Membrane Protein-Lipid Interactions Probed Using Mass Spectrometry.

Membrane proteins that exist in lipid bilayers are not isolated molecular entities. The lipid molecules that surround them play crucial roles in maintaining their full structural and functional integrity. Research directed at investigating these critical lipid-protein interactions is developing rapidly. Advancements in both instrumentation and software, as well as in key biophysical and biochemical techniques, are accelerating the field. In this review, we provide a brief outline of structural techniques used to probe protein-lipid interactions and focus on the molecular aspects of these interactions obtained from native mass spectrometry (native MS). We highlight examples in which lipids have been shown to modulate membrane protein structure and show how native MS has emerged as a complementary technique to X-ray crystallography and cryo-electron microscopy. We conclude with a short perspective on future developments that aim to better understand protein-lipid interactions in the native environment.

Two hearts beat as one: the debate over RAS dimers continues.

A recent report by Yun et al. describes the detection of RAS dimers using intact mass spectrometry and investigates the role that membrane lipids, nucleotide state, and binding partners have in their formation.

Structural Basis of Tail-Anchored Membrane Protein Biogenesis by the GET Insertase Complex.

Membrane protein biogenesis faces the challenge of chaperoning hydrophobic transmembrane helices for faithful membrane insertion. The guided entry of tail-anchored proteins (GET) pathway targets and inserts tail-anchored (TA) proteins into the endoplasmic reticulum (ER) membrane with an insertase (yeast Get1/Get2 or mammalian WRB/CAML) that captures the TA from a cytoplasmic chaperone (Get3 or TRC40, respectively). Here, we present cryo-electron microscopy reconstructions, native mass spectrometry, and structure-based mutagenesis of human WRB/CAML/TRC40 and yeast Get1/Get2/Get3 complexes. Get3 binding to the membrane insertase supports heterotetramer formation, and phosphatidylinositol binding at the heterotetramer interface stabilizes the insertase for efficient TA insertion in vivo. We identify a Get2/CAML cytoplasmic helix that forms a "gating" interaction with Get3/TRC40 important for TA insertion. Structural homology with YidC and the ER membrane protein complex (EMC) implicates an evolutionarily conserved insertion mechanism for divergent substrates utilizing a hydrophilic groove. Thus, we provide a detailed structural and mechanistic framework to understand TA membrane insertion.

Lipopeptide antibiotics disrupt interactions of undecaprenyl phosphate with UptA.

The peptidoglycan pathway represents one of the most successful antibacterial targets with the last critical step being the flipping of carrier lipid, undecaprenyl phosphate (C55-P), across the membrane to reenter the pathway. This translocation of C55-P is facilitated by DedA and DUF368 domain-containing family membrane proteins via unknown mechanisms. Here, we employ native mass spectrometry to investigate the interactions of UptA, a member of the DedA family of membrane protein from Bacillus subtilis, with C55-P, membrane phospholipids, and cell wall-targeting antibiotics. Our results show that UptA, expressed and purified in Escherichia coli, forms monomer-dimer equilibria, and binds to C55-P in a pH-dependent fashion. Specifically, we show that UptA interacts more favorably with C55-P over shorter-chain analogs and membrane phospholipids. Moreover, we demonstrate that lipopeptide antibiotics, amphomycin and aspartocin D, can directly inhibit UptA function by out-competing the substrate for the protein binding, in addition to their propensity to form complex with free C55-P. Overall, this study shows that UptA-mediated translocation of C55-P is potentially mediated by pH and anionic phospholipids and provides insights for future development of antibiotics targeting carrier lipid recycling.

Are Internal Fragments Observable in Electron Based Top-Down Mass Spectrometry?

