Characterization of Amyloid β Peptides in Cerebrospinal Fluid by an Automated Immunoprecipitation Procedure Followed by Mass Spectrometry

Abstract

Pathogenic events in Alzheimer's disease are believed to involve an imbalance between the production and clearance of the neurotoxic 42 amino acid form of the beta-amyloid peptide (Abeta1-42). Although much is known about the production of Abeta1-42, many questions remain about its degradation. Here, we describe an optimized automated immunoprecipitation mass spectrometry method that enables accurate and rapid monitoring of the major Abeta isoforms in cerebrospinal fluid. Furthermore, we describe a technique of antibody immobilization, minimizing background signals. The identities of these Abeta products were confirmed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and nanoflow liquid chromatography and tandem mass spectrometry with a hybrid linear trap Fourier transform ion cyclotron resonance mass spectrometer. Finally, we report the finding of two novel Abeta peptides (Abeta2-17 and Abeta3-17).

Site-specific characterization of threonine, serine, and tyrosine glycosylations of amyloid precursor protein/amyloid β-peptides in human cerebrospinal fluid

“…All the major peaks were identified as multiple charged ions of previously described Aβ peptides in CSF (20,21) with a dominance of the Aβ1-40 and Aβ1-38 isoforms. Altogether 33 different Aβ peptides were identified (SI Appendix, Table S1) some of which, e.g., Aβ1-15, previously identified as a positive biomarker of γ-secretase inhibition (22,23), constituted less than 1% of the total ion current chromatogram.…”

“…We propose that in the presence of a Tyr10 O-glycosylation γ-secretase-dependent cleavage is modified, such that instead of cleavage at amino acid residues 40-42, the residues at 15-20 become the preferred cleavage sites (9,46). This hypothesis is based on the presence and structures of the Aβ1-X series of Tyr10 O-glycosylated peptides (Aβ1-15, 1-16, 1-17, 1-18, 1-19, and [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] and the lack of glycosylated Aβ1-40/42 peptides in CSF. However, the appearance of Aβ peptides in CSF is the result of both complex production and clearance mechanisms (17).…”

“…Immunopurification of Aβ peptides from CSF using the 6E10 antibody (2-10 μg antibody∕mL of CSF, Signet Laboratories) was performed as described (20). Capture-andrelease of sialylated glycopeptides was performed as published (27) with a few modifications.…”

Pyroglutamate Amyloid β (Aβ) Aggravates Behavioral Deficits in Transgenic Amyloid Mouse Model for Alzheimer Disease

“…IP using the KingFisher magnetic particle processor (Thermo Scientific) and mass spectrometric analysis using MALDI TOF/TOF MS were performed as described previously (29). Briefly, an aliquot (4 l, 1 mg/ml) of the A␤-specific antibodies 6E10 and 4G8 was separately added to 50 l of Dynabeads M-280 sheep anti-mouse IgG (Invitrogen) according to the manufacturer's instructions.…”

Second Generation γ-Secretase Modulators Exhibit Different Modulation of Notch β and Aβ Production

“…For tandem mass spectrometry (MS/MS) analysis, F-N␤ peptides were immunoprecipitated using the ␣-FLAG M2 antibody (Sigma) coupled to magnetic beads (2). Briefly, 10 g of the antibody was added to 50 l of magnetic Dynabeads M-280 sheep anti-mouse IgG (Invitrogen).…”