Introduction
Use of Lucid ProteinChips with immobilized antibodies specific to the N-terminus region of Beta Amyloid fragments allowed for simultaneous detection and profiling of these fragments in post-mortem human brain tissue of Alzheimer’s patients and not in non-Alzheimer’s patients. Identification of fragments of mass less than 4,000 Daltons was demonstrated via direct MS/MS analysis from a Lucid ProteinChip using a Bruker Ultraflextreme ToF-ToF mass spectrometer. Dose response curves were generated using calibration materials in artificial CSF, demonstrating quantitative capabilities for multiple fragments simultaneously. The use of an internal standard to improve mass accuracy as well as reproducibility was also shown.
Materials/Methods
6E10 monoclonal antibody was immobilized to a carbodimide reactive ProteinChip surface, using an overnight reaction at 4ºC. The ProteinChips were exhaustively washed in PBS and de-salted using de-ionized water. Large quantities of ProteinChips were prepared at the same time and stored at -20 degrees C. Six post mortem frontal cortex brain samples were obtained, three from non-Alzheimer’s and three from Alzheimer’s diagnosed subjects. Ages ranged from 63 through 101 years old across all samples, with both genders represented. Brain tissue was homogenized in the presence of TUC with DTT and protease inhibitors, followed by centrifugation. Supernatant was diluted into artificial CSF using a 1-10 dilution and spiked with internal standard. The samples were profiled on ProteinChips with immobilized 6E10.
Lyophilized fragments 1-33, 1-38, 1-40, and 1-42 were combined and serially diluted in artificial CSF and spiked with internal standard. Concentrations of 2.5, 5.0, 10, and 20 nM were assayed in duplicate on a Lucid ProteinChip with immobilized 6E10.
Results
Beta-amyloid fragments 1-36, 1-38, 1-40, 2-20, 1-42, and 2-42 were detected from Alzheimer’s afflicted human brain tissue, while no detection of these or other fragments was seen in non-Alzheimer’s brain tissue samples, which included the brain sample from the 101 year old patient. Negative controls on Bovine IgG showed no nonspecific binding in the Alzheimer’s samples.
Calibration data presented clearly show the ability to generate reproducible dose response data using a Lucid ProteinChip on a Bruker Ultraflextreme mass spectrometer. The data also shows the benefits of normalizing data based on a spiked internal standard.
Conclusion
Multiple Beta-amyloid fragments were simultaneously profiled in Alzheimer’s patients only. No fragments were seen in non-Alzheimer’s patients or in negative controls. The data supports that Alzheimer’s disease is strongly associated with Beta-amyloid fragments due to plaque formation of the fragments in the brain. That the 101 year old brain sample from a non-Alzheimer’s patient contained no Beta-amyloid fragments suggests that the presence of those fragments is not inherently age-related.
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