Ultrasonication-dependent formation and degradation of α-synuclein amyloid fibrils.

Ultrasonication can be used to break the supersaturation of α-synuclein, a protein associated with Parkinson's disease, at pH7.4 above the critical concentration of fibrillation, thereby inducing the formation of amyloid fibrils. We speculated that ultrasonication could also be used to depolymerize preformed fibrils below the critical concentration. However, extensive ultrasonic irradiation transformed preformed fibrils into amorphous aggregates even above the critical concentration. Exposing preformed fibrils to the hydrophobic air-water interface of cavitation bubbles may have destabilized the fibrils and stabilized amorphous aggregates. Upon extensive ultrasonic irradiation, the accompanying decomposition of chemical structures was suggested when monitored by analytical ultracentrifugation. Amorphous aggregates produced by extensive ultrasonication showed higher cytotoxicity, suggesting that, although ultrasonication might be a useful approach for inactivating amyloid fibrils, potential cytotoxicity of amorphous aggregates should be considered.

The early events of alpha-synuclein oligomerization revealed by photo-induced cross-linking.

Assembly of alpha-synuclein (alpha-Syn) into neurotoxic oligomers and fibrils is an important pathogenic feature of Parkinson's disease. Studying the early events of alpha-Syn aggregation, such as oligomerization and nucleation, is indispensable to understanding the complicated process. Here, photo-induced cross-linking of unmodified proteins (PICUP) technique is applied to elucidate the early-stage oligomerization of alpha-Syn. Results show that alpha-Syn in solution exhibits a mixture of various species, including at least monomers, dimers and trimers. Aggregation of alpha-Syn probably originates from the dimeric and trimeric seeds. Furthermore, the N-terminal amphipathic region is proposed to be required for the oligomerization (dimerization and trimerization) process. This observation may extend our knowledge on the early events of alpha-Syn aggregation and the neurotoxic aggregation species.

Identification and affinity-quantification of ß-amyloid and α-synuclein polypeptides using on-line SAW-biosensor-mass spectrometry.

Bioaffinity analysis using a variety of biosensors has become an established tool for detection and quantification of biomolecular interactions. Biosensors, however, are generally limited by the lack of chemical structure information of affinity-bound ligands. On-line bioaffinity-mass spectrometry using a surface-acoustic wave biosensor (SAW-MS) is a new combination providing the simultaneous affinity detection, quantification, and mass spectrometric structural characterization of ligands. We describe here an on-line SAW-MS combination for direct identification and affinity determination, using a new interface for MS of the affinity-isolated ligand eluate. Key element of the SAW-MS combination is a microfluidic interface that integrates affinity-isolation on a gold chip, in-situ sample concentration, and desalting with a microcolumn for MS of the ligand eluate from the biosensor. Suitable MS-acquisition software has been developed that provides coupling of the SAW-MS interface to a Bruker Daltonics ion trap-MS, FTICR-MS, and Waters Synapt-QTOF- MS systems. Applications are presented for mass spectrometric identifications and affinity (K(D)) determinations of the neurodegenerative polypeptides, ß-amyloid (Aß), and pathophysiological and physiological synucleins (α- and ß-synucleins), two key polypeptide systems for Alzheimer's disease and Parkinson's disease, respectively. Moreover, first in vivo applications of αSyn polypeptides from brain homogenate show the feasibility of on-line affinity-MS to the direct analysis of biological material. These results demonstrate on-line SAW-bioaffinity-MS as a powerful tool for structural and quantitative analysis of biopolymer interactions.