Purified ANGPTL41–159 preparations (wt and E15K) were diluted to 10 µM with D2O-phosphate buffer at pH 7.1, and hydrogen–deuterium exchange was monitored for 10, 100, and 1000 s at 25°C. Panel A provides a heat map of the deuterium uptake values (relative to a fully exchanged control) for peptic peptides from wt and E15K ANGPTL41–159 as assessed by HDX-MS. The relative deuterium uptake values for these peptides are plotted on the primary protein sequence [ranging from blue (no deuterium uptake) to red (full deuterium uptake)]. The position of the specific epitope 1 (SE1) defined by the neutralizing mAb 14D12 is shown by the light green cylinder (Desai et al., 2007; Lee et al., 2009), and the sequence representing an active synthetic peptide is shown by the dark green cylinder (Yau et al., 2009). The secondary structure of ANGPTL4 wt was predicted by PSIPRED (Jones, 1999) and the positions of the two α-helices are shown by red cylinders. The position of E15K polymorphism is shown by the blue letter. Panel B shows the isotope envelopes recorded for peptide 20–41 of ANGPTL4 wt and E15K; it shows progressive deuterium uptake as a function of labeling time. Panel C visualizes the differences in the dynamics of wt and E15K ANGPTL41–159 with a butterfly plot, showing differential deuterium uptake for the two proteins recorded after 10 s (orange), 100 s (red), and 1000 s (blue). The differential uptake for the full-deuterium exchange controls is shown for comparison (black). Peptides including residues 18–30 are highlighted by the transparent green box. The shaded gray area corresponds to the largest standard deviation in the data sets recorded for each peptide (triplicates). In panel A, note that the primary sequence starts with a methionine for the bacterial expression, but this is not included in the peptide numbering. In panel C, the presence of the methionine in a peptic peptide is denoted as +1.