1. Amyloid-beta oligomerization is associated with the generation of a typical peptide fragment fingerprint 
Nikita Rudinskiy, Christophe Fuerer, Davide Demurtas, Sebastian Zamorano, Cyntia De Piano, Abigail G. Herrmann, Tara L. Spires- Jones, Patrick Oeckl, Markus Otto, Matthew P. Frosch, Marc Moniatte, Bradley T. Hyman, Adrien W. Schmid 
Alzheimer's & Dementia: The Journal of the Alzheimer's Association 
Volume 12, Issue 9, Pages (September 2016)
DOI: /j.jalz
Copyright © 2016 The Alzheimer's Association Terms and Conditions

2. Fig. 1
Targeted MS analysis of Aβ peptide aggregation. (A) MALDI-TOF-TOF MS analysis of Aβ42 peptide aggregation in heavy (H218O) labeled (top) and normal (bottom) water. Inset right: zoom of fragment Aβ1–25 showing the change in isotopic distribution because of an 18O atom incorporation. The red line indicates the mass of the theoretical monoisotopic peak of Aβ1–25. (B) MALDI-TOF-TOF analysis of aggregated Aβ42: C-terminal amidation can be observed in some peptide fragments (arrows). (C) High resolution LC-MS analysis of Aβ42 aggregation in heavy water. The MS spectrum shows the 18O-associated change in isotopic distribution observed with Aβ1–25 (left) and its complementary C-terminal fragment Aβ26–42 (right). Top spectrum: measured isotopic distribution of heavy labeled Aβ1–25; bottom spectrum: isotopic distribution of Aβ1–25 after aggregation in normal water. Residual amounts of C-terminal amidated (CONH2) peptide can be observed within the same isotopic cluster where the mass difference between the unmodified monoisotopic peak (m/z =  ) and the amidated monoisotopic peak (arrows; m/z =  ) is 0.989 Da (theoretical Δ mass = 0.984 Da). Fragment Aβ1–25 represents one typical example of a large N-terminal peptide where the monoisotopic peak (red) was found to be shifted by 2 Da (m/z =  [M+4H]4+) when compared with the normal (light) monoisotopic peak (m/z =  [M+4H]4+) or its theoretical mass (m/z =  [M+4H]4+). No changes in monoisotopic masses were observed for the C-terminal fragment Aβ26–42 (right). (D) LC-MS analysis of aggregated Aβ42 in normal water (as in C: bottom spectrum) but subjected to Lys-N digestion and spiking with heavy lysine labeled surrogate peptides corresponding to the Lys-N cleavage products of residues 16–27 and 16–25. The MS spectrum shows a zoom of the autocleaved peptide fragment of residues 16–25 ([M+2H]2+ m/z =  ) and the spiked-in surrogate (heavy: [M+2H]2+ m/z =  ) as well as peptide fragment 16–27 (light: m/z =  [M+2H]2+) and the heavy labeled surrogate peptide: m/z =  [M+2H]2+), which corresponds to Lys-N cleavage product of full-length Aβ42. The total ion chromatogram (TIC) of fragment 16–25 (m/z =  ) indicates that relative abundance of this accounts of approximately 5% of full-length Aβ (16–27: m/z =  ). (E) LC-MS: Comparison of the normal, light (top), and heavy oxygen (bottom) isotopic distributions of Lys-N fragment 16–25 (as in C). The spectrum of the heavy 18O incorporated fragment bears an overlap of two isotopic envelopes, of which the third isotope of m/z =  , ([M+2H]2+) accounts for the population bearing 18O at its C-terminal. (F) MS/MS (CID) fragmentation produced identical fragment ions for light (black) and 18O-incorporated (red) peptides, and the CID fragmentation signature allowed for unambiguous identification of an 18O atom incorporation at the peptide's C-terminal residue Gly25. Abbreviations: MS, mass spectrometry; Aβ, amyloid-beta; MALDI-TOF/TOF, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; CID, collision-induced dissociation.
Alzheimer's & Dementia: The Journal of the Alzheimer's Association  , DOI: ( /j.jalz )
Copyright © 2016 The Alzheimer's Association Terms and Conditions

