Mass spectrometry to characterize the binding of a peptide to a lipid surface

The binding of an amphipathic alpha-helical peptide to small unilamellar li pid vesicles has been examined using chemical derivitization and mass spect rometry. The peptide is derived from the sequence of human apolipoprotein C -II: (apoC-II), the protein activator of Lipoprotein lipase (LpL), ApoC-II1 9-39 forms approximately 60% alpha-helix upon binding to model egg yolk. ph osphatidylcholine small unilamellar vesicles. Measurement of the affinity o f the peptide for lipid by spectrophotometric methods is complicated by the contribution of scattered light to optical signals. Instead, we characteri ze the binding event using the differential labeling of lysine residues by the lipid and aqueous-phase cross-linkers, disuccinimidyl suberate (DSS) an d bis(sulfosuccinimidyl) suberate (BS3), respectively. In aqueous solution, the three lysine residues of the peptide are accessible to both cross-link ers. In the presence of lipid, the C-terminal lysine residue becomes inacce ssible to the lipid-phase crosslinker DSS, but remains accessible to the aq ueous-phase cross-linker, BS3. We use mass spectrometry to characterize thi s binding event and to derive a dissociation constant for the interaction ( K-d = 5 mu M). We also provide evidence for the formation of dimeric cross- linked peptide when high densities of peptide are bound to the lipid surfac e. (C) 1999 Academic Press.