Eye lens ß-crystallins are predicted by native ion mobility-mass spectrometry and computations to form compact higher-ordered heterooligomers.

Eye lens α- and ß-/γ-crystallin proteins are not replaced after fiber cell denucleation and maintain lens transparency and refractive properties. The exceptionally high (∼400-500 mg/mL) concentration of crystallins in mature lens tissue and multiple other factors impede precise characterization of ß-crystallin interactions, oligomer composition, size, and topology. Native ion mobility-mass spectrometry is used here to probe ß-crystallin association and provide insight into homo- and heterooligomerization kinetics for these proteins. These experiments include separation and characterization of higher-order ß-crystallin oligomers and illustrate the unique advantages of native IM-MS. Recombinantly expressed ßB1, ßB2, and ßA3 isoforms are found to have different homodimerization propensities, and only ßA3 forms larger homooligomers. Heterodimerization of ßB2 with ßA3 occurs ∼3 times as fast as that of ßB1 with ßA3, and ßB1 and ßB2 heterodimerize less readily. Ion mobility experiments, molecular dynamics simulations, and PISA analysis together reveal that observed oligomers are consistent with predominantly compact, ring-like topologies.

MZA: A Data Conversion Tool to Facilitate Software Development and Artificial Intelligence Research in Multidimensional Mass Spectrometry.

Modern mass spectrometry-based workflows employing hybrid instrumentation and orthogonal separations collect multidimensional data, potentially allowing deeper understanding in omics studies through adoption of artificial intelligence methods. However, the large volume of these rich spectra challenges existing data storage and access technologies, therefore precluding informatics advancements. We present MZA (pronounced m-za), the mass-to-charge (m/z) generic data storage and access tool designed to facilitate software development and artificial intelligence research in multidimensional mass spectrometry measurements. Composed of a data conversion tool and a simple file structure based on the HDF5 format, MZA provides easy, cross-platform and cross-programming language access to raw MS-data, enabling fast development of new tools in data science programming languages such as Python and R. The software executable, example MS-data and example Python and R scripts are freely available at https://github.com/PNNL-m-q/mza.

Protein shape sampled by ion mobility mass spectrometry consistently improves protein structure prediction.

Ion mobility (IM) mass spectrometry provides structural information about protein shape and size in the form of an orientationally-averaged collision cross-section (CCSIM). While IM data have been used with various computational methods, they have not yet been utilized to predict monomeric protein structure from sequence. Here, we show that IM data can significantly improve protein structure determination using the modelling suite Rosetta. We develop the Rosetta Projection Approximation using Rough Circular Shapes (PARCS) algorithm that allows for fast and accurate prediction of CCSIM from structure. Following successful testing of the PARCS algorithm, we use an integrative modelling approach to utilize IM data for protein structure prediction. Additionally, we propose a confidence metric that identifies near native models in the absence of a known structure. The results of this study demonstrate the ability of IM data to consistently improve protein structure prediction.

Measurement and Theory of Gas-Phase Ion Mobility Shifts Resulting from Isotopomer Mass Distribution Changes.

The unanticipated discovery of recent ultra-high-resolution ion mobility spectrometry (IMS) measurements revealing that isotopomers─compounds that differ only in the isotopic substitution sites─can be separated has raised questions as to the physical basis for their separation. A study comparing IMS separations for two isotopomer sets in conjunction with theory and simulations accounting for ion rotational effects provides the first-ever prediction of rotation-mediated shifts. The simulations produce observable mobility shifts due to differences in gas-ion collision frequency and translational-to-rotational energy transfer. These differences can be attributed to distinct changes in the moment of inertia and center of mass between isotopomers. The simulations are in broad agreement with the observed experiments and consistent with relative mobility differences between isotopomers. These results provide a basis for refining IMS theory and a new foundation to obtain additional structural insights through IMS.

Lipid Head Group Adduction to Soluble Proteins Follows Gas-Phase Basicity Predictions: Dissociation Barriers and Charge Abstraction.

