Determination of the Aggregate Binding Site of Amyloid Protofibrils Using Electron Capture Dissociation Tandem Mass Spectrometry.
J Am Soc Mass Spectrom
; 31(2): 267-276, 2020 Feb 05.
Article
en En
| MEDLINE
| ID: mdl-31922736
ABSTRACT
Amyloid fibril formation is a hallmark in a range of human diseases. Analysis of the molecular details of amyloid aggregation, however, is limited by the difficulties in solubilizing, separating, and identifying the aggregated biomolecules. Additional labeling or protein modification is required in many current analytical techniques in order to provide molecular details of amyloid protein aggregation, but these modifications may result in protein structure disruption. Herein, ultrahigh resolution mass spectrometry (MS) with electron capture dissociation tandem MS (ECD MS/MS) has been applied to monitor the formation of early oligomers of human islet amyloid polypeptide (hIAPP), which aggregate rapidly in the pancreas of type II diabetes (T2D) patients. ECD MS/MS results show the aggregation region of the early oligomers is at the Ser-28/Ser-29 residue of a hIAPP unit and at the Asn-35 residue of another hIAPP unit near the C-terminus in the gas phase. These data contribute to the understanding of the binding site between hIAPP units which may help for specific target region therapeutic development in the future. Furthermore, MS has also been applied to quantify the amount of soluble amyloid protein remaining in the incubated solutions, which can be used to estimate the aggregation rate of amyloid protein during incubation (28 days). These data are further correlated with the results obtained using fluorescence spectroscopy and transmission electron microscopy (TEM) to generate a general overview of amyloid protein aggregation. The methods demonstrated in this article not only explore the aggregation site of hIAPP down to an amino acid residue level, but are also applicable to many amyloid protein aggregation studies.
Palabras clave
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Sitios de Unión
/
Espectrometría de Masas en Tándem
/
Amiloide
Límite:
Humans
Idioma:
En
Revista:
J Am Soc Mass Spectrom
Año:
2020
Tipo del documento:
Article
País de afiliación:
Reino Unido