23 research outputs found
Ion mobility mass spectrometry uncovers the impact of the patterning of oppositely charged residues on the conformational distributions of intrinsically disordered proteins
The global dimensions and amplitudes of conformational fluctuations of intrinsically disordered proteins are governed, in part, by the linear segregation versus clustering of oppositely charged residues within the primary sequence. Ion mobility-mass spectrometry (IM-MS) affords unique advantages for probing the conformational consequences of the linear patterning of oppositely charged residues because it measures and separates proteins electrosprayed from solution on the basis of charge and shape. Here, we use IM-MS to measure the conformational consequences of charge patterning on the C-terminal intrinsically disordered region (p27 IDR) of the cell cycle inhibitory protein p27Kip1. We report the range of charge states and accompanying collisional cross section distributions for wild-type p27 IDR and two variants with identical amino acid compositions, Îș14 and Îș56, distinguished by the extent of linear mixing versus segregation of oppositely charged residues. Wild-type p27 IDR (Îș31) and Îș14, where the oppositely charged residues are more evenly distributed, exhibit a broad distribution of charge states. This is concordant with high degrees of conformational heterogeneity in solution. By contrast, Îș56 with linear segregation of oppositely charged residues leads to limited conformational heterogeneity and a narrow distribution of charged states. Gas-phase molecular dynamics simulations demonstrate that the interplay between chain solvation and intrachain interactions (self-solvation) leads to conformational distributions that are modulated by salt concentration, with the wild-type sequence showing the most sensitivity to changes in salt concentration. These results suggest that the charge patterning within the wild-type p27 IDR may be optimized to sample both highly solvated and self-solvated conformational states
Lysolipids are prominent in subretinal drusenoid deposits, a high-risk phenotype in age-related macular degeneration
IntroductionAge related macular degeneration (AMD) causes legal blindness worldwide, with few therapeutic targets in early disease and no treatments for 80% of cases. Extracellular deposits, including drusen and subretinal drusenoid deposits (SDD; also called reticular pseudodrusen), disrupt cone and rod photoreceptor functions and strongly confer risk for advanced disease. Due to the differential cholesterol composition of drusen and SDD, lipid transfer and cycling between photoreceptors and support cells are candidate dysregulated pathways leading to deposit formation. The current study explores this hypothesis through a comprehensive lipid compositional analysis of SDD.MethodsHistology and transmission electron microscopy were used to characterize the morphology of SDD. Highly sensitive tools of imaging mass spectrometry (IMS) and nano liquid chromatography tandem mass spectrometry (nLC-MS/MS) in positive and negative ion modes were used to spatially map and identify SDD lipids, respectively. An interpretable supervised machine learning approach was utilized to compare the lipid composition of SDD to regions of uninvolved retina across 1873 IMS features and to automatically discern candidate markers for SDD. Immunohistochemistry (IHC) was used to localize secretory phospholipase A2 group 5 (PLA2G5). ResultsAmong the 1873 detected features in IMS data, three lipid classes, including lysophosphatidylcholine (LysoPC), lysophosphatidylethanolamine (LysoPE) and lysophosphatidic acid (LysoPA) were observed nearly exclusively in SDD while presumed precursors, including phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidic acid (PA) lipids were detected in SDD and adjacent photoreceptor outer segments. Molecular signals specific to SDD were found in central retina and elsewhere. IHC results indicated abundant PLA2G5 in photoreceptors and retinal pigment epithelium (RPE). DiscussionThe abundance of lysolipids in SDD implicates lipid remodeling or degradation in deposit formation, consistent with ultrastructural evidence of electron dense lipid-containing structures distinct from photoreceptor outer segment disks and immunolocalization of secretory PLA2G5 in photoreceptors and RPE. Further studies are required to understand the role of lipid signals observed in and around SDD
The potential of ion mobility mass spectrometry for tuning synthetic host guest systems: A case study using novel zinc(II)dipicolylamine anion sensors
Ion Mobility Mass Spectrometry Measures the Conformational Landscape of p27 and its Domains and how this is Modulated upon Interaction with Cdk2/cyclinâ A
Intrinsically disordered proteins have been reported to undergo âdisorder to orderâ transitions upon binding to their partners in the cell. The extent of the ordering on binding and the lack of order prior to binding is difficult to visualize with classical structure determination methods. Binding of p27 to the Cdk2/cyclin A complex is accompanied by partial folding of p27 in the KID domain, with the retention of dynamic behaviour for function, particularly in the C-terminal half of the protein, positioning it as an exemplary system to probe conformational diversity. Here we employ native ion mobility with mass spectrometry (IM-MS) to measure the intrinsic dynamic properties of p27, both in isolation and within the trimeric complex with Cdk2/cyclin A. This stepwise approach reveals the conformational distributions of the constituent proteins and how they are restructured on complex formation; the trimeric Cdk2/cyclin A/p27-KID complex possesses significant structural heterogeneity cf. Cdk2/cyclin A. These findings support the formation of a fuzzy complex in which both the N and C termini of p27 interact with Cdk2/cyclin A in multiple closely associated states
Using Collision Cross Section Distributions to Assess the Distribution of Collision Cross Section Values
The potential of ion mobility mass spectrometry for tuning synthetic host guest systems::A case study using novel zinc(II)dipicolylamine anion sensors
Hybrid Mass Spectrometry Methods Reveal Lot-to-Lot Differences and Delineate the Effects of Glycosylation on the Structure of HerceptinÂź
To consider the
measurable variations in biopharmaceuticals we use mass spectrometry and systematically
evaluate three lots of HerceptinÂź, two mAb standards and an intact Fc-hinge
fragment. Each mAb is examined in three states; glycan intact, truncated (following
endoS2 treatment) and fully deglycosylated. Despite equivalence at the protein level, each
lot of HerceptinÂź gives a distinctive signature in three different mass
spectrometry analyses. Ion mobility mass
spectrometry (IM-MS) shows that in the API, the attached N-glycans reduce the
conformational spread of each mAb by 10.5 â 25 %. Hydrogen/deuterium exchange
mass spectrometry (HDX-MS) data supports this, with lower global deuterium
uptake in solution when comparing intact to the fully deglycosylated protein. HDX-MS
and activated IM-MS map the influence of glycans on the mAb and reveal
allosteric effects which extend far beyond the Fc domains into the Fab region. Taken
together these findings, and the supplied interactive data sets could be used
to provide acceptance criteria with application for MS based characterisation of
biosimilars and novel therapeutic mAbs. </p
Hybrid Mass Spectrometry Methods Reveal Lot-to-Lot Differences and Delineate the Effects of Glycosylation on the Tertiary Structure of Herceptin
Initial Steps of Amyloidogenic Peptide Assembly Revealed by Cold-Ion Spectroscopy
The early stages of fibril formation are difficult to capture in solution. We use cold-ion spectroscopy to examine an 11-residue peptide derived from the protein transthyretin and clusters of this fibre-forming peptide containing up to five units in the gas phase. For each oligomer, the UV spectra exhibit distinct changes in the electronic environment of aromatic residues in this peptide compared to that of the monomer and in the bulk solution. The UV spectra of the tetra- and pentamer are superimposable but differ significantly from the spectra of the monomer and trimer. Such a spectral evolution suggests that a common structural motif is formed as early as the tetramer. The presence of this stable motif is further supported by the low conformational heterogeneity of the tetra- and pentamer, revealed from their IR spectra. From comparison of the IR-spectra in the gas and condensed phases, we propose putative assignments for the dominant motif in the oligomers