Fig 1.
Anti-Aβ/APP antibodies used in this study.
The epitopes targeted by antibodies are labeled in the upper panel. The APP695 isoform lacks the Kunitz-like protease inhibitor (KPI) domain and the OX-2 antigen domain, and the APP751 isoform lacks only the OX-2 domain. Detailed information of these antibodies are shown in the lower panel.
Fig 2.
6E10-immunoreactive proteins in human CSF.
(A-C) Representative IP/WB. Monoclonal antibody 6E10 was used to immunoprecipitate Aβ/APP metabolites from human CSF samples (250 μL per sample). Immunocaptured proteins were separated by SDS-PAGE, and detected by biotinylated 6E10. Note the proteins migrating at ~55 kDa (arrowhead) and at ~15 kDa (arrows), which are further characterized in this study. Proteins migrating at ~100 kDa and at <10 kDa likely represent the soluble, α-secretase-cleaved APP metabolites (sAPPα) and monomeric canonical Aβ1-40/42, respectively. Proteins migrating at ~70 kDa likely represent APP metabolites whose identities are beyond the scope of investigation of this study. Three sets of samples (A, 250 μL of CSF per lane; B, 240 μL of CSF per lane; C, 160 μL of CSF) were immunoprecipitated by monoclonal antibody 6E10; the eluates were separated by SDS-PAGE using 10.5–14% Tris-HCl gels and detected by biotinylated 6E10. The IP/WB experiments were performed by different experimenters at three different times spanning a five-year period; nonetheless, Fig 2A, 2B and 2C show a similar WB pattern. (D) WB showing fractionation of 6E10-immunoreactive species by SEC. The ~100- and ~70- kDa proteins were eluted together in fractions 49–75, the ~55-kDa proteins in fractions 57–63, the ~15-kDa proteins in fractions 65–69, and the <10-kDa proteins in fractions 79–83. No <10-kDa proteins were detected in the early fractions containing the ~55- and the ~15- kDa species, and no ~55 kDa or ~15 kDa species were detected in the late fractions containing the <10-kDa species. Five percent of the volume of the injected CSF sample is shown in the first lane (In). Vertical arrows indicate the fractions in which globular protein standards of the indicated molecular weights were eluted. The mismatch between the predicted elution fraction and molecular weights estimated by SDS-PAGE suggests that the 6E10-immunoreactive Aβ/APP metabolites do not migrate through the column as globular proteins. LP CSF, CSF obtained by lumbar puncture from living providers.
Fig 3.
Immunological characterization of the ~55- and the ~15- kDa, 6E10-immunoreactive proteins in CSF.
(A) A series of antibodies directed against various epitopes of Aβ were used to immunoprecipitate target proteins from CSF, and blots were probed with biotinylated- 6E10 or 82E1. Capture antibodies are indicated by the labels above each lane. Top panels show short exposure (30 sec), and bottom panels show longer exposure (3 min). Note that the ~55-kDa proteins (arrowheads) were immunoprecipitated by monoclonal antibodies targeting the N-terminal region (6E10 and 42–5), mid-region (4G8) and the C-terminal end of Aβ (anti-Aβx-40/42); whereas the ~15-kDa proteins (arrows) were only immunoprecipitated by monoclonal antibodies targeting the N-terminal region of Aβ. (B) A series of antibodies directed against various epitopes of APP were used to immunoprecipitate target proteins from CSF, and blots were probed with biotinylated 6E10. Capture antibodies are indicated by the labels above each lane. Both the ~55- and the ~15- kDa species were immunoprecipitated by monoclonal antibody 1G6 directed against an APP epitope N-terminally adjacent to Aβ, but not by antibodies directed against the N- (“NT”) or C- (“CT”) termini of APP. Mouse and rabbit IgGs (msIgG, rbtIgG) served as negative control capture reagents. * = non-specific bands.
Fig 4.
The C-termini of Aβ were not detected in the ~15-kDa, 6E10-immunoreactive proteins of human CSF.