Protein tandem mass spectrometry (MS/MS) often generates sequence-informative fragments from backbone bond cleavages near the termini. This lack of fragmentation in the protein interior is particularly apparent in native top-down mass spectrometry (MS). Improved sequence coverage, critical for reliable annotation of posttranslational modifications and sequence variants, may be obtained from internal fragments generated by multiple backbone cleavage events. However, internal fragment assignments can be error prone due to isomeric/isobaric fragments from different parts of a protein sequence. Also, internal fragment generation propensity depends on the chosen MS/MS activation strategy. Here, we examine internal fragment formation in electron capture dissociation (ECD) and electron transfer dissociation (ETD) following native and denaturing MS, as well as LC/MS of several proteins. Experiments were undertaken on multiple instruments, including quadrupole time-of-flight, Orbitrap, and high-field Fourier-transform ion cyclotron resonance (FT-ICR) across four laboratories. ECD was performed at both ultrahigh vacuum and at similar pressure to ETD conditions. Two complementary software packages were used for data analysis. When feasible, ETD-higher energy collision dissociation MS3 was performed to validate/refute potential internal fragment assignments, including differentiating MS3 fragmentation behavior of radical versus even-electron primary fragments. We show that, under typical operating conditions, internal fragments cannot be confidently assigned in ECD or ETD. On the other hand, such fragments, along with some b-type terminal fragments (not typically observed in ECD/ETD spectra) appear at atypical ECD operating conditions, suggesting they originate from a separate ion-electron activation process. Furthermore, atypical fragment ion types, e.g., x ions, are observed at such conditions as well as upon EThcD, presumably due to vibrational activation of radical z-type ions.

Probing differences among Aß oligomers with two triangular trimers derived from Aß.

The assembly of the ß-amyloid peptide (Aß) to form oligomers and fibrils is closely associated with the pathogenesis and progression of Alzheimer's disease. Aß is a shape-shifting peptide capable of adopting many conformations and folds within the multitude of oligomers and fibrils the peptide forms. These properties have precluded detailed structural elucidation and biological characterization of homogeneous, well-defined Aß oligomers. In this paper, we compare the structural, biophysical, and biological characteristics of two different covalently stabilized isomorphic trimers derived from the central and C-terminal regions Aß. X-ray crystallography reveals the structures of the trimers and shows that each trimer forms a ball-shaped dodecamer. Solution-phase and cell-based studies demonstrate that the two trimers exhibit markedly different assembly and biological properties. One trimer forms small soluble oligomers that enter cells through endocytosis and activate capase-3/7-mediated apoptosis, while the other trimer forms large insoluble aggregates that accumulate on the outer plasma membrane and elicit cellular toxicity through an apoptosis-independent mechanism. The two trimers also exhibit different effects on the aggregation, toxicity, and cellular interaction of full-length Aß, with one trimer showing a greater propensity to interact with Aß than the other. The studies described in this paper indicate that the two trimers share structural, biophysical, and biological characteristics with oligomers of full-length Aß. The varying structural, assembly, and biological characteristics of the two trimers provide a working model for how different Aß trimers can assemble and lead to different biological effects, which may help shed light on the differences among Aß oligomers.

Effects of charge on protein ion structure: Lessons from cation-to-anion, proton-transfer reactions.

Collision cross-section values, which can be determined using ion mobility experiments, are sensitive to the structures of protein ions and useful for applications to structural biology and biophysics. Protein ions with different charge states can exhibit very different collision cross-section values, but a comprehensive understanding of this relationship remains elusive. Here, we review cation-to-anion, proton-transfer reactions (CAPTR), a method for generating a series of charge-reduced protein cations by reacting quadrupole-selected cations with even-electron monoanions. The resulting CAPTR products are analyzed using a combination of ion mobility, mass spectrometry, and collisional activation. We compare CAPTR to other charge-manipulation strategies and review the results of various CAPTR-based experiments, exploring their contribution to a deeper understanding of the relationship between protein ion structure and charge state.

Toward Organism-scale Structural Biology: S-layer Reined in by Bacterial LPS.

Technical developments are unifying molecular and cellular biology. A recent electron cryotomography study by von Kügelgen et al. highlights the bright future for such studies, seamlessly integrating near-atomic resolution protein structures, organism-scale architecture, native mass spectrometry, and molecular dynamic simulations to clarify how the Caulobacter crescentus S-layer assembles on the lipopolysaccharides (LPS) of the cell surface.

Combining Native Mass Spectrometry and Proteomics to Differentiate and Map the Metalloform Landscape in Metallothioneins.

Within the intricate landscape of the proteome, approximately 30% of all proteins bind metal ions. This repertoire is even larger when considering all the different forms of a protein, known as proteoforms. Here, we propose the term "metalloforms" to refer to different structural or functional variations of a protein resulting from the binding of various hetero- or homogeneous metal ions. Using human Cu(I)/Zn(II)-metallothionein-3 as a representative model, we developed a chemical proteomics strategy to simultaneously differentiate and map Zn(II) and Cu(I) metal binding sites. In the first labeling step, N-ethylmaleimide reacts with Cysteine (Cys), resulting in the dissociation of all Zn(II) ions while Cu(I) remains bound to the protein. In the second labeling step, iodoacetamide is utilized to label Cu(I)-bound Cys residues. Native mass spectrometry (MS) was used to determine the metal/labeling protein stoichiometries, while bottom-up/top-down MS was used to map the Cys-labeled residues. Next, we used a developed methodology to interrogate an isolated rabbit liver metallothionein fraction containing three metallothionein-2 isoforms and multiple Cd(II)/Zn(II) metalloforms. The approach detailed in this study thus holds the potential to decode the metalloproteoform diversity within other proteins.

