skip to main content

This content will become publicly available on January 28, 2023

Title: Characterizing Soluble Protein Aggregates Using Native Mass Spectrometry Coupled with Temperature-Controlled Electrospray Ionization and Size-Exclusion Chromatography
Characterization of soluble protein aggregates provides valuable information for revealing mechanisms of protein aggregation process and assessing the activity and safety of protein therapeutics. However, the noncovalent interaction, the transient nature and higher degree of structural heterogeneity of the soluble aggregation system hinders precise characterization at the molecular level. Here, we describe methods using native mass spectrometry coupled with temperature-control electrospray ionization and size-exclusion chromatography to monitor the aggregation process and profile the aggregates in detail.
; ;
Garcia Fruitós, E.; Arís Giralt, A.
Award ID(s):
Publication Date:
Journal Name:
Methods in molecular biology
Page Range or eLocation-ID:
Sponsoring Org:
National Science Foundation
More Like this
  1. Abstract

    Amyloid fibril formation is central to the etiology of a wide range of serious human diseases, such as Alzheimer’s disease and prion diseases. Despite an ever growing collection of amyloid fibril structures found in the Protein Data Bank (PDB) and numerous clinical trials, therapeutic strategies remain elusive. One contributing factor to the lack of progress on this challenging problem is incomplete understanding of the mechanisms by which these locally ordered protein aggregates self-assemble in solution. Many current models of amyloid deposition diseases posit that the most toxic species are oligomers that form either along the pathway to forming fibrilsmore »or in competition with their formation, making it even more critical to understand the kinetics of fibrillization. A recently introduced topological model for aggregation based on network Hamiltonians is capable of recapitulating the entire process of amyloid fibril formation, beginning with thousands of free monomers and ending with kinetically accessible and thermodynamically stable amyloid fibril structures. The model can be parameterized to match the five topological classes encompassing all amyloid fibril structures so far discovered in the PDB. This paper introduces a set of network statistical and topological metrics for quantitative analysis and characterization of the fibrillization mechanisms predicted by the network Hamiltonian model. The results not only provide insight into different mechanisms leading to similar fibril structures, but also offer targets for future experimental exploration into the mechanisms by which fibrils form.

    « less
  2. Amyloids are fibrous cross-β protein aggregates that are capable of proliferation via nucleated polymerization. Amyloid conformation likely represents an ancient protein fold and is linked to various biological or pathological manifestations. Self-perpetuating amyloid-based protein conformers provide a molecular basis for transmissible (infectious or heritable) protein isoforms, termed prions. Amyloids and prions, as well as other types of misfolded aggregated proteins are associated with a variety of devastating mammalian and human diseases, such as Alzheimer's, Parkinson's and Huntington's diseases, transmissible spongiform encephalopathies (TSEs), amyotrophic lateral sclerosis (ALS) and transthyretinopathies. In yeast and fungi, amyloid-based prions control phenotypically detectable heritable traits. Simplicitymore »of cultivation requirements and availability of powerful genetic approaches makes yeast Saccharomyces cerevisiae an excellent model system for studying molecular and cellular mechanisms governing amyloid formation and propagation. Genetic techniques allowing for the expression of mammalian or human amyloidogenic and prionogenic proteins in yeast enable researchers to capitalize on yeast advantages for characterization of the properties of disease-related proteins. Chimeric constructs employing mammalian and human aggregation-prone proteins or domains, fused to fluorophores or to endogenous yeast proteins allow for cytological or phenotypic detection of disease-related protein aggregation in yeast cells. Yeast systems are amenable to high-throughput screening for antagonists of amyloid formation, propagation and/or toxicity. This review summarizes up to date achievements of yeast assays in application to studying mammalian and human disease-related aggregating proteins, and discusses both limitations and further perspectives of yeast-based strategies.« less
  3. Abstract The founding principles of protein folding introduced by Christian Anfinsen, together with the numerous mechanistic investigations that followed, assume that protein folding is a thermodynamically controlled process. On the other hand, this review underscores the fact that thermodynamic control is far from being the norm in protein folding, as long as one considers an extended chemical-potential landscape encompassing aggregates, in addition to native, unfolded and intermediate states. Here, we highlight the key role of kinetic trapping of the protein native state relative to unfolded, intermediate and, most importantly, aggregated states. We propose that kinetic trapping serves an important rolemore »in biology by protecting the bioactive states of a large number of proteins from deleterious aggregation. In the event that undesired aggregates were somehow formed, specialized intracellular disaggregation machines have evolved to convert any aberrant populations back to the native state, thus restoring a fully bioactive and aggregation-protected protein cohort.« less
  4. Aggregation of misfolded oligomeric amyloid-beta (Aβ) peptides on lipid membranes has been identified as a primary event in Alzheimer's pathogenesis. However, the structural and dynamical features of this membrane assisted Aβ aggregation have not been well characterized. The microscopic characterization of dynamic molecular-level interactions in peptide aggregation pathways has been challenging both computationally and experimentally. In this work, we explore differential patterns of membrane-induced Aβ 16–22 (K–L–V–F–F–A–E) aggregation from the microscopic perspective of molecular interactions. Physics-based coarse-grained molecular dynamics (CG-MD) simulations were employed to investigate the effect of lipid headgroup charge – zwitterionic (1-palmitoyl-2-oleoyl- sn-glycero -3-phosphocholine: POPC) and anionic (1-palmitoyl-2-oleoyl-more »sn-glycero -3-phospho- l -serine: POPS) – on Aβ 16–22 peptide aggregation. Our analyses present an extensive overview of multiple pathways for peptide absorption and biomechanical forces governing peptide folding and aggregation. In agreement with experimental observations, anionic POPS molecules promote extended configurations in Aβ peptides that contribute towards faster emergence of ordered β-sheet-rich peptide assemblies compared to POPC, suggesting faster fibrillation. In addition, lower cumulative rates of peptide aggregation in POPS due to higher peptide–lipid interactions and slower lipid diffusion result in multiple distinct ordered peptide aggregates that can serve as nucleation seeds for subsequent Aβ aggregation. This study provides an in-silico assessment of experimentally observed aggregation patterns, presents new morphological insights and highlights the importance of lipid headgroup chemistry in modulating the peptide absorption and aggregation process.« less
  5. During the past decade, cellulose nanofibrils (CNFs) have shown tremendous potential as a building block to fabricate new advanced materials that are both biocompatible and biodegradable. The excellent mechanical properties of the individual CNF can be transferred to macroscale fibers through careful control in hydrodynamic alignment and assembly processes. The optimization of such processes relies on the understanding of nanofibril dynamics during the process, which in turn requires in situ characterization. Here, we use a shear-free mixing experiment combined with scanning small-angle X-ray scattering (scanning-SAXS) to provide time-resolved nanoscale kinetics during the in situ assembly of dispersed cellulose nanofibrils (CNFs)more »upon mixing with a sodium chloride solution. The addition of monovalent ions led to the transition to a volume-spanning arrested (gel) state. The transition of CNFs is associated with segmental aggregation of the particles, leading to a connected network and reduced Brownian motion, whereby an aligned structure can be preserved. Furthermore, we find that the extensional flow seems to enhance the formation of these segmental aggregates, which in turn provides a comprehensible explanation for the superior material properties obtained in shear-free processes used for spinning filaments from CNFs. This observation clearly highlights the need for different assembly strategies depending on morphology and interactions of the dispersed nanoparticles, where this work can be used as a guide for improved nanomaterial processes.« less