Extended Data Fig. 2: A flexible tag-based approach to assessing variant effects on secretion.
From: Multiplex and multimodal mapping of variant effects in secreted proteins via MultiSTEP
a, Heatmap showing strep tag secretion scores for missense FIX variants. Color indicates MultiSTEP score from 0 (blue, lowest 5% of scores) to white (1, WT) to red (increased). Black dots indicate the WT amino acid. Missing data are gray. b, Density distributions of strep tag secretion scores for FIX missense variants (orange) and synonymous variants (blue). Dashed line denotes the 5th percentile of the synonymous variant distribution. c, Scatter plot comparing MultiSTEP-derived strep tag secretion scores for seven different FIX variants (p.C28Y, p.A37T, p.G58E, p.E67K, p.C134R, p.S220T, and p.H267L), WT, and an unrecombined negative control to the geometric mean of Alexa Fluor-647 fluorescence measured using flow cytometry individually (n = 3 replicates). Error bars show standard error of the mean. d-e, Scatter plots of median MultiSTEP-derived strep tag secretion scores and heavy chain (d) or light chain (e) at each position in FIX (n = 3 replicates). Points are colored by chain architecture, using the color scheme as Fig. 2a. Black dashed line indicates the line of perfect correlation between secretion scores. Gray background indicates <0.3 point deviation from perfect correlation. f, Density plots of MultiSTEP-derived synonymous variant scores generated with the indicated antibody. The dashed vertical line shows WT score.
