Enrichment and analysis of Alzheimer's Abeta1-42 peptide in human plasma and whole blood.

The Abeta1-42 fragment from the Amyloid Precursor Protein (APP) has presented considerable challenges from an analytical perspective. It is present at low levels in the circulation and can bind to proteins which mask its presence in assays. A number of therapeutic strategies target the lowering of this peptide, necessitating more robust and sensitive methods for its measurement. In this study, conditions for extracting and enriching Abeta1-42 using solid-phase extraction (SPE) and reverse-phase HPLC (RP-HPLC) were optimized. The new process provided reproducible recovery of Abeta1-42 of about 80% and allowed for concentration of the peptide prior to assay. Radiolabeled Abeta1-42 and ELISA for Abeta1-42 were used to determine the recovery and distribution of the peptide from whole blood collected in the presence of potassium-EDTA. Endogenous Abeta1-42 yielded a cell pellet:plasma ratio near 40:60 while exogenously added peptide distributed with a ratio of about 27:73. Additionally, the Abeta1-42 in the plasma and cell pellet fractions maintained stability over many hours. Comparing the measurement of Abeta1-42 using a commercial ELISA before and after enrichment demonstrated noticeable improvement of signal in samples enriched for the peptide. The current study also showed that conspicuous amounts of Abeta1-42 partition to the cell pellet but that this fraction can be robustly recovered and measured with SPE and HPLC. The process utilized established chromatographic techniques and is suitable for automation. It is also compatible with other detection methods including mass spectrometry.

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.