Gábor Transform-Based Signal Isolation, Rapid Deconvolution, and Quantitation of Intact Protein Ions with Mass Spectrometry.

High-resolution mass spectrometry (HRMS) is a powerful technique for the characterization and quantitation of complex biological mixtures, with several applications including clinical monitoring and tissue imaging. However, these medical and pharmaceutical applications are pushing the analytical limits of modern HRMS techniques, requiring either further development in instrumentation or data processing methods. Here, we demonstrate new developments in the interactive Fourier-transform analysis for mass spectrometry (iFAMS) software including the first application of Gábor transform (GT) to protein quantitation. Newly added automation tools detect signals from minimal user input and apply thresholds for signal selection, deconvolution, and baseline correction to improve the objectivity and reproducibility of deconvolution. Additional tools were added to improve the deconvolution of highly complex or congested mass spectra and are demonstrated here for the first time. The "Gábor Slicer" enables the user to explore trends in the Gábor spectrogram with instantaneous ion mass estimates accurate to 10 Da. The charge adjuster allows for easy visual confirmation of accurate charge state assignments and quick adjustment if necessary. Deconvolution refinement utilizes a second GT of isotopically resolved data to remove common deconvolution artifacts. To assess the quality of deconvolution from iFAMS, several comparisons are made to deconvolutions using other algorithms such as UniDec and an implementation of MaxEnt in Agilent MassHunter BioConfirm. Lastly, the newly added batch processing and quantitation capabilities of iFAMS are demonstrated and compared to a common extracted ion chromatogram approach.

The complete assembly of human LAT1-4F2hc complex provides insights into its regulation, function and localisation.

The LAT1-4F2hc complex (SLC7A5-SLC3A2) facilitates uptake of essential amino acids, hormones and drugs. Its dysfunction is associated with many cancers and immune/neurological disorders. Here, we apply native mass spectrometry (MS)-based approaches to provide evidence of super-dimer formation (LAT1-4F2hc)2. When combined with lipidomics, and site-directed mutagenesis, we discover four endogenous phosphatidylethanolamine (PE) molecules at the interface and C-terminus of both LAT1 subunits. We find that interfacial PE binding is regulated by 4F2hc-R183 and is critical for regulation of palmitoylation on neighbouring LAT1-C187. Combining native MS with mass photometry (MP), we reveal that super-dimerization is sensitive to pH, and modulated by complex N-glycans on the 4F2hc subunit. We further validate the dynamic assemblies of LAT1-4F2hc on plasma membrane and in the lysosome. Together our results link PTM and lipid binding with regulation and localisation of the LAT1-4F2hc super-dimer.

Label-Free Composition Analysis of Supramolecular Polymer-Nanoparticle Hydrogels by Reversed-Phase Liquid Chromatography Coupled with a Charged Aerosol Detector.

Supramolecular hydrogels formed through polymer-nanoparticle interactions are promising biocompatible materials for translational medicines. This class of hydrogels exhibits shear-thinning behavior and rapid recovery of mechanical properties, providing desirable attributes for formulating sprayable and injectable therapeutics. Characterization of hydrogel composition and loading of encapsulated drugs is critical to achieving the desired rheological behavior as well as tunable in vitro and in vivo payload release kinetics. However, quantitation of hydrogel composition is challenging due to material complexity, heterogeneity, high molecular weight, and the lack of chromophores. Here, we present a label-free approach to simultaneously determine hydrogel polymeric components and encapsulated payloads by coupling a reversed phase liquid chromatographic method with a charged aerosol detector (RPLC-CAD). The hydrogel studied consists of modified hydroxypropylmethylcellulose, self-assembled PEG-b-PLA nanoparticles, and a therapeutic compound, bimatoprost. The three components were resolved and quantitated using the RPLC-CAD method with a C4 stationary phase. The method demonstrated robust performance, applicability to alternative cargos (i.e., proteins) and was suitable for composition analysis as well as for evaluating in vitro release of cargos from the hydrogel. Moreover, this method can be used to monitor polymer degradation and material stability, which can be further elucidated by coupling the RPLC method with (1) a multi-angle light scattering detector (RPLC-MALS) or (2) high resolution mass spectrometry (RPLC-MS) and a Fourier-transform based deconvolution algorithm. We envision that this analytical strategy could be generalized to characterize critical quality attributes of other classes of supramolecular hydrogels, establish structure-property relationships, and provide rational design guidance in hydrogel drug product development.