(A) The monoclonal antibody 6E10 was used to immunoprecipitate proteins from CSF, and WB was probed with C-terminal end-specific monoclonal antibodies targeting Aβ40 and Aβ42. No ~15-kDa proteins were detected. To serve as negative controls, mouse IgG replaced 6E10 as the capture reagent (msIgG + CSF) or 6E10 was incubated with immunoprecipitation dilution buffer in the absence of CSF (6E10 only). Syn Aβ, synthetic Aβ1–40 (5 ng) and Aβ1–42 (5 ng). (B) The same blot shown in (A) was stripped with Restore PLUS stripping buffer (Thermo Fisher Scientific) and reprobed with biotinylated 6E10, and the ~15-kDa proteins (arrows) were then detected. * = non-specific bands.
Fig 5.
The ~55- and the ~15- kDa, 6E10-immunoreactive species contain APP metabolites located N-terminally of Aβ.
(A) CSF samples were first immunodepleted using the antibodies noted above each lane, and then subjected to immunoprecipitation with 6E10, and WB was probed with biotinylated 6E10. Immunodepletion with 6E10 removed both the ~55- (arrowhead) and the ~15- kDa (arrows) proteins as expected. Immunodepletion with the 1G6 antibody fully removed the ~15-kDa species and partially removed the ~55-kDa species, indicating the presence of APP fragments located N-terminally of Aβ in these bands. In contrast, an antibody directed against the C-terminus (CT) of APP and the antibody 4G8 failed to deplete the ~55- and the ~15- kDa, 6E10-immunoreactive proteins. Note that the blot was transferred from a 10–20% Tris-Tricine gel. (B) In different CSF samples, immunodepletion with the antibody 1G6 did not reduce levels of the ~55-kDa band, but almost fully eliminated the ~15-kDa band. Immunodepletion with mouse IgG (msIgG) served as a negative control. Note that the blot was transferred from a 10.5–14% Tris-HCl gel.
Fig 6.
Mapping the N- and C-termini of the ~55- and the ~15- kDa, 6E10-immunoreactive proteins.
(A) CSF samples were immunoprecipitated using either 22C11 or 1G7, directed against APP66-81 and APP380-665, respectively, and WB was probed with biotinylated 6E10. Neither antibody captured the ~55- or the ~15- kDa proteins. (B) CSF samples were immunoprecipitated using 6E10, and immunocaptured proteins were detected using biotinylated 2B3, directed against the α-secretase cleavage site of APP. Neither the ~55- nor the ~15- kDa proteins were detected. In both experiments, mouse IgG (msIgG) was used as a negative control capture reagent. * = non-specific bands.
Fig 7.
In-gel trypsin digestion/MS analysis of the ~55-kDa, 6E10-immunoreactive proteins of human CSF samples.
The location of the ~55-kDa target proteins was identified in unstained gels by overlaying the analytic lanes on the film record of the Western blot of the reference lane (shown at left, an arrowhead pointing to the ~55-kDa, 6E10-immunoreactive species). After alignment by the molecular weight standards, the pieces of unstained gel overlaying the bands of interest were excised. The isolated bands were subjected to in-gel trypsin digestion followed by MS analysis. The MS/MS spectra of the identified peptide fragments are shown in the right panel.
Fig 8.
In-gel trypsin digestion/MS analysis of the ~15-kDa, 6E10-immunoreactive proteins of human CSF samples.
The location of the ~15-kDa target proteins was identified in unstained gels by overlaying the analytic lanes on the film record of the Western blot of the reference lane (shown at left, arrows pointing to ~15-kDa, 6E10-immunoreactive bands from which peptides were identified by MS). After alignment by the molecular weight standards, the pieces of unstained gel overlaying the bands of interest were excised. The isolated bands were subjected to in-gel trypsin digestion followed by MS analysis. The MS/MS spectrum of the identified peptide fragment is shown in the right panel.
Fig 9.
Aβ/APP fragments in the ~55- and the ~15- kDa, 6E10-immunoreactive proteins identified by mass spectrometry.
Schematic diagrams showing peptide fragments (striated blocks) in the ~55- or the ~15- kDa species. The sequences of MS-identified peptide fragments are listed in the table below the diagram. KPI = the Kunitz-like protease inhibitor domain, present in the APP770 and APP751 isoforms but absent in the APP695 isoform; OX-2 = the OX-2 antigen domain, present in the APP770 isoform but absent in the APP751 and APP695 isoforms.