A high-yield method to extract peptides from rat brain tissue.
A process to extract and enrich extracellular peptides and proteins from tissues should have broad utility in the burgeoning proteomics field. To address this need, a novel three-step protocol was developed to extract polypeptides from whole tissue samples and enrich the extracellular components. The initial homogenization of rat brain was carried out at neutral pH to optimize protein and peptide stability and solubility. Subsequent covalent chromatography on an activated thiopropyl resin was employed to debulk the tissue extract by selectively removing a substantial fraction of the intracellular protein component under nondenaturing conditions. Finally, extraction with 0.1% trifluoroacetic acid was used to selectively precipitate large proteins while enhancing the solubility of smaller proteins and peptides. The fractions from each step in the process were compared to a single extract obtained by homogenization in 0.5 M acetic acid. The recovery and yields of endogenous neuropeptides and an exogenously added peptide were evaluated by enzyme immunoassay and Western blotting, respectively. In summary, the three-step protocol was superior to the extraction of tissue with 0.5 M acetic acid in terms of peptide recovery, enrichment, and sample stability. Enrichment of the extracellular protein compartment from tissues should be valuable in proteomics experiments aimed at identifying biomarkers that can partition into serum.
Kline
Tiffany R
TR
Department of Proteomics, Bristol-Myers Squibb Pharmaceutical Research Institute, PO Box 5400, Princeton, NJ 08543-5400, USA.
Pang
James
J
Hefta
Stanley A
SA
Opiteck
Gregory J
GJ
Kiefer
Susan E
SE
Scheffler
Julie E
JE
D000818:Animals; D001923:Brain Chemistry; D002352:Carrier Proteins / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D047908:Intracellular Signaling Peptides and Proteins; D009479:Neuropeptides / Q000302:isolation & purification*; D000068797:Orexins; D051381:Rats
D002352:Carrier Proteins; D047908:Intracellular Signaling Peptides and Proteins; D009479:Neuropeptides; D000068797:Orexins