Effects of Nano-Electrospray Ionization Emitter Position on Unintentional In-Source Activation of Peptide and Protein Ions.

Native ion mobility-mass spectrometry (IM-MS) typically introduces protein ions into the gas phase through nano-electrospray ionization (nESI). Many nESI setups have mobile stages for tuning the ion signal and extent of co-solute and salt adduction. However, tuning the position of the emitter capillary in nESI can have unintended downstream consequences for collision-induced unfolding or collision-induced dissociation (CIU/D) experiments. Here, we show that relatively small variations in the nESI emitter position can shift the midpoint (commonly called the "CID50" or "CIU50") potential of CID breakdown curves and CIU transitions by as much as 8 V on commercial instruments. A spatial "map" of the shift in CID50 for the loss of heme from holomyoglobin onto the emitter position on a Waters Synapt G2-Si mass spectrometer shows that emitter positions closer to the instrument inlet can result in significantly greater in-source activation, whereas different effects are found on an Agilent 6545XT instrument for the ions studied. A similar effect is observed for CID of the singly protonated leucine enkephalin peptide and Shiga toxin 1 subunit B homopentamer on the Waters Synapt G2-Si instrument. In-source activation effects on a Waters Synapt G2-Si are also investigated by examining the RMSD between CIU fingerprints acquired at different emitter positions and the shifts in CIU50 for structural transitions of bovine serum albumin and NIST monoclonal antibody.

Label-Free Composition Analysis of Supramolecular Polymer - Nanoparticle Hydrogels by Reversed-Phase Liquid Chromatography Coupled with a Charged Aerosol Detector.

Supramolecular hydrogels formed through polymer-nanoparticle interactions are promising biocompatible materials for translational medicines. This class of hydrogels exhibits shear-thinning behavior and rapid recovery of mechanical properties following applied stresses, providing desirable attributes for formulating sprayable and injectable therapeutics. Characterization of hydrogel composition and loading of encapsulated drugs is critical to achieving desired rheological behavior as well as tunable in vitro and in vivo payload release kinetics. However, quantitation of hydrogel compositions is challenging due to material complexity, heterogeneity, high molecular weight, and the lack of chromophores. Here, we present a label-free approach to simultaneously determine hydrogel polymeric components and encapsulated payloads by coupling a reversed phase liquid chromatographic method with a charged aerosol detector (RPLC-CAD). The hydrogel studied consists of modified hydroxypropylmethylcellulose, self-assembled PEG-b-PLA nanoparticles, and a therapeutic compound, Bimatoprost. The three components were resolved and quantitated using the RPLC-CAD method with a C4 stationary phase. The method demonstrated robust performance, applicability to alternative cargos (i.e. proteins), and was suitable for composition analysis as well as for evaluating in vitro release of cargos from the hydrogel. Moreover, this method can be used to monitor polymer degradation and material stability, which can be further elucidated by coupling the RPLC method with high resolution mass spectrometry and a Fourier-transform based deconvolution algorithm. To our knowledge, this is the first RPLC-CAD method for characterizing the critical quality attributes of supramolecular hydrogels. We envision this analytical strategy could be generalized to characterize other classes of supramolecular hydrogels, establish structure-property relationships, and provide rational design guidance in hydrogel drug product development.

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.

Moderate-Affinity Affibodies Modulate the Delivery and Bioactivity of Bone Morphogenetic Protein-2.

