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.
Warder
Scott E
SE
Advanced Technology, Global Pharmaceutical Research and Development, Abbott Laboratories, 100 Abbott Park Road, Abbott Park, IL 60064, USA. scott.warder@abbott.com
Tucker
Lora A
LA
Strelitzer
Tamara J
TJ
McKeegan
Evelyn M
EM
Meuth
Joseph L
JL
Jung
Paul M
PM
Saraf
Anita
A
Singh
Bhawani
B
Lai-Zhang
Jie
J
Gagne
Gerard
G
Rogers
John C
JC
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
D001798:Blood Proteins; D019163:Reducing Agents; D010978:Platelet Factor 4