No abstract

We describe here experiments designed to characterize the secondary structure of amyloid fibrils of the Alzheimer's amyloid plaque peptide A␤, using hydrogen-deuterium exchange measurements evaluated by mass spectrometry. The results show that Ϸ50% of the amide protons of the polypeptide backbone of A␤(1-40) resist exchange in aqueous, neutral pH buffer even after more than 1,000 h of incubation at room temperature. We attribute this extensive, strong protection to H-bonding by residues in core regions of ␤-sheet structure within the fibril. The backbone amide hydrogens exchange at variable rates, suggesting different degrees of protection within the fibril. These data suggest that it is unlikely that the entire A␤ sequence is involved in H-bonded secondary structure within the amyloid fibril. Future studies using the methods described here should reveal further details of A␤ fibril structure and assembly. These methods also should be amenable to studies of other amyloid fibrils and protein aggregates.

No abstract

The term "wrong-way-round ionization" has been used in studies of electrospray ionization to describe the observation of protonated or deprotonated ions when sampling strongly basic or acidic solutions (respectively) where such ions are not expected to exist in appreciable concentrations in solution. Study of the dependence of ionization of the weak base caffeine on the electrospray capillary potential reveals three distinct contributors to wrong-way-round ionization. At near-neutral pH in solutions of low ionic strength, protonation of caffeine results from the surface enrichment of electrolytically produced protons in the surface layer of the droplets from which ions are desorbed. For solutions made strongly basic with ammonia, gas-phase proton transfer from ammonium ions can create protonated caffeine. These two mechanisms have been discussed previously elsewhere. For solutions of high ionic strength at neutral or high pH, the data suggest that discharge-induced ionization is responsible for the production of protonated caffeine. This mechanism probably accounts for some of the wrong-way-round ionization reported elsewhere.

Metastable oligomeric and protofibrillar forms of amyloidogenic proteins have been implicated as on-pathway assembly intermediates in amyloid formation and as the major toxic species in a number of amyloid diseases including Alzheimer's disease. We describe here a chemical biology approach to structural analysis of A␤ protofibrils. Library screening yielded several molecules that stimulate A␤ aggregation. One of these compounds, calmidazolium chloride (CLC), rapidly and efficiently converts A␤(1-40) monomers into clusters of protofibrils. As monitored by electron microscopy, these protofibrils persist for days when incubated in PBS at 37°C, with a slow transition to fibrillar structures apparent only after several weeks. Like normal protofibrils, the CLC-A␤ aggregates exhibit a low thioflavin T response. Like A␤ fibrils, the clustered protofibrils bind the anti-amyloid Ab WO1. The CLC-A␤ aggregates exhibit the same protection from hydrogen-deuterium exchange as do protofibrils isolated from a spontaneous A␤ fibril formation reaction: Ϸ12 of the 39 A␤(1-40) backbone amide protons are protected from exchange in the protofibril, compared with approximately twice that number in amyloid fibrils. Scanning proline mutagenesis analysis shows that the A␤ molecule in these protofibrillar assemblies exhibits the same flexible N and C termini as do mature amyloid fibrils. The major difference in A␤ conformation between fibrils and protofibrils is added structural definition in the 22-29 segment in the fibril. Besides aiding structural analysis, compounds capable of facilitating oligomer and protofibril formation might have therapeutic potential, if they act to sequester A␤ in a form and͞or location that cannot engage the toxic pathway.amyloid ͉ chemical biology ͉ hydrogen exchange ͉ proline scanning

A simple procedure for preparing gold-coated silica capillaries for use in electrospray ionization mass spectrometry is described. The tip of the capillary is mechanically tapered to a fine point, and a thin film of gold is vapor deposited on the outer surface following treatment with an organofunctional silane. The performance characteristics of these durable capillaries as continuous infusion sources are examined, and their utility in on-line capillary electrophoresis mass spectrometry is demonstrated.

Gradual corrosion of stainless steel electrospray emitters under conditions of normal use generates surface irregularities that can promote electrical discharge. The increased emission current affects the electrochemical reactions associated with the spray process. When sampling the peptide Aβ(1-40), this is manifest by oxidation of methionine at position 35 to methionine sulfoxide. The resultant mass shift and reduced sensitivity can adversely affect H/D exchange experiments. These effects can be avoided by adding a redox buffer or (preferably) by re-polishing the emitter, especially to a rounded geometry.

A model has been developed to account qualitatively for the effects of ion pairing, surface activity, and electrophoretic mobility in electrospray mass spectrometry. The model is tested with various salt and amino acid mixtures. The data suggest that the axial charge gradient arising from electrophoretic separation at droplet genesis may persist within the electrosprayed droplets at least until the first droplet fission, accounting for the field dependence of detected ion clustering of quaternary ammonium salts and for the relatively field-invariant charge distribution of horse heart myoglobin samples.