Fig. 5

SEC fractionation confirms the presence of two distinct pools of serum IgA1 clones. A SEC fractionation of pooled serum from healthy donors, where each fraction was analyzed for its protein content by bottom-up proteomics. The horizontal axis depicts each analyzed fraction, and the y-axis depicts the normalized abundance of the proteins. IgA1 (red trace) has two peaks in the SEC chromatogram. The first, higher MW, peak coincides with the elution profile of J-chain, indicating that the first peak contains dimeric J-chain coupled IgA1. The second IgA1 peak does not contain J-chain, is of lower MW, and contains monomeric IgA1, as indicated by the cartoons. The J-chain SEC profile also has an additional peak. The higher MW peak corresponds with the elution of IgM, the other serum Ig complex that contains J-chain. B SEC fractionation of IgA captured from the serum of Donor 1 (C1). Fractions F1 – F4 were collected across the chromatogram, indicated by the grey dashed lines. As observed in panel (A), the serum profile contains two peaks corresponding to dimeric J-chain coupled IgA1 and monomeric IgA, in similar ratios as observed in (A). These ratios are similar to the ratios measured by MP (Fig. 4). C Deconvoluted IgA1 Fab mass profiles of the four serum fractions from (B). Each peak represents a unique Fab (based on unique RT/mass pair) and the height of each peak represents the concentration of that clone. Red peaks represent clones unique to serum, while purple peaks indicate clones shared between serum and milk. On the right of the mass profiles, pie charts are shown depicting the percentage of unique shared clones in each repertoire. In the mass profile, two peaks are highlighted by red and purple numbers