3. Fig. 2
N-terminal peptide fragments have increased aggregation properties. (A) In vitro aggregation (t = 1 week) of N-terminal peptide fragments: Changes in peptide morphology are evidenced by significant shifts in SDS-PAGE migration behavior as seen by the appearance of several bands at the migration level of 6 kDa–14 kDa. WB probing with 4G8 (reprobed after 6E10) was more specific for all peptides, particularly for fragment Aβ1–23 (lane 8) when compared with 6E10 (lane 3). The C-terminal amidated form of Aβ1–25 appeared to have an increased tendency to form SDS-PAGE stable HMW oligomers centered at the migration level of ≤62 kDa (lane: 6). (The inset on the right shows a zoom of lane: 6). (B) SEC (Superdex 75, Tris-NaCl) and WB analysis of in vitro aggregated Aβ1–25 showing the elution fractions of stable Aβ1–25 trimers and dimers (12 mL) and monomers (15 mL; inset: WB analysis of aggregated Aβ1–25 before SEC). Abbreviations: SDS-PAGE, sodium dodecylsulfate polyacrylamide gel electrophoresis; Aβ, amyloid-beta; HMW, high molecular weight; SEC, size exclusion chromatography.
Alzheimer's & Dementia: The Journal of the Alzheimer's Association  , DOI: ( /j.jalz )
Copyright © 2016 The Alzheimer's Association Terms and Conditions

4. Fig. 3
Validation of neoepitope antibody specificity. (A) N-5ns recognizes an epitope of residues ending with Gly25 and Asp23 (blot A′, lanes 3–6) with low affinity for the amidated C-terminal residues Val24 or Gly25. (blots A through A′ and B through B′ were reblotted with different antibodies). (B) Antibody N-4 showed high specificity for the amidated C-teminal residue Val24, without cross reacting with full-length Aβ40 or any other shorter peptide fragments. (C) Neoepitope antibodies show high selectivity for immunoprecipitation-mass spectrometry (IP-MS) (MADI-TOF/TOF) applications. A peptide mixture of fragments: Aβ1–22, Aβ1–23, Aβ1–24-NH2, Aβ1–25 (all 20 ng/mL), and full-length Aβ40 (50 ng/mL) was used. (note: the lower signal intensity observed for 1–40 is due to ion signal suppression caused by shorter fragments during MALDI-TOF-TOF analysis). (D) Validation of N-5nsC-terminal specificity using direct ELISA. Antibody concentrations at ≤0.28 μg/mL (dashed line at: 5000× dilution) were typically used for immunoblotting analysis. N-5ns showed high selectivity for C-terminal Gly25, but at the shown working concentration, N-5ns showed significantly decreased binding to the amidated peptide form (Aβ1–25-NH2). (E) Validation of neoepitope antibody binding specificity using WB. No cross reactivity was observed for the shorter peptide fragment Aβ1–22 (lane 1) or full-length Aβ40 (lane 7) using N-3s, N-5s (blots: A–A′) and N-5ns (blot: B″). (immunoblots A through A″ and B through B″ were sequentially reblotted using different antibodies and scanned at the indicated different wavelengths: blots A and A′ (700 nm), blot A′′ (800 nm), blots B and B′′ (700 nm), and blot B′ (800 nm), respectively. (F) Immunoblotting using antibody N-4 revealed high specificity for the amidated peptide fragment Aβ1–24 (-NH2). (blots A through A″ were reblotted with different antibodies). Abbreviations: Aβ, amyloid-beta; MALDI-TOF/TOF, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.
Alzheimer's & Dementia: The Journal of the Alzheimer's Association  , DOI: ( /j.jalz )
Copyright © 2016 The Alzheimer's Association Terms and Conditions