Online Mixed-Bed Ion Exchange Chromatography for Native Top-Down Proteomics of Complex Mixtures.

Native top-down mass spectrometry (nTDMS) allows characterization of protein structure and noncovalent interactions with simultaneous sequence mapping and proteoform characterization. The majority of nTDMS studies utilize purified recombinant proteins, with significant challenges hindering application to endogenous systems. To perform native top-down proteomics (nTDP), where endogenous proteins from complex biological systems are analyzed by nTDMS, it is essential to separate proteins under nondenaturing conditions. However, it remains difficult to achieve high resolution with MS-compatible online chromatography while preserving protein tertiary structure and noncovalent interactions. Herein, we report the use of online mixed-bed ion exchange chromatography (IEC) to enable separation of endogenous proteins from complex mixtures under nondenaturing conditions, preserving noncovalent interactions for nTDP analysis. We have successfully detected large proteins (>146 kDa) and identified endogenous metal-binding and oligomeric protein complexes in human heart tissue lysate. The use of a mixed-bed stationary phase allowed retention and elution of proteins over a wide range of isoelectric points without altering the sample or mobile phase pH. Overall, our method provides a simple online IEC-MS platform that can effectively separate proteins from complex mixtures under nondenaturing conditions and preserve higher-order structure for nTDP applications.

Imaged Capillary Isoelectric Focusing Coupled to High-Resolution Mass Spectrometry (icIEF-MS) for Cysteine-Linked Antibody-Drug Conjugate (ADC) Heterogeneity Characterization Under Native Condition.

Native mass spectrometry (nMS) is a cutting-edge technique that leverages electrospray ionization MS (ESI-MS) to investigate large biomolecules and their complexes in solution. The goal of nMS is to retain the native structural features and interactions of the analytes during the transition to the gas phase, providing insights into their natural conformations. In biopharmaceutical development, nMS serves as a powerful tool for analyzing complex protein heterogeneity, allowing for the examination of non-covalently bonded assemblies in a state that closely resembles their natural folded form. Herein, we present an imaged capillary isoelectric focusing-MS (icIEF-MS) workflow to characterize cysteine-linked antibody-drug conjugate (ADC) under native conditions. Two ADCs were analyzed: a latest generation cysteine-linked ADC polatuzumab vedotin and the first FDA-approved cysteine-linked ADC brentuximab vedotin. This workflow benefits from a recently developed icIEF system that is MS-friendly and capable of directly coupling to a high-sensitivity MS instrument. Results show that the icIEF separation is influenced by both drug payloads and the post-translational modifications (PTMs), which are then promptly identified by MS. Overall, this native icIEF-MS method demonstrates the potential to understand and control the critical quality attributes (CQAs) that are essential for the safe and effective use of ADCs.

What's the defect? Using mass defects to study oligomerization of membrane proteins and peptides in nanodiscs with native mass spectrometry.

Many membrane proteins form functional complexes that are either homo- or hetero-oligomeric. However, it is challenging to characterize membrane protein oligomerization in intact lipid bilayers, especially for polydisperse mixtures. Native mass spectrometry of membrane proteins and peptides inserted in lipid nanodiscs provides a unique method to study the oligomeric state distribution and lipid preferences of oligomeric assemblies. To interpret these complex spectra, we developed novel data analysis methods using macromolecular mass defect analysis. Here, we provide an overview of how mass defect analysis can be used to study oligomerization in nanodiscs, discuss potential limitations in interpretation, and explore strategies to resolve these ambiguities. Finally, we review recent work applying this technique to studying formation of antimicrobial peptide, amyloid protein, and viroporin complexes with lipid membranes.

Revealing the Fates of Proteins in the Gas Phase.

