Premium
P2‐225: Differential expression of beta‐amyloid fragments in crude brain lysate samples using immunoassay TOF mass spectrometry
Author(s) -
Rusa Mariana,
Roth Steve,
Bulman Amanda,
Hammond Mathew,
Thulasiraman Vanitha,
Liu Ning,
Schuerenberg Martin,
Plows Fiona
Publication year - 2010
Publication title -
alzheimer's and dementia
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 6.713
H-Index - 118
eISSN - 1552-5279
pISSN - 1552-5260
DOI - 10.1016/j.jalz.2010.05.1274
Subject(s) - chemistry , lysis , mass spectrometry , immunoassay , chromatography , amyloid beta , monoclonal antibody , microbiology and biotechnology , amyloid (mycology) , antibody , biochemistry , peptide , biology , immunology , inorganic chemistry
Background: Beta amyloid fragment deposits in the brain are associated with the onset of neurodegenerative diseases, including Alzheimer’s disease. The use of TOF mass spectrometry in measuring these fragments has the advantage of eliminating cross-reactivity errors associated with other methods such as ELISA, and allows for simultaneous detection of numerous fragments from any sample. Methods: Immuno-specific surfaces were prepared using ProteinChip array functionalized with amino groups. These reactive surfaces were used to immobilize 6E10 monoclonal antibody, specific to N-terminus beta-amyloid fragments. Negative control surfaces were prepared by using non-specific bovine IgG in place of 6E10 on the same reactive surfaces. Artificial CSF was used to prepare calibration material and internal standard. Fragments 1-33, 1-38, 1-40, and 1-42 were spiked across a range of concentration to prepare calibration material. Non-naturally occurring fragment Cys 1-24 was selected to prepare internal standard, as it is recognized by 6E10 Mab. Six human brain frontal cortex samples were analyzed, three from Alzheimer’s diagnosed and three from non Alzheimer’s sources. These samples were lysed in the presence of TUC, DTT, and protease inhibitors. The lysed samples were diluted in artificial CSF and applied directly to both 6E10 and Bovine IgG immobilized surfaces. After wash and desalting steps, alpha-cyano-4-hydroxycinnamic acid was applied and the targets were assayed in a Bruker ultrafleXtreme mass spectrometer. Results: Differential expression of Beta Amyloid fragments was observed. In known Alzheimer’s frontal brain cortex lysate multiple fragments were detected on the 6E10 surface and not on the control Bovine IgG surface. The non-Alzheimers frontal brain cortex lysate samples showed no beta-amyloid fragments on either surface type. Quantitative results were reported using calibration generated in artificial CSF. The use of an internal standard was shown to help minimize variability and improve results. The use of pre-coupled antibody arrays further reduces assay time and increases assay reproducibility. Conclusions: TOF mass spectrometry has been used to successfully develop a method for the detection of beta-amyloid fragments from human brain tissue. Quantitative results are obtained using calibration materials and internal standard. Multiple fragments are simultaneously monitored and differences are detected in diseased vs. normal brains.