title: Reducing agent-mediated precipitation of high-abundance plasma proteins.
abstract: 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.
authors.affiliation: Advanced Technology, Global Pharmaceutical Research and Development, Abbott Laboratories, 100 Abbott Park Road, Abbott Park, IL 60064, USA. scott.warder@abbott.com
authors.forename: Scott E
authors.initials: SE
authors.lastname: Warder
authors.forename: Lora A
authors.initials: LA
authors.lastname: Tucker
authors.forename: Tamara J
authors.initials: TJ
authors.lastname: Strelitzer
authors.forename: Evelyn M
authors.initials: EM
authors.lastname: McKeegan
authors.forename: Joseph L
authors.initials: JL
authors.lastname: Meuth
authors.forename: Paul M
authors.initials: PM
authors.lastname: Jung
authors.forename: Anita
authors.initials: A
authors.lastname: Saraf
authors.forename: Bhawani
authors.initials: B
authors.lastname: Singh
authors.forename: Jie
authors.initials: J
authors.lastname: Lai-Zhang
authors.forename: Gerard
authors.initials: G
authors.lastname: Gagne
authors.forename: John C
authors.initials: JC
authors.lastname: Rogers
mesh_terms: D000818:Animals; D001798:Blood Proteins / Q000737:chemistry*; D011232:Chemical Precipitation; D000882:Haplorhini; D006801:Humans; D010978:Platelet Factor 4 / Q000097:blood; D019163:Reducing Agents / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013696:Temperature
chemical_list: D001798:Blood Proteins; D019163:Reducing Agents; D010978:Platelet Factor 4