5. Fig. 4
Correlation of Aβ peptide aggregation and secondary structure conformation with the generation of N-terminal fragment fingerprints. (A) Circular dichroism (CD) spectrum of Aβ40 secondary structure in PBS and TFE (20%) solution. In PBS solution, Aβ40 has a typical random coil structure, whereas in TFE, the peptide formed a stable helical structure. (B) MALDI-TOF/TOF analysis of Aβ40 fragments observed in Tris (40 mM; top), PBS (center), and TFE (20%; bottom) solution (t = 5 days). (C) Heat map of relative peptide fragment intensities observed by MS after 5 days incubation in different solutions. (D) Monitoring Aβ42 aggregation using DB and probed with N-5ns (blot A) or 6E10 (blot B). A gradual increase in N-5ns signal can be observed. Insets: TEM imaging of Aβ42 peptide morphologies observed. (E) Aβ40 aggregation is significantly increased in the presence of tTGase as compared with Aβ40 alone, and this shift in aggregation can be probed using N-5ns. (F) IP-MS (N-5ns; MALDI-TOF/TOF) analysis of different aggregation conditions (as shown in E: t = 24 hours), Aβ40 (top), Aβ40&TGase (center), and Aβ42 (bottom) highlights the presence of fragments Aβ1–23 and Aβ1–25. (Samples were centrifuged, and the supernatant (30%–50% of total volume) was recovered and filtered (100 kDa cutoff membrane) to reduce the amount of monomeric Aβ before IP). Abbreviations: Aβ, amyloid-beta; MS, mass spectrometry; DB, dotblot; TEM, transmission electron microscopy; MALDI-TOF/TOF, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.
Alzheimer's & Dementia: The Journal of the Alzheimer's Association  , DOI: ( /j.jalz )
Copyright © 2016 The Alzheimer's Association Terms and Conditions

6. Fig. 5
Targeting Aβ Oligomers using neo-epitope antibodies. (A) SEC analysis of Aβ42 ADDL's. The gradual increase in dotblot (N-5ns) signal correlates with a time-dependent peptide accumulation of cleaved Aβ fragments. SEC fractions were probed with either the N-5ns or 6E10 antibody. (B) Immunoblotting (left: N-5ns and right: 6E10) of Aβ oligomer and monomer elution in SEC fractions 8.5 mL–12.5 mL. N-5ns probing indicated that oligomeric fractions were highly enriched in truncated Aβ isoforms. (C) MALDI-TOF/TOF analysis of the “crude” oligomeric fraction (volume 8.0 mL) showing the identification of fragments Aβ1–23, Aβ1–24-NH2, and Aβ1–25 along with some shorter fragments of Aβ1–17 to Aβ1–22 as well as full-length Aβ42 (top spectrum). The inset shows a zoom of the DB analysis and the isotopic envelope of fragment Aβ1–25, where the monoisotopic peak of Aβ1–25-NH2 is highlighted (inset; arrow). Middle spectrum: IP-MS analysis of the same SEC fraction using antibody N-4 or N-5ns (bottom spectrum). Only trace amounts of the amidated form Aβ–25-NH2 (inset; arrow) were detected after IP with N-5ns. (D) IP-MS (N-5ns) analysis of SEC fractions containing oligomers; fraction volume 11.5 mL (top spectrum), 14 mL (middle spectrum), and monomers; volume 18 mL (bottom spectrum). IP-MS analysis with N-5ns allowed identification of fragments Aβ1–25 and full-length Aβ42 in all fractions, however, with different relative abundances. Insets show a zoom of the corresponding SEC fractions analyzed by DB (as shown in A) using antibody N-5ns and 6E10, respectively. (E) Quantitative SRM analysis of oligomeric and monomeric fractions showing that 70% FA dissociation of oligomers significantly reduced the amount of full-length Aβ42 stably associated with fragment Aβ1–25. (F) IP-MS (MALDI-TOF/TOF) of the monomeric fraction (SEC vol: 18 mL) using a combination of three antibodies (N-3s, N-4, and N-5ns). (G) Sequential IP-MS of the same sample as shown in (F) but denatured in 90% FA before the second IP. Abbreviations: Aβ, amyloid-beta; SEC, size exclusion chromatography; ADDL, amyloid-derived diffusible ligand; DB, dotblot; SRM, selected reaction monitoring; FA, formic acid; IP, immunoprecipitation; MALDI-TOF/TOF, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.
Alzheimer's & Dementia: The Journal of the Alzheimer's Association  , DOI: ( /j.jalz )
Copyright © 2016 The Alzheimer's Association Terms and Conditions

