RESUMO
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.
Assuntos
Peptídeos beta-Amiloides/análise , alfa-Sinucleína/análise , Sequência de Aminoácidos , Substituição de Aminoácidos , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/efeitos da radiação , Animais , Anticorpos Monoclonais/metabolismo , Afinidade de Anticorpos , Técnicas Biossensoriais , Encéfalo/metabolismo , Ciclotrons , Epitopos , Estudos de Viabilidade , Análise de Fourier , Humanos , Espectrometria de Massas , Camundongos Transgênicos , Técnicas Analíticas Microfluídicas , Peso Molecular , Proteínas Mutantes/análise , Proteínas Mutantes/química , Proteínas Mutantes/efeitos da radiação , Neurônios/química , Proteínas Recombinantes/análise , Proteínas Recombinantes/química , Proteínas Recombinantes/efeitos da radiação , Som , alfa-Sinucleína/química , alfa-Sinucleína/genética , alfa-Sinucleína/efeitos da radiaçãoRESUMO
Protein amyloidogenesis is generally considered to be a major cause of two most severe neurodegenerative disorders, Parkinson's disease (PD) and Alzheimer's disease (AD). Formation and accumulation of fibrillar aggregates and plaques derived from α-synuclein (α-Syn) and ß-amyloid (Aß) polypeptide in brain have been recognized as characteristics of Parkinson's disease and Alzheimer's disease. Oligomeric aggregates of α-Syn and Aß are considered as neurotoxic intermediate products leading to progressive neurodegeneration. However, molecular details of the oligomerization and aggregation pathway(s) and the molecular structure details are still unclear. We describe here the application of ion-mobility mass spectrometry (IMS-MS) to the identification of α-Syn and Aß oligomerization-aggregation products, and to the characterization of different conformational forms. IMS-MS is an analytical technique capable of separating gaseous ions based on their size, shape, and topography. IMS-MS studies of soluble α-Syn and Aß-aggregates prepared by in vitro incubation over several days were performed on a quadrupole time of flight mass spectrometer equipped with a "travelling wave" ion mobility cell, and revealed the presence of different conformational states and, remarkably, truncation and proteolytic products of high aggregating reactivity. These results suggest that different polypeptide sequences may contribute to the formation of oligomeric aggregates of heterogeneous composition and distinct biochemical properties.
Assuntos
Peptídeos beta-Amiloides/química , Espectrometria de Massas/métodos , Fragmentos de Peptídeos/química , Multimerização Proteica , alfa-Sinucleína/química , Sequência de Aminoácidos , Dados de Sequência Molecular , Estrutura Secundária de ProteínaRESUMO
We describe here an on-line combination of a surface acoustic wave (SAW) biosensor with electrospray ionization mass spectrometry (SAW-ESI-MS) that enables the direct detection, identification, and quantification of affinity-bound ligands from a protein-ligand complex on a biosensor chip. A trapping column was used between the SAW-biosensor and the electrospray mass spectrometer equipped with a micro-guard column, which provides simultaneous sample concentration and desalting for the mass spectrometric analysis of the dissociated ligand. First applications of the on-line SAW-ESI-MS combination include (1), differentiation of ß-amyloid (Aß) epitope peptides bound to anti-Aß antibodies; (2), the identification of immobilized Substance P peptide-calmodulin complex; (3), identification and quantification of the interaction of 3-nitrotyrosine-modified peptides with nitrotyrosine-specific antibodies; and (4), identification of immobilized anti-α-synuclein-human α-synuclein complex. Quantitative determinations of protein-ligand complexes by SAW yielded dissociation constants (K(D)) from micro-to low nanomolar sample concentrations. The on-line bioaffinity-ESI-MS combination presented here is expected to enable broad bioanalytical application to the simultaneous, label-free determination and quantification of biopolymer-ligand interactions, as diverse as antigen-antibody and lectin-carbohydrate complexes.