Reducing agent-mediated precipitation of high-abundance plasma proteins

Depletion of high-abundance proteins is regarded as a critical sample preparation step for most plasma proteomic analyses and profiling strategies. This report describes a process that rapidly and reproducibly precipitates high-abundance disulfide-rich proteins, including albumin and transferrin, from serum and plasma. A low volume of concentrated reducing agent, viz. dithiothreitol (DTT) or tris(2-carboxyethyl)phosphine (TCEP), was added directly to plasma followed by a brief incubation at ambient temperature. Removal of the precipitate via centrifugation and identification of the protein content revealed an albumin-enriched pellet. Direct analysis of the supernatant by MALDI-TOF-MS afforded peptidome and small protein profiles with enhanced features and minimal ionization of full-length albumin. The reproducible and quantitative nature of the method has been demonstrated by monitoring the plasma levels of an antiangiogenic protein biologic, rKringle5 (rK5). The 10.5-kDa analyte was only reliably detected in plasma after treatment with reducing agent, ionizing linearly from 150 to 1200 fmol (on-target) with a mean CV of 7%. This method distinguishes itself from immunoaffinity resin-based approaches since it can be scaled to large milliliter quantities and it is compatible with plasma from all species tested.