Uncontrolled bone morphogenetic protein-2 (BMP-2) release can lead to off-target bone growth and other adverse events. To tackle this challenge, yeast surface display is used to identify unique BMP-2-specific protein binders known as affibodies that bind to BMP-2 with different affinities. Biolayer interferometry reveals an equilibrium dissociation constant of 10.7 nm for the interaction between BMP-2 and high-affinity affibody and 34.8 nm for the interaction between BMP-2 and the low-affinity affibody. The low-affinity affibody-BMP-2 interaction also exhibits an off-rate constant that is an order of magnitude higher. Computational modeling of affibody-BMP-2 binding predicts that the high- and low-affinity affibodies bind to two distinct sites on BMP-2 that function as different cell-receptor binding sites. BMP-2 binding to affibodies reduces expression of the osteogenic marker alkaline phosphatase (ALP) in C2C12 myoblasts. Affibody-conjugated polyethylene glycol-maleimide hydrogels increase uptake of BMP-2 compared to affibody-free hydrogels, and high-affinity hydrogels exhibit lower BMP-2 release into serum compared to low-affinity hydrogels and affibody-free hydrogels over four weeks. Loading BMP-2 into affibody-conjugated hydrogels prolongs ALP activity of C2C12 myoblasts compared to soluble BMP-2. This work demonstrates that affibodies with different affinities can modulate BMP-2 delivery and activity, creating a promising approach for controlling BMP-2 delivery in clinical applications.

Linker Length Drives Heterogeneity of Multivalent Complexes of Hub Protein LC8 and Transcription Factor ASCIZ.

LC8, a ubiquitous and highly conserved hub protein, binds over 100 proteins involved in numerous cellular functions, including cell death, signaling, tumor suppression, and viral infection. LC8 binds intrinsically disordered proteins (IDPs), and although several of these contain multiple LC8 binding motifs, the effects of multivalency on complex formation are unclear. Drosophila ASCIZ has seven motifs that vary in sequence and inter-motif linker lengths, especially within subdomain QT2-4 containing the second, third, and fourth LC8 motifs. Using isothermal-titration calorimetry, analytical-ultracentrifugation, and native mass-spectrometry of QT2-4 variants, with methodically deactivated motifs, we show that inter-motif spacing and specific motif sequences combine to control binding affinity and compositional heterogeneity of multivalent duplexes. A short linker separating strong and weak motifs results in stable duplexes but forms off-register structures at high LC8 concentrations. Contrastingly, long linkers engender lower cooperativity and heterogeneous complexation at low LC8 concentrations. Accordingly, two-mers, rather than the expected three-mers, dominate negative-stain electron-microscopy images of QT2-4. Comparing variants containing weak-strong and strong-strong motif combinations demonstrates sequence also regulates IDP/LC8 assembly. The observed trends persist for trivalent ASCIZ subdomains: QT2-4, with long and short linkers, forms heterogeneous complexes, whereas QT4-6, with similar mid-length linkers, forms homogeneous complexes. Implications of linker length variations for function are discussed.

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.

Investigation of Charge-State-Dependent Compaction of Protein Ions with Native Ion Mobility-Mass Spectrometry and Theory.

The precise relationship between native gas-phase protein ion structure, charge, desolvation, and activation remains elusive. Much evidence supports the Charge Residue Model for native protein ions formed by electrospray ionization, but scaling laws derived from it relate only to overall ion size. Closer examination of drift tube CCSs across individual native protein ion charge state distributions (CSDs) reveals deviations from global trends. To investigate whether this is due to structure variation across CSDs or contributions of long-range charge-dipole interactions, we performed in vacuo force field molecular dynamics (MD) simulations of multiple charge conformers of three proteins representing a variety of physical and structural features: ß-lactoglobulin, concanavalin A, and glutamate dehydrogenase. Results from these simulated ions indicate subtle structure variation across their native CSDs, although effects of these structural differences and long-range charge-dependent interactions on CCS are small. The structure and CCS of smaller proteins may be more sensitive to charge due to their low surface-to-volume ratios and reduced capacity to compact. Secondary and higher order structure from condensed-phase structures is largely retained in these simulations, supporting the use of the term "native-like" to describe results from native ion mobility-mass spectrometry experiments, although, notably, the most compact structure can be the most different from the condensed-phase structure. Collapse of surface side chains to self-solvate through formation of new hydrogen bonds is a major feature of gas-phase compaction and likely occurs during the desolvation process. Results from these MD simulations provide new insight into the relationship of gas-phase protein ion structure, charge, and CCS.

