/MS spectrum of the CAPE-Cys133 adduct was acquired using a LTQ linear ion trap mass spectrometer. MS/MS spectra of mouse MD2 sequences (A, Arg132-Phe147, RC¹³³VAEAIAGDTEEKLF; B, Ile80-Leu108, SVNSIELPKRKEVLC⁹⁵HGHDDDYSFC¹⁰⁵RAL) were selected and the fragment ions were assigned as b (red colour) or y (blue colour) series ions. Mass accuracy of ±0.5 Da was used in the assignments. * denotes fragment ions with one CAPE. The proposed structure of CAPE-Cys133 MD2 adduct is presented in (A).