Structural Analysis of [Cu(II)-amyloidogenic peptide] Complexes

Abstract

Studies on the interactions of amyloidogenic proteins with trace metals, such as copper, have indicated that the metal ions perform a critical function in the early oligomerization process. Herein, we investigate the effects of Cu(II) ions on the active sequence regions of amyloidogenic proteins using electrospray ionization mass spectrometry (ESI-MS) and collision induced dissociation tandem MS (CID-MS/MS). We chose three amyloidogenic peptides NNQQNY, LYQLEN, and VQIVYK from yeast prion like protein Sup35, insulin chain A, and tau protein, respectively. [Cu-peptide] complexes for all three peptides were observed in the mass spectra. The mass spectra also show that increasing Cu(II) concentrations decrease the population of existing peptide oligomers. The tandem mass spectrum of NNQQNY shows preferential binding for the N-terminal region. All three peptides are likely to appear to be in a Cu-monomer-monomer (Cu-M-M) structure instead of a monomer-Cu-monomer (M-Cu-M) structure.

Figure 1. Comparison of full mass spectra at a) 1:0, b) 1:0.1, and c) 1:1 ratios of [peptide]:[Cu] of the active sequences NNQQNY. Allpeaks with m/z values greater than 1000 were magnified by a factor of 20. Magnified NNQQNY [2M+H]1+ peaks are presented asinsets.

Figure 2. MS/MS spectra of singly and doubly charged monomer complexes of NNQQNY, LYQLEN, and VQIVYK peptides with abound Cu(II) ion. [M+Cu-H]1+ of a) NNQQNY, c) LYQLEN, and e) VQIVYK. [M+Cu]2+ of b) NNQQNY, d) LYQLEN, and f)VQIVYK. Peaks labeled -18 likely result from the loss of an H2O moiety. Peaks labeled -28 result from the loss of an additional COmoiety and would conventionally be labeled as an ions. Peaks labeled -44 likely result from the loss of a CO2 moiety. bt fragments areexpressed in blue and bold and yn fragments are expressed in red and bold.

Figure 3. MS/MS spectra of singly and doubly charged dimer complexes of NNQQNY with a bound Cu(II) ion. a) [2M+Cu-H]1+ and b)[2M+Cu]2+. MS/MS/MS spectra of (bt+Cu) 1+ fragments observed in b) spectrum. c) (b5+Cu) 1+ and d) (b4+Cu) 1+.

Table 1. Summary of the nomenclature used for MS/MS spectra. Italics indicate conventional notation as proposed by Roepstorff andFohlman.28 Non-italicized bt would conventionally be represented as a neutral species [bt]0. Non-italicized yn would be represented as[yn”-2H]0.

Table 2. Comparison of [Cu-peptide] MS/MS fragmentationpatterns for NNQQNY, LYQLEN, and VQIVYK.