7. Fig. 6
Immunohistochemical detection of truncated Aβ fragments in postmortem human frontal cortex tissue. (A) Representative photomicrographs of N-5ns (3 μg/mL) staining of paraffin-embedded human frontal cortex sections from AD patients (n = 6) and age-matched non-AD controls (n = 3) using horseradish peroxidase detection system. N-5ns specifically labels extracellular amyloid plaques and CAA. AD samples without incubation with primary antibody (no 1ry), as well as control (from nondemented patients) samples, show no significant staining. (B) Representative photomicrographs of immunofluorescent labeling of thioflavin-S-positive extracellular amyloid plaques and CAA in human AD frontal cortex with N-5ns (3 μg/mL). No N-5ns-positive Aβ fragments are detected in the thioflavin-S-positive intraneuronal tau tangles. (C) No nonspecific signal is detected when the incubation with primary antibody is omitted (exposure and brightness/contrast levels are matched between B and C to permit comparison). (D) Array tomography: N-5ns stains plaques that are colabeled with 6E10 and thioflavin-S in the superior temporal gyrus of human brain. (E) Array tomography: Costaining with synaptic markers PSD-95 and synaptophysin reveals a subset of both presynapses (turquoise, arrowhead) and postsynapses (yellow, arrows) around N-5ns-positive plaques that contain truncated Aβ. Scale bars: 500 μm (A, top panels); 100 μm (A, bottom panels; B and C); 5 μm (D and E); 2 μm (inset in E). Abbreviations: Aβ, amyloid-beta; AD, Alzheimer's disease.
Alzheimer's & Dementia: The Journal of the Alzheimer's Association  , DOI: ( /j.jalz )
Copyright © 2016 The Alzheimer's Association Terms and Conditions

8. Fig. 7
Mass spectrometry analysis of Aβ fragment signatures in human AD brains. (A) Representative WB analysis of two AD brain samples subjected to a hybrid IP (6E10/4G8) (lanes: 1 and 4) or IP using a combination of neoepitope antibodies (N-3s/N-4/N-5ns; lanes: 2 and 6). IP with 6E10/4G8 revealed a band centered at the migration level of the monomer (top membrane, lanes 1 and 4), whereas IP with neoepitope antibodies resulted in a pulldown of a band centered at the 6 kDa level in one AD brain sample (lane: 5). Reblotting (bottom membrane) with the neoepitope antibody mixture revealed a band at the migration level of ≥4 kDa in both AD brain samples (lanes: 2 and 5). (lane: 3 = neoepitope antibody mixture alone). (B) Hybrid IP-MS (6E10 and 4G8) analysis (MALDI-TOF/TOF) of an AD brain extract (FA) showing the presence of full-length Aβ40 and truncated Aβ peptides. (+ indicates Na adduct of preceding peak, * indicates nonidentified peaks). (C) Combined IP-MS (N-3s, N-4, N-5ns) analysis (MADLI-TOF/TOF) of the same brain sample resulted in a pulldown of truncated Aβ fragments corresponding to the neoepitope targets. (D) High resolution LC-MS (Orbitrap) analysis of Aβ fragments after IP with neoepitope antibodies (as in C). The spectrum shows two isotopic populations of quadruply and triply charged ions of different truncated Aβ isoforms. (E) MS/MS analysis (Orbitrap) of Aβ1–25, Aβ1–24(-NH2), Aβ2–24(-NH2), and Aβ1–23 as shown in spectrum (D). Abbreviations: Aβ, amyloid-beta; AD, Alzheimer's disease; IP, immunoprecipitation; MALDI-TOF/TOF, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.
Alzheimer's & Dementia: The Journal of the Alzheimer's Association  , DOI: ( /j.jalz )
Copyright © 2016 The Alzheimer's Association Terms and Conditions