Native mass spectrometry analysis of membrane proteins has yielded many useful insights in recent years with respect to membrane protein-lipid interactions, including identifying specific interactions and even measuring binding affinities based on observed abundances of lipid-bound ions after collision-induced dissociation (CID). However, the behavior of non-covalent complexes subjected to extensive CID can in principle be affected by numerous factors related to gas-phase chemistry, including gas-phase basicity (GB) and acidity, shared-proton bonds, and other factors. A recent report from our group showed that common lipids span a wide range of GB values. Notably, phosphatidylcholine (PC) and sphingomyelin lipids are more basic than arginine, suggesting they may strip charge upon dissociation in positive ion mode, while phosphoserine lipids are slightly less basic than arginine and may form especially strong shared-proton bonds. Here, we use CID to probe the strength of non-specific gas-phase interactions between lipid head groups and several soluble proteins, used to deliberately avoid possible physiological protein-lipid interactions. The strengths of the protein-head group interactions follow the trend predicted based solely on lipid and amino acid GBs: phosphoserine (PS) head group forms the strongest bonds with these proteins and out-competes the other head groups studied, while glycerophosphocholine (GPC) head groups form the weakest interactions and dissociate carrying away a positive charge. These results indicate that gas-phase thermochemistry can play an important role in determining which head groups remain bound to protein ions with native-like structures and charge states in positive ion mode upon extensive collisional activation.

Dynamic Time-Warping Correction for Shifts in Ultrahigh Resolving Power Ion Mobility Spectrometry and Structures for Lossless Ion Manipulations.

Detection of arrival time shifts between ion mobility spectrometry (IMS) separations can limit achievable resolving power (Rp), particularly when multiple separations are summed or averaged, as commonly practiced in IMS. Such variations can be apparent in higher Rp measurements and are particularly evident in long path length traveling wave structures for lossless ion manipulations (SLIM) IMS due to their typically much longer separation times. Here, we explore data processing approaches employing single value alignment (SVA) and nonlinear dynamic time warping (DTW) to correct for variations between IMS separations, such as due to pressure fluctuations, to enable more effective spectrum summation for improving Rp and detection of low-intensity species. For multipass SLIM IMS separations, where narrow mobility range measurements have arrival times that can extend to several seconds, the SVA approach effectively corrected for such variations and significantly improved Rp for summed separations. However, SVA was much less effective for broad mobility range separations, such as obtained with multilevel SLIM IMS. Changes in ions' arrival times were observed to be correlated with small pressure changes, with approximately 0.6% relative arrival time shifts being common, sufficient to result in a loss of Rp for summed separations. Comparison of the approaches showed that DTW alignment performed similarly to SVA when used over a narrow mobility range but was significantly better (providing narrower peaks and higher signal intensities) for wide mobility range data. We found that the DTW approach increased Rp by as much as 115% for measurements in which 50 IMS separations over 2 s were summed. We conclude that DTW is superior to SVA for ultra-high-resolution broad mobility range SLIM IMS separations and leads to a large improvement in effective Rp, correcting for ion arrival time shifts regardless of the cause, as well as improving the detectability of low-abundance species. Our tool is publicly available for use with universal ion mobility format (.UIMF) and text (.txt) files.

Increasing Collisional Activation of Protein Complexes Using Smaller Aperture Source Sampling Cones on a Synapt Q-IM-TOF Instrument with a Stepwave Source.

Quadrupole ion mobility time-of-flight (Q-IM-TOF) mass spectrometers have revolutionized investigation of native biomolecular complexes. High pressures in the sources of these instruments aid transmission of protein complexes through damping of kinetic energy by collisional cooling. As adducts are removed through collisional heating (declustering), excessive collisional cooling can prevent removal of nonspecific adducts from protein ions, leading to inaccurate mass measurements, broad mass spectral peaks, and obfuscation of ligand binding. We show that reducing the source pressure using smaller aperture source sampling cones (SC) in a Waters Synapt G2-Si instrument increases protein ion heating by decreasing collisional cooling, providing a simple way to enhance removal of adducted salts from soluble proteins (GroEL 14-mer) and detergents from a transmembrane protein complex (heptameric Staphylococcus aureus α-hemolysin, αHL). These experiments are supported by ion heating and cooling simulations which demonstrate reduced collisional cooling at lower source pressures. Using these easily swapped sample cones of different apertures is a facile approach to reproducibly extend the range of activation in Synapt-type instruments.