Approaches to Heterogeneity in Native Mass Spectrometry.

Native mass spectrometry (MS) is aimed at preserving and determining the native structure, composition, and stoichiometry of biomolecules and their complexes from solution after they are transferred into the gas phase. Major improvements in native MS instrumentation and experimental methods over the past few decades have led to a concomitant increase in the complexity and heterogeneity of samples that can be analyzed, including protein-ligand complexes, protein complexes with multiple coexisting stoichiometries, and membrane protein-lipid assemblies. Heterogeneous features of these biomolecular samples can be important for understanding structure and function. However, sample heterogeneity can make assignment of ion mass, charge, composition, and structure very challenging due to the overlap of tens or even hundreds of peaks in the mass spectrum. In this review, we cover data analysis, experimental, and instrumental advances and strategies aimed at solving this problem, with an in-depth discussion of theoretical and practical aspects of the use of available deconvolution algorithms and tools. We also reflect upon current challenges and provide a view of the future of this exciting field.

Fourier-Transform Approach for Reconstructing Macromolecular Mass Defect Profiles.

State-of-the-art native mass spectrometry (MS) methods have been developed for analysis of highly heterogeneous intact complexes and have provided much insight into the structure and properties of noncovalent assemblies that can be difficult to study using denatured proteins. These native MS methods can often be used to study even highly polydisperse membrane proteins embedded in detergent micelles, nanodiscs, and other membrane mimics. However, characterizing highly polydisperse native complexes which are also heterogeneous presents additional challenges for native MS. Macromolecular mass defect (MMD) analysis aims to characterize heterogeneous ion populations obfuscated by adduct polydispersity and reveal the distribution of "base" masses, and was recently implemented in the Bayesian analysis software UniDec. Here, we illustrate an alternative, orthogonal MMD analysis method implemented in the deconvolution program iFAMS, which takes advantage of Fourier transform (FT) to deconvolve low-resolution data with few user-input parameters and which can provide high quality results even for mass spectra with a signal-to-noise ratio of ∼5:1. Agreement between this method, which is based on frequency-domain data, and the mass-domain algorithm of UniDec provides strong evidence that both methods can accurately characterize highly polydisperse and heterogeneous ion populations. The FT algorithm is expected to be very useful in characterizing many types of analytes ranging from membrane proteins to polymer-conjugated proteins, branched polymers, and other large analytes, as well as for reconstructing isotope profiles for highly complex but still isotope-resolved mass spectra.

Influenza AM2 Channel Oligomerization Is Sensitive to Its Chemical Environment.

Viroporins are small viral ion channels that play important roles in the viral infection cycle and are proven antiviral drug targets. Matrix protein 2 from influenza A (AM2) is the best-characterized viroporin, and the current paradigm is that AM2 forms monodisperse tetramers. Here, we used native mass spectrometry and other techniques to characterize the oligomeric state of both the full-length and transmembrane (TM) domain of AM2 in a variety of different pH and detergent conditions. Unexpectedly, we discovered that AM2 formed a range of different oligomeric complexes that were strongly influenced by the local chemical environment. Native mass spectrometry of AM2 in nanodiscs with different lipids showed that lipids also affected the oligomeric states of AM2. Finally, nanodiscs uniquely enabled the measurement of amantadine binding stoichiometries to AM2 in the intact lipid bilayer. These unexpected results reveal that AM2 can form a wider range of oligomeric states than previously thought possible, which may provide new potential mechanisms of influenza pathology and pharmacology.

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.

Multivalent binding of the partially disordered SARS-CoV-2 nucleocapsid phosphoprotein dimer to RNA.

