781: Protein enrichment using Congo red (CR) affinity enhances characterization of the urine misfoldome in preeclampsia (PE)

High abundance proteins (i.e., albumin) are routinely removed during proteomics sample processing, as they can mask lower abundance peptides of biological/clinical interest. In PE, the urine misfoldome sums all the proteins with propensity to aggregate. Experimental albumin depletion can be detrimental to PE discovery because it alters the structure of urine misfoldome by eliminating protein identities co-aggregated with albumin. Our objective was to provide an alternative experimental approach by studying the effect of CR precipitation on enhancing identification of biomarkers that are constituents of the PE urine misfoldome. We analyzed representative urine samples from pregnant control (CRL, n=5) and severe PE (sPE, n=5) women using 3 different techniques aimed at misfoldome enrichment: CR-affinity, sodium-dodecyl-sulfate (SDS), and acetone precipitation. Native urine and their precipitates were profiled using MALDI-TOF MS and UPLC/MS/MS for peak areas measurement and ID confirmation of albumin and co-aggregated proteins (e.g. A1AT). Spectral counts and exponentially modified protein abundance index were calculated using Sequest and Mascot software. The calculated enrichment factor (EF) was expressed as the ratio of A1AT/albumin from precipitate over that of native urine. Urine CR precipitate profiles are different compared to that of native urine, SDS and acetone precipitates. The A1AT/albumin peak ratios for the CR, SDS, acetone precipitates and for native urine were: [mean±SE] 69.3±103.4, 0.091±0.03, 1.01±1.00, and 0.071±0.02, respectively (P<0.001). The CR urine analysis yielded the higher proteome EFs (3-100 fold), with clear identification of many biomarkers (A1AT shown in Figure). CR-affinity precipitation resulted in the largest Shannon index (measure of protein diversity) compared with native urine and its supernatant. In PE, identification and discovery of clinically relevant urine biomarkers in the misfoldome is highly enhanced using CR-affinity precipitation given its superiority over other laboratory precipitation tools.