Rapid Determination of Activation Energies for Gas-Phase Protein Unfolding and Dissociation in a Q-IM-ToF Mass Spectrometer.

Ion mobility-mass spectrometry has emerged as a powerful tool for interrogating a wide variety of chemical systems. Collision-induced unfolding (CIU), typically performed in time-of-flight instruments, has been utilized to obtain valuable qualitative insight into protein structure and illuminate subtle differences between related species. CIU experiments can be performed relatively quickly, but unfolding energy information obtained from them has not yet been interpreted quantitatively. While several methods can determine quantitative dissociation energetics for small molecules, clusters, and peptides, these methods have rarely been applied to proteins, and never to study unfolding. Here, we present a method to rapidly determine activation energies for protein unfolding and dissociation, built on a model for energy deposition during collisional activation. The method is validated by comparing activation energies for dissociation of three complexes with those obtained using blackbody infrared radiative dissociation (BIRD); values from the two methods are in agreement. Several protein monomers were unfolded using CIU, including multiple charge states of both cations and anions, and activation energies determined. ΔH⧧ and ΔS⧧ values are found to be correlated, leading to ΔG⧧ values that lie within a narrow range (∼70-80 kJ/mol) and vary more with charge state than with protein identity. ΔG⧧ is anticorrelated with charge density, highlighting the key role of Coulombic repulsion in gas-phase unfolding. Measured ΔG⧧ values are similar to those computed for proton transfer within small peptides, suggesting that proton transfer is the rate-limiting step in gas-phase unfolding and providing evidence of a link between the Mobile Proton model and CIU.

Experimental and theoretical investigation of overall energy deposition in surface-induced unfolding of protein ions.

Recent advances in native mass spectrometry have enabled its use to probe the structure of and interactions within biomolecular complexes. Surface-induced dissociation, in which inter- and intramolecular interactions are disrupted following an energetic ion-surface collision, is a method that can directly interrogate the topology of protein complexes. However, a quantitative relationship between the ion kinetic energy at the moment of surface collision and the internal energy deposited into the ion has not yet been established for proteins. The factors affecting energy deposition in surface-induced unfolding (SIU) of protein monomers were investigated and a calibration relating laboratory-frame kinetic energy to internal energy developed. Protein monomers were unfolded by SIU and by collision-induced unfolding (CIU). CIU and SIU cause proteins to undergo the same unfolding transitions at different values of laboratory-frame kinetic energy. There is a strong correlation between the SIU and CIU energies, demonstrating that SIU, like CIU, can largely be understood as a thermal process. The change in internal energy in CIU was modeled using a Monte Carlo approach and theory. Computed values of the overall efficiency were found to be approximately 25% and used to rescale the CIU energy axis and relate nominal SIU energies to internal energy. The energy deposition efficiency in SIU increases with mass and kinetic energy from a low of ∼20% to a high of ∼68%, indicating that the effective mass of the surface increases along with the mass of the ion. The effect of ion structure on energy deposition was probed using multiple stages of ion activation. Energy deposition in SIU strongly depends on structure, decreasing as the protein is elongated, due to decreased effective protein-surface collisional cross section and increased transfer to rotational modes.

Extended Protein Ions Are Formed by the Chain Ejection Model in Chemical Supercharging Electrospray Ionization.