The nucleocapsid phosphoprotein N plays critical roles in multiple processes of the severe acute respiratory syndrome coronavirus 2 infection cycle: it protects and packages viral RNA in N assembly, interacts with the inner domain of spike protein, binds to structural membrane (M) protein during virion packaging and maturation, and to proteases causing replication of infective virus particle. Even with its importance, very limited biophysical studies are available on the N protein because of its high level of disorder, high propensity for aggregation, and high susceptibility for autoproteolysis. Here, we successfully prepare the N protein and a 1000-nucleotide fragment of viral RNA in large quantities and purity suitable for biophysical studies. A combination of biophysical and biochemical techniques demonstrates that the N protein is partially disordered and consists of an independently folded RNA-binding domain and a dimerization domain, flanked by disordered linkers. The protein assembles as a tight dimer with a dimerization constant of sub-micromolar but can also form transient interactions with other N proteins, facilitating larger oligomers. NMR studies on the ∼100-kDa dimeric protein identify a specific domain that binds 1-1000-nt RNA and show that the N-RNA complex remains highly disordered. Analytical ultracentrifugation, isothermal titration calorimetry, multiangle light scattering, and cross-linking experiments identify a heterogeneous mixture of complexes with a core corresponding to at least 70 dimers of N bound to 1-1000 RNA. In contrast, very weak binding is detected with a smaller construct corresponding to the RNA-binding domain using similar experiments. A model that explains the importance of the bivalent structure of N to its binding on multivalent sites of the viral RNA is presented.

Experimental Determination of Activation Energies for Covalent Bond Formation via Ion/Ion Reactions and Competing Processes.

The combination of ion/ion chemistry with commercially available ion mobility/mass spectrometry systems has allowed rich structural information to be obtained for gaseous protein ions. Recently, the simple modification of such an instrument with an electrospray reagent source has allowed three-dimensional gas-phase interrogation of protein structures through covalent and noncovalent interactions coupled with collision cross section measurements. However, the energetics of these processes have not yet been studied quantitatively. In this work, previously developed Monte Carlo simulations of ion temperatures inside traveling wave ion guides are used to characterize the energetics of the transition state of activated ubiquitin cation/sulfo-benzoyl-HOAt reagent anion long-lived complexes formed via ion/ion reactions. The ΔH‡ and ΔS‡ of major processes observed from collisional activation of long-lived gas-phase ion/ion complexes, namely collision induced unfolding (CIU), covalent bond formation, or neutral loss of the anionic reagent via intramolecular proton transfer, were determined. Covalent bond formation via ion/ion complexes was found to be significantly lower energy compared to unfolding and bond cleavage. The ΔG‡ values of activation of all three processes lie between 55 and 75 kJ/mol, easily accessible with moderate collisional activation. Bond formation is favored over reagent loss at lower activation energies, whereas reagent loss becomes competitive at higher collision energies. Though the ΔG‡ values between CIU of a precursor ion and covalent bond formation of its ion/ion product complex are comparable, our data suggest covalent bond formation does not require extensive isomerization.

Yorkie-Warts Complexes are an Ensemble of Interconverting Conformers Formed by Multivalent Interactions.

Multiple copies of WW domains and PPXY motif sequences are often reciprocally presented by regulatory proteins that interact at crucial regulatory steps in the cell life cycle. While biophysical studies of single WW domain-single PPXY motif complexes abound in the literature, the molecular mechanisms of multivalent WW domain-PPXY assemblies are still poorly understood. By way of investigating such assemblies, we characterized the multivalent association of the entire cognate binding domains, two WW sequences and five PPXY motifs respectively, of the Yorkie transcription coactivator and the Warts tumor suppressor. Isothermal titration calorimetry, sedimentation velocity, size-exclusion chromatography coupled to multi-angle light scattering and native-state mass spectrometry of Yorkie WW domains interactions with the full-length Warts PPXY domain, and numerous PPXY motif variants of Warts show that the two proteins assemble via binding of tandem WW domains to adjacent PPXY pairs to produce an ensemble of interconverting complexes of variable stoichiometries, binding energetics and WW domain occupancy. Apparently, the Yorkie tandem WW domains first target the two adjacent PPXY motifs at the C-terminus of the Warts polypeptide and additional WW domains bind unoccupied motifs. Similar ensembles of interconverting conformers may be common in multivalent WW domain-PPXY interactions to promote the adaptability and versatility of WW domain-PPXY mediated cellular processes.