The ability to observe intact proteins by native mass spectrometry allows measurements of size, oligomeric state, numbers and types of ligands and post translational modifications bound, among many other characteristics. These studies have the potential to, and in some cases are, advancing our understanding of the role of structure in protein biology and biochemistry. However, there are some long-unresolved questions about to what extent solution-like structures persist without solvent in the vacuum of the mass spectrometer. Strong evidence from multiple sources over the years has demonstrated that well-folded proteins maintain native-like states if care is taken during sample preparation, ionization, and transmission through the gas phase. For partially unfolded states, dynamic and disordered proteins, and other important landmarks along the protein folding/unfolding pathway, caution has been urged in the interpretation of the results of native ion mobility/mass spectrometric data. New gas-phase tools allow us to provide insight into these questions with in situ, in vacuo labeling reactions delivered through ion/ion chemistry. This Young Scientist Perspective demonstrates the robustness of these tools in describing native-like structure as well as possible deviations from native-like structure during native ion mobility/mass spectrometry. This Perspective illustrates some of the changes in structure produced by the removal of solvent and details some of the challenges and potential of the field.

The Complementarity of Nuclear Magnetic Resonance and Native Mass Spectrometry in Probing Protein-Protein Interactions.

Nuclear magnetic resonance (NMR) and native mass spectrometry (MS) are mature physicochemical techniques with long histories and important applications. NMR spectroscopy provides detailed information about the structure, dynamics, interactions, and chemical environment of biomolecules. MS is an effective approach for determining the mass of biomolecules with high accuracy, sensitivity, and speed. The two techniques offer unique advantages and provide solid tools for structural biology. In the present review, we discuss their individual merits in the context of their applications to structural studies in biology with specific focus on protein interactions and evaluate their limitations. We provide specific examples in which these techniques can complement each other, providing new information on the same scientific case. We discuss how the field may develop and what challenges are expected in the future. Overall, the combination of NMR and MS plays an increasingly important role in integrative structural biology, assisting scientists in deciphering the three-dimensional structure of composite macromolecular assemblies.

Molecular assemblies of the catalytic domain of SOS with KRas and oncogenic mutants.

Ras is regulated by a specific guanine nucleotide exchange factor Son of Sevenless (SOS), which facilitates the exchange of inactive, GDP-bound Ras with GTP. The catalytic activity of SOS is also allosterically modulated by an active Ras (Ras-GTP). However, it remains poorly understood how oncogenic Ras mutants interact with SOS and modulate its activity. Here, native ion mobility-mass spectrometry is employed to monitor the assembly of the catalytic domain of SOS (SOScat) with KRas and three cancer-associated mutants (G12C, G13D, and Q61H), leading to the discovery of different molecular assemblies and distinct conformers of SOScat engaging KRas. We also find KRasG13D exhibits high affinity for SOScat and is a potent allosteric modulator of its activity. A structure of the KRasG13D•SOScat complex was determined using cryogenic electron microscopy providing insight into the enhanced affinity of the mutant protein. In addition, we find that KRasG13D-GTP can allosterically increase the nucleotide exchange rate of KRas at the active site more than twofold compared to KRas-GTP. Furthermore, small-molecule Ras•SOS disruptors fail to dissociate KRasG13D•SOScat complexes, underscoring the need for more potent disruptors. Taken together, a better understanding of the interaction between oncogenic Ras mutants and SOS will provide avenues for improved therapeutic interventions.

Liquid-Liquid Phase Separation Primes Spider Silk Proteins for Fiber Formation via a Conditional Sticker Domain.

Many protein condensates can convert to fibrillar aggregates, but the underlying mechanisms are unclear. Liquid-liquid phase separation (LLPS) of spider silk proteins, spidroins, suggests a regulatory switch between both states. Here, we combine microscopy and native mass spectrometry to investigate the influence of protein sequence, ions, and regulatory domains on spidroin LLPS. We find that salting out-effects drive LLPS via low-affinity stickers in the repeat domains. Interestingly, conditions that enable LLPS simultaneously cause dissociation of the dimeric C-terminal domain (CTD), priming it for aggregation. Since the CTD enhances LLPS of spidroins but is also required for their conversion into amyloid-like fibers, we expand the stickers and spacers-model of phase separation with the concept of folded domains as conditional stickers that represent regulatory units.

A Hybrid Orbitrap-Nanoelectromechanical Systems Approach for the Analysis of Individual, Intact Proteins in Real Time.