Supercharging electrospray ionization can be a powerful tool for increasing charge states in mass spectra and generating unfolded ion structures, yet key details of its mechanism remain unclear. The structures of highly extended protein ions and the mechanism of supercharging were investigated using ion mobility-mass spectrometry. Head-to-tail-linked polyubiquitins (Ubq1-11) were used to determine size and charge state scaling laws for unfolded protein ions formed by supercharging while eliminating amino acid composition as a potential confounding factor. Collisional cross section was found to scale linearly with mass for these ions and several other monomeric proteins, and the maximum observed charge state for each analyte scales with mass in agreement with an analytical charge state scaling law for protein ions with highly extended structures that is supported by experimental gas-phase basicities. These results indicate that these highly unfolded ions can be considered quasi-one-dimensional, and collisional cross sections modeled with the Trajectory Method in Collidoscope show that these ions are significantly more extended than linear α-helices but less extended than straight chains. The effect of internal disulfide bonds on the extent of supercharging was probed using bovine serum albumin, ß-lactoglobulin, and lysozyme, each of which contains multiple internal disulfide bonds. Reduction of the disulfide bonds led to a marked increase in charge state upon supercharging without significantly altering folding in solution. This evidence supports a supercharging mechanism in which these proteins unfold before or during evaporation of the electrospray droplet and ionization occurs by the Chain Ejection Model.

Collidoscope: An Improved Tool for Computing Collisional Cross-Sections with the Trajectory Method.

Ion mobility-mass spectrometry (IM-MS) can be a powerful tool for determining structural information about ions in the gas phase, from small covalent analytes to large, native-like or denatured proteins and complexes. For large biomolecular ions, which may have a wide variety of possible gas-phase conformations and multiple charge sites, quantitative, physically explicit modeling of collisional cross sections (CCSs) for comparison to IMS data can be challenging and time-consuming. We present a "trajectory method" (TM) based CCS calculator, named "Collidoscope," which utilizes parallel processing and optimized trajectory sampling, and implements both He and N2 as collision gas options. Also included is a charge-placement algorithm for determining probable charge site configurations for protonated protein ions given an input geometry in pdb file format. Results from Collidoscope are compared with those from the current state-of-the-art CCS simulation suite, IMoS. Collidoscope CCSs are within 4% of IMoS values for ions with masses from ~18 Da to ~800 kDa. Collidoscope CCSs using X-ray crystal geometries are typically within a few percent of IM-MS experimental values for ions with mass up to ~3.5 kDa (melittin), and discrepancies for larger ions up to ~800 kDa (GroEL) are attributed in large part to changes in ion structure during and after the electrospray process. Due to its physically explicit modeling of scattering, computational efficiency, and accuracy, Collidoscope can be a valuable tool for IM-MS research, especially for large biomolecular ions. Graphical Abstract ᅟ.

Multiple Evolutionary Origins of Ubiquitous Cu2+ and Zn2+ Binding in the S100 Protein Family.

The S100 proteins are a large family of signaling proteins that play critical roles in biology and disease. Many S100 proteins bind Zn2+, Cu2+, and/or Mn2+ as part of their biological functions; however, the evolutionary origins of binding remain obscure. One key question is whether divalent transition metal binding is ancestral, or instead arose independently on multiple lineages. To tackle this question, we combined phylogenetics with biophysical characterization of modern S100 proteins. We demonstrate an earlier origin for established S100 subfamilies than previously believed, and reveal that transition metal binding is widely distributed across the tree. Using isothermal titration calorimetry, we found that Cu2+ and Zn2+ binding are common features of the family: the full breadth of human S100 paralogs-as well as two early-branching S100 proteins found in the tunicate Oikopleura dioica-bind these metals with µM affinity and stoichiometries ranging from 1:1 to 3:1 (metal:protein). While binding is consistent across the tree, structural responses to binding are quite variable. Further, mutational analysis and structural modeling revealed that transition metal binding occurs at different sites in different S100 proteins. This is consistent with multiple origins of transition metal binding over the evolution of this protein family. Our work reveals an evolutionary pattern in which the overall phenotype of binding is a constant feature of S100 proteins, even while the site and mechanism of binding is evolutionarily labile.

A simple bioluminescent method for measuring D-amino acid oxidase activity.

D-Amino acid oxidase (DAO) plays important roles in regulating D-amino acid neurotransmitters and was recently identified as a key enzyme integral to hydrogen sulfide production from D-Cys. We report here the development of a simple biocompatible, bioluminescent method for measuring DAO activity based on the highly selective condensation of D-Cys with 6-hydroxy-2-cyanobenzothiazole (CBT-OH) to form D-luciferin.