Structural Differences in Aβ Amyloid Protofibrils and Fibrils Mapped by Hydrogen Exchange – Mass Spectrometry with On-line Proteolytic Fragmentation

Kheterpal, Indu; Chen, Maolian; Cook, Kelsey D.; Wetzel, Ronald

doi:10.1016/j.jmb.2006.06.066

Cited by 125 publications

(152 citation statements)

References 63 publications

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“…Size-exclusion chromatography revealed that the gammabodies only bind to Aβ prefibrillar oligomers if they are added before Aβ oligomerization, suggesting that the Aβ12-21 and Aβ15-24 gammabodies intercalate into onpathway Aβ oligomers during nucleation. Previous work has established that the central hydrophobic region of Aβ (residues 17-21) undergoes conformational changes when fibrillar intermediates convert into amyloid fibrils (32,33). Thus, we hypothesize that the conformations of the grafted Aβ12-21 and Aβ15-24 loops are incompatible with the β-sheet conformation formed by the central Aβ motif when fibrillar intermediates convert into amyloid fibrils, which leads to destabilization of Aβ fibrils and formation of small gammabody-Aβ complexes.…”

Section: Discussionmentioning

confidence: 84%

Rational design of potent domain antibody inhibitors of amyloid fibril assembly

Ladiwala

Bhattacharya

Perchiacca

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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Fig. 7. IAPP and α-Synuclein gammabodies potently inhibit amyloid formation in a sequence-specific manner. IAPP (32 μM) and α-Synuclein (residues 1-115, 50 μM) were incubated in the absence (control) and presence of gammabodies (1:10 gammabody:monomer molar ratio), and fibrillization was monitored via (A) immunoblotting and (B) AFM. In B, IAPP and α-Synuclein fibrillization was also monitored in the presence of sequence-specific (R10/99, IAPP residues 7-17; 5C2, α-Synuclein residues 61-95) and conformation-specific (A11, prefibrillar oligomers; OC, fibrillar conformers) antibodies (1:10 antibody:monomer molar ratio). In B, the AFM images are 3 × 3 μm, and the blank images are samples with heights <1 nm. The heights of the IAPP and α-Synuclein aggregates are 21 ± 3 and 25 ± 4 nm, respectively.

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Section: Discussionmentioning

confidence: 84%

Rational design of potent domain antibody inhibitors of amyloid fibril assembly

Ladiwala

Bhattacharya

Perchiacca

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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“…In one such case, two amyloidogenic variants of lysozyme were compared with wild-type protein and were found to have unfolded regions in the ␤-domain and adjacent C-helix [67]. Structural differences between A␤1-40 protofibrils and fibrils have also been reported [68]: although the Cand N-terminal fragments in both the fibrils and proto- fibrils were found to be highly exposed to HDX, the internal peptide fragment consisting of residues 20 -34 was found to be highly protected in fibrils, but not so in protofibrils, suggesting that although the ␤-sheet elements in the final fibrils may be present to some extent in the protofibrils, the transition from protofibril to fibril does involve significant ordering of this central region.…”

Section: What Can We Learn About Amyloid Fibrils?mentioning

confidence: 99%

Mass spectrometry and the amyloid problem—How far can we go in the gas phase?

Ashcroft

2010

J. Am. Soc. Mass Spectrom.

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A number of proteins are capable of converting from their soluble, monomeric form into highlyordered, insoluble aggregates known as amyloid fibrils. In vivo, these fibrils, which accumulate in organs and tissues, are associated with a wide range of amyloid diseases for which there are currently no therapeutic solutions. The molecular details of the pathway from native monomer through oligomeric intermediates to the final amyloid fibril remain a challenging enigma. Over the past few years, mass spectrometry has been applied to investigate the various stages of amyloid fibril formation, and this report summarizes the key steps achieved to date. (J Am Soc Mass Spectrom 2010, 21, 1087-1096

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“…Hydrolysis of A␤ (1-40) using pepsin has been previously reported [3,9]. Preliminary experiments (data not shown) with unpurified alternative enzymes gave poor signal/background (S/B) and required very high enzyme concentrations (enzyme:substrate Ͼ 40:1) to achieve hydrolysis.…”

Section: Resultsmentioning

confidence: 87%

“…Both spectra in Figure 3 contain signals for intact A␤ (1-40) plus six protons (5% of the base peak for protease XIII and 20% for protease XVIII), reflecting While some of the additional peaks derive from relatively large fragments that may be further digested in the monomer experiments, this cannot account for all differences. For example, while the base peak for protease XVIII is a larger fragment in the fibril spectrum ( [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19] 5ϩ in Figure 3b versus [34-40] ϩ in Figure 1b), the average number of residues in an assigned ion (not weighted by relative intensity) is actually less in Figure 3b (12.3) than in Figure 1b (13.8). This difference is even larger (11.7 versus 14.1) for protease XIII (Figure 3a and Figure 1a, respectively).…”

Section: Resultsmentioning

confidence: 99%

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Complementary peptide sequence coverage using alternative enzymes for on-line digestion with a triaxial electrospray probe

Dykstra

Chen²,

Cook³

2009

J. Am. Soc. Mass Spectrom.

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Using alternative enzymes for on-line digestion with a triaxial electrospray probe extends sequence coverage. This is the first report of utilization of our triaxial probe for on-line analysis with enzymes other than pepsin, suggesting potential for broader application. The probe allows access to processes occurring on a timescale and/or involving substrate conformations complementary to those for conventional (off-line) digestion. Some of the features observed in application to A␤ fibrils are suggestive of unique reactive intermediates during dissolution. Data obtained with enzyme mixtures suggest synergistic effects. (J Am Soc Mass Spectrom 2009, 20, 1983-1987) © 2009 American Society for Mass Spectrometry H ydrolysis followed by tandem electrospray (ES) mass spectrometry is widely used for "bottom up" sequencing and localization of hydrogen/ deuterium exchange (HDX). For HDX studies, pepsin is a preferred enzyme because it tolerates the low pH that minimizes artifactual exchange during sample processing. There are cases where sequence coverage using pepsin is suboptimal, especially in the short hydrolysis times used to reduce HDX artifacts. This challenge was amplified in HDX studies of A␤ (1-40) fibrils [1,2] because of the need to accomplish both hydrolysis and rapid fibril dissolution with minimal scrambling and artifactual exchange. A triaxial probe [3] was designed and proven to accomplish this by on-line mixing of a fibril suspension with a solution that quenched HDX and initiated dissolution and hydrolysis, followed ϳ12 s later by addition of acetonitrile to provide a strong and stable ES signal. Stocks and Konermann [4] employed a similar mixing apparatus to probe denaturation via oxidative labeling, but coupled it with conventional LC/MS rather than attempting on-line hydrolysis. Our probe was designed for the specialized application requiring on-line hydrolysis; it was therefore of interest to determine whether it might be usable with other enzymes compatible with low pH constraints. We report here the efficacy of using alternative pepsin-like enzymes and their mixtures for improving sequence coverage with on-line hydrolysis.

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Structural Differences in Aβ Amyloid Protofibrils and Fibrils Mapped by Hydrogen Exchange – Mass Spectrometry with On-line Proteolytic Fragmentation

Cited by 125 publications

References 63 publications

Rational design of potent domain antibody inhibitors of amyloid fibril assembly

Rational design of potent domain antibody inhibitors of amyloid fibril assembly

Mass spectrometry and the amyloid problem—How far can we go in the gas phase?

Complementary peptide sequence coverage using alternative enzymes for on-line digestion with a triaxial electrospray probe

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