A Mass-Spectrometry-Based Framework To Define the Extent of Disorder in Proteins

Abstract

In the past decade, mass spectrometry (MS) coupled with electrospray ionization (ESI) has been extensively applied to the study of intact proteins and their complexes, often without the requirement of labels. Solvent conditions (for example, pH, ionic strength, and concentration) affect the observed desolvated species; the ease of altering such extrinsic factors renders ESI-MS an appropriate method by which to consider the range of conformational states that proteins may occupy, including natively folded, disordered and amyloid. Rotationally averaged collision cross sections of the ionized forms of proteins, provided by the combination of mass spectrometry and ion mobility (IM-MS), are also instructive in exploring conformational landscapes in the absence of solvent. Here, we ask the following question: "If the only technique you had was ESI-IM-MS, what information would it provide on the structural preferences of an unknown protein?" We have selected 20 different proteins, both monomeric and multimeric, ranging in mass from 2846 Da (melittin) to 150 kDa (Immunoglobulin G), and we consider how they are presented to a mass spectrometer under different solvent conditions. Mass spectrometery allows us to distinguish which of these proteins are structured (melittin, human beta defensin 1, truncated human lymphotactin, Cytochrome C, holo hemoglobin-α, ovalbumin, human transthyretin, avidin, bovine serum albumin, concanavalin, human serum amyloid protein, and Immunoglobulin G) from those that contain at least some regions of disorder (human lymphotactin, N-terminal p53, α-Synuclein, N-terminal MDM2, and p53 DNA binding domain) or denatured due to solvent conditions (ubiquitin, apo hemoglobin-α, apo hemoglobin-β) by considering two experimental parameters: the range of charge states occupied by the protein (Δz) and the range of collision cross sections in which the protein is observed (ΔCCS). We also provide a simple model to predict the difference between the collision cross sections of the most compact and the most extended form of a given protein, based on the volume of the amino acids it contains. We compare these calculated parameters with experimental values. In addition, we consider the occupancy of conformations based on the intensities of ions in the mass spectra. This allows us to qualitatively predict the potential energy landscape of each protein. Our empirical approach to assess order or disorder is shown to be more accurate than the use of charge hydropathy plots, which are frequently used to predict disorder, and could provide an initial route to characterization. Finally, we present an ESI-IM-MS methodology to determine if a given protein is structured or disordered.

Distinct higher-order α-synuclein oligomers induce intracellular aggregation

“…Increasing the activation energies also resulted in increased as the protein unfolded (results not shown), demonstrating that the proteins retain a native like conformation within our experiments. The monomeric form of α-syn is considered a natively disordered protein as it displays considerable conformational heterogeneity [35,36]. Analysis of WT α-syn before the addition of cofactors and ethanol revealed a primarily extended population (charge state ions + 8 to + 18) and a sub-population of a more compact conformational series (charge state ions + 6 to + 8) with multiple overlapping features consistent with previous data [34,37] (Figure 1).…”

Structural characterization of encapsulated ferritin provides insight into iron storage in bacterial nanocompartments

“…In addition, many of the charge states observed have higher charge than the theoretical maximal charge on spherical globular protein, as determined by the De La Mora relationship ( Z R = 0.0778$\surd$ m ; for the EncFtn sH monomer Z R = 8.9) Fernandez (Fernandez de la Mora, 2000). As described by Beveridge et al, all these factors are indicative of a disordered protein (Beveridge et al, 2014). DOI: http://dx.doi.org/10.7554/eLife.18972.019…”

“…By contrast, IM-MS measurements of the monomeric EncFtn sH at pH 8.0 under the same instrumental conditions revealed that the metal-free protein monomer exists in a wide range of charge states (+6 to +16) and adopts many conformations in the gas phase with collision cross sections ranging from 12 nm 2  to 26 nm 2 (Figure 7—figure supplement 1). These observations are indicative of an unstructured protein with little secondary or tertiary structure (Beveridge et al, 2014). Thus, IM-MS studies highlight that higher order structure in EncFtn sH is mediated/stabilized by metal binding, an observation that is in agreement with our solution studies.…”

Ensemble Methods Enable a New Definition for the Solution to Gas-Phase Transfer of Intrinsically Disordered Proteins

“…19 Overall, gas-phase methods appear uniquely suited to meet the challenge of describing the conformations and dynamics of flexible proteins as evidenced by the increasing number of publications in this area in recent years. [20][21][22][23][24] Nevertheless, recent studies describing the collapse of disordered structures in the absence of solvent have raised questions over the fidelity to which gas-phase methods can capture the solution conformations of these proteins. 25,26 Recent shifts in our understanding of how flexible species escape electrospray droplets during ESI also add to the complexity of describing the solution to gas-phase transfer of IDPs.…”