Nanoelectromechanical systems (NEMS)-based mass spectrometry (MS) is an emerging technique that enables determination of the mass of individual adsorbed particles by driving nanomechanical devices at resonance and monitoring the real-time changes in their resonance frequencies induced by each single molecule adsorption event. We incorporate NEMS into an Orbitrap mass spectrometer and report our progress towards leveraging the single-molecule capabilities of the NEMS to enhance the dynamic range of conventional MS instrumentation and to offer new capabilities for performing deep proteomic analysis of clinically relevant samples. We use the hybrid instrument to deliver E. coli GroEL molecules (801 kDa) to the NEMS devices in their native, intact state. Custom ion optics are used to focus the beam down to 40 µm diameter with a maximum flux of 25 molecules/second. The mass spectrum obtained with NEMS-MS shows good agreement with the known mass of GroEL.

Native Proteomics by Capillary Zone Electrophoresis-Mass Spectrometry.

Native proteomics measures endogenous proteoforms and protein complexes under a near physiological condition using native mass spectrometry (nMS) coupled with liquid-phase separations. Native proteomics should provide the most accurate bird's-eye view of proteome dynamics within cells, which is fundamental for understanding almost all biological processes. nMS has been widely employed to characterize well-purified protein complexes. However, there are only very few trials of utilizing nMS to measure proteoforms and protein complexes in a complex sample (i.e., a whole cell lysate). Here, we pioneer the native proteomics measurement of large proteoforms or protein complexes up to 400 kDa from a complex proteome via online coupling of native capillary zone electrophoresis (nCZE) to an ultra-high mass range (UHMR) Orbitrap mass spectrometer. The nCZE-MS technique enabled the measurement of a 115-kDa standard protein complex while consuming only about 0.1 ng of protein material. nCZE-MS analysis of an E.coli cell lysate detected 72 proteoforms or protein complexes in a mass range of 30-400 kDa in a single run while consuming only 50-ng protein material. The mass distribution of detected proteoforms or protein complexes agreed well with that from mass photometry measurement. This work represents a technical breakthrough in native proteomics for measuring complex proteomes.

ProSight Native: Defining Protein Complex Composition from Native Top-Down Mass Spectrometry Data.

Native mass spectrometry has recently moved alongside traditional structural biology techniques in its ability to provide clear insights into the composition of protein complexes. However, to date, limited software tools are available for the comprehensive analysis of native mass spectrometry data on protein complexes, particularly for experiments aimed at elucidating the composition of an intact protein complex. Here, we introduce ProSight Native as a start-to-finish informatics platform for analyzing native protein and protein complex data. Combining mass determination via spectral deconvolution with a top-down database search and stoichiometry calculations, ProSight Native can determine the complete composition of protein complexes. To demonstrate its features, we used ProSight Native to successfully determine the composition of the homotetrameric membrane complex Aquaporin Z. We also revisited previously published spectra and were able to decipher the composition of a heterodimer complex bound with two noncovalently associated ligands. In addition to determining complex composition, we developed new tools in the software for validating native mass spectrometry fragment ions and mapping top-down fragmentation data onto three-dimensional protein structures. Taken together, ProSight Native will reduce the informatics burden on the growing field of native mass spectrometry, enabling the technology to further its reach.

Normal Alpha-1-Antitrypsin Variants Display in Serum Allele-Specific Protein Levels.

Alpha-1-antitrypsin (A1AT or SERPINA1) has been proposed as a putative biomarker distinguishing healthy from diseased donors throughout several proteomics studies. However, the SERPINA1 gene displays high variability of frequent occurring genotypes among the general population. These different genotypes may affect A1AT expression and serum protein concentrations, and this is often not known, ignored, and/or not reported in serum proteomics studies. Here, we address allele-specific protein serum levels of A1AT in donors carrying the normal M variants of A1AT by measuring the proteoform profiles of purified A1AT from 81 serum samples, originating from 52 donors. When focusing on heterozygous donors, our data clearly reveal a statistically relevant difference in allele-specific protein serum levels of A1AT. In donors with genotype PI*M1VM1A, the experimentally observed ratio was approximately 1:1 (M1V/M1A, 1.00:0.96 ± 0.07, n = 17). For individuals with genotype PI*M1VM2, this ratio was 1:1.28 (M1V/M2, 1.00:1.31, ±0.19, n = 7). For genotypes PI*M1VM3 and PI*M1AM3, a significant higher amount of M3 was observed compared to the M1-subtypes (M1V/M3, 1.00:1.84 ± 0.35, n = 8; M1A/M3, 1.00:1.61 ± 0.33, n = 5). We argue that these observations are important and should be considered when analyzing serum A1AT levels before proposing A1AT as a putative serum biomarker.