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.

Gas-Phase Protonation Thermodynamics of Biological Lipids: Experiment, Theory, and Implications.

Phospholipids are important to cellular function and are a vital structural component of plasma and organelle membranes. These membranes isolate the cell from its environment, allow regulation of the internal concentrations of ions and small molecules, and host diverse types of membrane proteins. It remains extremely challenging to identify specific membrane protein-lipid interactions and their relative strengths. Native mass spectrometry, an intrinsically gas-phase method, has recently been demonstrated as a promising tool for identifying endogenous protein-lipid interactions. However, to what extent the identified interactions reflect solution- versus gas-phase binding strengths is not known. Here, the "Extended" Kinetic Method and ab initio computations at three different levels of theory are used to experimentally and theoretically determine intrinsic gas-phase basicities (GB, ΔG for deprotonation of the protonated base) and proton affinities (PA, ΔH for deprotonation of the protonated base) of six lipids representing common phospholipid types. Gas-phase acidities (ΔG and ΔH for deprotonation) of neutral phospholipids are also evaluated computationally and ranked experimentally. Intriguingly, it is found that two of these phospholipids, sphingomyelin and phosphatidylcholine, have the highest GB of any small, monomeric biomolecules measured to date and are more basic than arginine. Phosphatidylethanolamine and phosphatidylserine are found to be similar in GB to basic amino acids lysine and histidine, and phosphatidic acid and phosphatidylglycerol are the least basic of the six lipid types studied, though still more basic than alanine. Kinetic Method experiments and theory show that the gas-phase acidities of these phospholipids are high but less extreme than their GB values, with phosphatidylserine and phosphatidylglycerol being the most acidic. These results indicate that sphingomyelin and phosphatidylcholine lipids can act as charge-reducing agents when dissociated from native membrane protein-lipid complexes in the gas phase and provide a straightforward model to explain the results of several recent native mass spectrometry studies of protein-lipid complexes.

Distinct classes of multi-subunit heterogeneity: analysis using Fourier Transform methods and native mass spectrometry.

Native electrospray mass spectrometry is a powerful method for determining the native stoichiometry of many polydisperse multi-subunit biological complexes, including multi-subunit protein complexes and lipid-bound transmembrane proteins. However, when polydispersity results from incorporation of multiple copies of two or more different subunits, it can be difficult to analyze subunit stoichiometry using conventional mass spectrometry analysis methods, especially when m/z distributions for different charge states overlap in the mass spectrum. It was recently demonstrated by Marty and co-workers (K. K. Hoi, et al., Anal. Chem., 2016, 88, 6199-6204) that Fourier Transform (FT)-based methods can determine the bulk average lipid composition of protein-lipid Nanodiscs assembled with two different lipids, but a detailed statistical description of the composition of more general polydisperse two-subunit populations is still difficult to achieve. This results from the vast number of ways in which the two types of subunit can be distributed within the analyte ensemble. Here, we present a theoretical description of three common classes of heterogeneity for mixed-subunit analytes and demonstrate how to differentiate and analyze them using mass spectrometry and FT methods. First, we first describe FT-based analysis of mass spectra corresponding to simple superpositions, convolutions, and multinomial distributions for two or more different subunit types using model data sets. We then apply these principles with real samples, including mixtures of single-lipid Nanodiscs in the same solution (superposition), mixed-lipid Nanodiscs and copolymers (convolutions), and isotope distribution for ubiquitin (multinomial distribution). This classification scheme and the FT method used to study these analyte classes should be broadly useful in mass spectrometry as well as other techniques where overlapping, periodic signals arising from analyte mixtures are common.