Applications of multi-angle laser light-scattering detection in the analysis of peptides and proteins.
ABSTRACT The proliferation of new peptides and proteins requiring characterisation is a direct result of recent advances in genomics and proteomics, but protein aggregation is particular problem in the biotechnology industry, where aggregation is encountered throughout the lifetime of a therapeutic protein, including during refolding, purification, sterilization, shipping, and storage process. To ensure that it meets quality standards, the size, molecular weight and/or molecular weight distribution, and aggregate state must be accurately determined. Traditional analytical methods for determining molecular weight include size-exclusion chromatography (SEC), gel electrophoresis, analytical ultracentrifugation and time-of-flight mass spectrometry. These technologies are time-consuming (some take days), provide data based on relative standards, or cannot characterise very high molecular weight aggregates. Laser light-scattering (LS) detection coupled with SEC system have been used for over a decade to determine the size and molecular weight of bio-molecules such as proteins, peptides, polysaccharides, oligonucleotides, and antibodies, the method of choice being for molar mass determinations and the study of self-association and heterogeneous interaction under native, equilibrium conditions in solution. The purpose of the current review is to describe and discuss the capability of the SEC/LS system to determine absolute molecular weight of proteins and their complexes and the association state of the conjugate, either with itself or with protein receptor/ligands. For this, the "two or three detector" methods, each with its advantages and limitations, can be used to calculate the molecular weight of a simple protein or glycoprotein, and the stoichiometry of their complexes. Also, some alternative techniques for determining the molecular weight are discussed in this review. Applications of all these methodologies are described.
Article: Structure-function relationships of pre-fibrillar protein assemblies in Alzheimer's disease and related disorders.[show abstract] [hide abstract]
ABSTRACT: Several neurodegenerative diseases, including Alzheimer's, Parkinson's, Huntington's and prion diseases, are characterized pathognomonically by the presence of intra- and/or extracellular lesions containing proteinaceous aggregates, and by extensive neuronal loss in selective brain regions. Related non-neuropathic systemic diseases, e.g., light-chain and senile systemic amyloidoses, and other organ-specific diseases, such as dialysis-related amyloidosis and type-2 diabetes mellitus, also are characterized by deposition of aberrantly folded, insoluble proteins. It is debated whether the hallmark pathologic lesions are causative. Substantial evidence suggests that these aggregates are the end state of aberrant protein folding whereas the actual culprits likely are transient, pre-fibrillar assemblies preceding the aggregates. In the context of neurodegenerative amyloidoses, the proteinaceous aggregates may eventuate as potentially neuroprotective sinks for the neurotoxic, oligomeric protein assemblies. The pre-fibrillar, oligomeric assemblies are believed to initiate the pathogenic mechanisms that lead to synaptic dysfunction, neuronal loss, and disease-specific regional brain atrophy. The amyloid beta-protein (Abeta), which is believed to cause Alzheimer's disease (AD), is considered an archetypal amyloidogenic protein. Intense studies have led to nominal, functional, and structural descriptions of oligomeric Abeta assemblies. However, the dynamic and metastable nature of Abeta oligomers renders their study difficult. Different results generated using different methodologies under different experimental settings further complicate this complex area of research and identification of the exact pathogenic assemblies in vivo seems daunting. Here we review structural, functional, and biological experiments used to produce and study pre-fibrillar Abeta assemblies, and highlight similar studies of proteins involved in related diseases. We discuss challenges that contemporary researchers are facing and future research prospects in this demanding yet highly important field.Current Alzheimer Research 07/2008; 5(3):319-41. · 3.95 Impact Factor