Qualitative and quantitative characterization of the amyloid beta peptide (Abeta) population in biological matrices using an immunoprecipitation-LC/MS assay.
Targeting the metabolism of amyloid beta peptides (Abeta) is currently the leading experimental approach to treatment of Alzheimer's disease (AD). Described here is an immunoprecipitation-liquid chromatography/mass spectrometry (ip-LC/MS) assay to simultaneously characterize and quantitate different forms of Abeta in biological samples. The 4G8 antibody, specific for the 17-24 amino acid epitope of Abeta was employed to selectively isolate Abeta from in vitro samples for subsequent LC-MS analysis. A high resolution accurate mass hybrid linear ion trap-Orbitrap, LTQ-Orbitrap mass spectrometer was used to identify forms of 12 Abeta in H4-APP751 swe cell extracts based on ab initio calculations, accurate mass measurements, isotopic modeling and by de novo peptide sequencing using tandem mass spectrometry. The quantitative LC-MS analysis was performed on a linear ion trap mass spectrometer, LTQ, in full scan mode, this mode of operation enables sensitive detection levels and post-acquisition data mining for different forms of Abeta for quantitative assessment. Dosing studies with three known inhibitors of Abeta production, sulindac sulfide (SSide), BMS-299897 ('897) and compound W (CW) are reported to demonstrate the utility and analytical characteristics of the assay. This assay has the potential to provide insight into the formation of Abeta; increase understanding of drug mechanisms; and to contribute to drug efficacy studies.
Ford
Michael J
MJ
Bristol-Myers Squibb Company, Research and Development, Bioanalytical and Discovery Analytical Sciences, Wallingford, CT 06492, United States. Michael.Ford@bms.com
Cantone
Joseph L
JL
Polson
Craig
C
Toyn
Jeremy H
JH
Meredith
Jere E
JE
Drexler
Dieter M
DM
D000595:Amino Acid Sequence; D016229:Amyloid beta-Peptides / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000494:pharmacology; D015153:Blotting, Western; D002087:Butyrates / Q000494:pharmacology; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D003627:Data Interpretation, Statistical; D004104:Diiodothyronines / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D006846:Hydrocarbons, Halogenated / Q000494:pharmacology; D047468:Immunoprecipitation; D016014:Linear Models; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D012015:Reference Standards; D015203:Reproducibility of Results; D013467:Sulindac / Q000494:pharmacology
C497115:4-(2-((1R)-1-(((4-chlorophenyl)sulfonyl)-2,5-difluoroanilino)ethyl)-5-fluorophenyl)butanoic acid; D016229:Amyloid beta-Peptides; D000894:Anti-Inflammatory Agents, Non-Steroidal; D002087:Butyrates; D004104:Diiodothyronines; D006846:Hydrocarbons, Halogenated; C404304:compound W; D013467:Sulindac