(372e) Size, Charge, and Affinity Fractionation of Hemolyzed Sera From a Neonatal Repository

Hemolysis of serum or plasma can exclude very valuable samples from longitudinal studies, particular over time course, and risks the loss of valuable data. In human sera, the concentration of albumin is nearly ten billion times greater than that of cell-signaling proteins like the interleukins. The relative proportions of such rare, but extremely important biomarkers is further decreased in hemolyzed samples in which hemogloblin may nearly double the total protein concentration. Further, hemogloblin removal alone does not significantly decrease sample complexity. Sample prefractionation ideally partitions high abundance proteins like albumin, IgG, and hemoglobin into fractions separate from those containing the protein(s) of interest, potentially enabling enrichment and increasing the sensitivity of downstream microassays attempting to quantify these critical molecules. Samples from a neonatal depository are hoped to provide valuable information on pro-inflammatory response during the first days of life. However, such samples are extremely rare and frequently hemolyzed. To derive meaningful data from hemolyzed whole bloods and sera, fractionation schemes based of protein molecular mass, charge, or affinity were investigated as means of hemoglobin depletion with concomitant biomarker enrichment. Size separations were performed by sequential ultrafiltration with decreasing molecular weight cutoffs (MWCOs) to produce distinct size fractions, or by electrophoresis in the GELFREE System. Charge fractions were evaluated in the OFFGEL Fractionator or a Digital Protein Chip (dPC) capable of producing fractions of 0.05 pI unit intervals as well as pH specific column binding and elution. For affinity based separations, The ProteoMiner immobilized hexapeptide ligand library was used to isolate low abundance proteins without requiring prior hemoglobin depletion.