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.2011 Apr 30;302(1-3):167-173.
doi: 10.1016/j.ijms.2010.10.001.

Hydrogen exchange mass spectrometry as an analytical tool for the analysis of amyloid fibrillogenesis

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Hydrogen exchange mass spectrometry as an analytical tool for the analysis of amyloid fibrillogenesis

Carsten Scavenius et al. Int J Mass Spectrom..

Abstract

In this study, we have used glucagon as a model system for analyzing amyloid fibrillogenesis by hydrogen exchange MALDI mass spectrometry (HXMS). The hydrogen exchange mass spectrometry data correlated well with the traditional method based on Thioflavin T fluorescence and provided quantitative information by measuring the fibrillating molecules directly. The hydrogen exchange mass spectrometry data collected during fibrillogenesis revealed that glucagon fibrillation was a two component system showing an on/off type of interaction where only monomeric and fibrils were present without any substantial amount of intermediate species. This was evident by the extensive deuteration of the monomer and protection of the entire 29 residue glucagon peptide upon fibrillation.. The method complements the traditional procedures and has the potential to provide new information with respect to the nature of transient species, the structure of the growing fibrils and the mechanism of formation.

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Figures

Figure 1
Figure 1. Schematic representation of the utilized protocol
Monomeric, fibrillated or a mixture of monomeric and fibrillated glucagon were diluted 24 times in D2O (1). The hydrogen deuterium exchange reaction was quench by lowering pH and temperature and fibrillated species were dissolved by urea (2). Full-length glucagon was micro purified and analyzed by MALDI-TOF (3). For the analysis of glucagon fragments the urea treated samples were digested with immobilized pepsin (4). The pepsin generated peptides were micro purified prior to MALDI-TOF analysis (5).
Figure 2
Figure 2. Relative Deuterium uptake in monomeric and fibrillated glucagon
Monomeric (◆) and fibrillated (■) glucagon were diluted in D2O and incubated at 25°C for different periods, before the exchange was quenched by lowering the pH to 2.5 and the temperature to 0°C. The samples were either full-length glucagon or peptic peptides covering the entire sequence. (A) Mass spectra of monomeric (top) and fibrillated (middle) full-length glucagon after 10 min hydrogen exchange, with an unlabelled sample shown for comparison (bottom). (B) Sequence and schematic representations of the peptides generated by pepsin treatment of glucagon. The relative deuterium uptakes are shown for (C) full-length glucagon, (D) residue 1–21, (E) residue 10–29 and (F) residue 10–21. Error bars show the standard deviation (non visual error bar indicates standard deviation within the size of the marker).
Figure 3
Figure 3. Fibrillation followed by HXMS
During the first 12 hrs of glucagon fibrillation aliquots from the same fibrillating sample were withdraw for HXMS analysis. Representative spectra obtain at different time points during the fibrillation are shown. From the bottom its is aliquots removed from a fibrillating sample after 0, 120, 210, 300, 480 and 540 minutes.
Figure 4
Figure 4. Relative amount of fibrillated glucagon
During the fibrillation process of glucagon aliquots from the same sample were withdraw for either ThT (A) or HXMS (B) analysis. The relative amount of fibrillated glucagon was calculated based on the intensity of the isotopic cluster corresponding to monomeric and fibrillated glucagon. All analysis were made in triplicates with 5 min hydrogen exchange. Error bars indicates the standard deviation.
Figure 5
Figure 5. Urea induced dissociation of glucagon fibrils
Centrifugation purified glucagon fibrils were analyzed with HXMS under increasing urea concentration. Spectra obtain at different urea concentrations are shown (A). (B) The relative amount of fibril during urea dependent unfolding of glucagon fibrils was calculated from the intensity of the isotopic cluster corresponding to monomeric and fibrillated glucagon. Error bars show the standard deviation.
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References

    1. Chiti F, Dobson CM. Protein misfolding, functional amyloid, and human disease. Annu Rev Biochem. 2006;75:333–366. - PubMed
    1. Frokjaer S, Otzen DE. Protein drug stability: a formulation challenge. Nat Rev Drug Discov. 2005;4:298–306. - PubMed
    1. Kolstoe SE, Wood SP. Drug targets for amyloidosis. Biochem Soc Trans. 2010;38:466–470. - PubMed
    1. Runager K, Enghild JJ, Klintworth GK. Focus on molecules: Transforming growth factor beta induced protein (TGFBIp) Exp Eye Res. 2008;87:298–299. - PMC - PubMed
    1. Sawaya MR, Sambashivan S, Nelson R, Ivanova MI, Sievers SA, Apostol MI, Thompson MJ, Balbirnie M, Wiltzius JJ, McFarlane HT, Madsen AO, Riekel C, Eisenberg D. Atomic structures of amyloid cross-beta spines reveal varied steric zippers. Nature. 2007;447:453–457. - PubMed

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