Characterization of bispecific antigen-binding biotherapeutic fragmentation sites using microfluidic capillary electrophoresis coupled to mass spectrometry (mCZE-MS).

Fragmentation of therapeutic proteins is a potential critical quality attribute (CQA) that can occur in vivo or during manufacturing or storage due to enzymatic and non-enzymatic degradation pathways, such as hydrolysis, peroxide mediation, and acid/metal catalysis. Characterization of the fragmentation pattern of a therapeutic protein is traditionally accomplished using capillary gel electrophoresis with UV detection under both non-reducing and reducing conditions (nrCGE and rCGE). However, such methods are incompatible with direct coupling to mass spectrometry (MS) due to the use of anionic surfactants, e.g., sodium dodecyl sulfate (SDS). Here, we present a novel method to characterize size-based fragmentation variants of a new biotherapeutic kind using microfluidic ZipChip® capillary zone electrophoresis (mCZE) system interfaced with mass spectrometry (MS) to determine the molecular masses of fragments. A new modality of immuno-oncology therapy, bispecific antigen-binding biotherapeutic, was chosen to investigate its fragmentation pattern using mCZE-MS for the first time, according to our knowledge. Bispecific antigen-binding biotherapeutic samples from different stages of downstream column purification and forced degradation conditions were analyzed. The results were cross-validated with denaturing size-exclusion chromatography-mass spectrometry and conventional rSDS-CGE. In this study, we demonstrated that mCZE-MS could separate and characterize 12-40 kDa bispecific antigen-binding biotherapeutic fragments rapidly (within ≤12 minutes), with higher resolution and better sensitivity than traditional LC-MS methods.

Classification and Analysis of Human Body Movement Characteristics Associated with Acrophobia Induced by Virtual Reality Scenes of Heights.

Acrophobia (fear of heights), a prevalent psychological disorder, elicits profound fear and evokes a range of adverse physiological responses in individuals when exposed to heights, which will lead to a very dangerous state for people in actual heights. In this paper, we explore the behavioral influences in terms of movements in people confronted with virtual reality scenes of extreme heights and develop an acrophobia classification model based on human movement characteristics. To this end, we used wireless miniaturized inertial navigation sensors (WMINS) network to obtain the information of limb movements in the virtual environment. Based on these data, we constructed a series of data feature processing processes, proposed a system model for the classification of acrophobia and non-acrophobia based on human motion feature analysis, and realized the classification recognition of acrophobia and non-acrophobia through the designed integrated learning model. The final accuracy of acrophobia dichotomous classification based on limb motion information reached 94.64%, which has higher accuracy and efficiency compared with other existing research models. Overall, our study demonstrates a strong correlation between people's mental state during fear of heights and their limb movements at that time.

The catalytic mechanism of vitamin K epoxide reduction in a cellular environment.

Vitamin K epoxide reductases (VKORs) constitute a major family of integral membrane thiol oxidoreductases. In humans, VKOR sustains blood coagulation and bone mineralization through the vitamin K cycle. Previous chemical models assumed that the catalysis of human VKOR (hVKOR) starts from a fully reduced active site. This state, however, constitutes only a minor cellular fraction (5.6%). Thus, the mechanism whereby hVKOR catalysis is carried out in the cellular environment remains largely unknown. Here we use quantitative mass spectrometry (MS) and electrophoretic mobility analyses to show that KO likely forms a covalent complex with a cysteine mutant mimicking hVKOR in a partially oxidized state. Trapping of this potential reaction intermediate suggests that the partially oxidized state is catalytically active in cells. To investigate this activity, we analyze the correlation between the cellular activity and the cellular cysteine status of hVKOR. We find that the partially oxidized hVKOR has considerably lower activity than hVKOR with a fully reduced active site. Although there are more partially oxidized hVKOR than fully reduced hVKOR in cells, these two reactive states contribute about equally to the overall hVKOR activity, and hVKOR catalysis can initiate from either of these states. Overall, the combination of MS quantification and biochemical analyses reveals the catalytic mechanism of this integral membrane enzyme in a cellular environment. Furthermore, these results implicate how hVKOR is inhibited by warfarin, one of the most commonly prescribed drugs.

Lactiplantibacillus plantarum HG20 attenuates II type collagen-induced rheumatoid arthritis in rats via anti-inflammatory and inhibition of apoptosis.

AIMS: This study aimed to explore the therapeutic effects of Lactiplantibacillus plantarum HG20 (HG20) on collagen-induced arthritis (CIA) rats and its mechanism. METHODS AND RESULTS: CIA rats were established by injecting bovine type II collagen for 7 days, and treated by intragastric administration HG20 for 21 days. The foot palm temperature and arthritis score were measured once a week. The pathological changes in the knee joint were observed by hematoxylin and eosin staining. The levels of cytokines were detected by enzyme linked immunosorbent assay, and the effects of HG20 on inflammatory and apoptosis pathway of spleen cells were detected by western blot analysis. The results indicated that HG20 reduced the joint swelling degree and foot palm temperature, inhibited the development of joint histopathology, decreased the levels of pro-inflammatory cytokines, down-regulate the expression of pro-inflammatory cytokines by nuclear factor kappa-B pathway, and inhibited the apoptosis of spleen cells by inhibiting phosphatidylinositol 3-kinase/protein kinase B pathway and regulating apoptosis pathways. CONCLUSIONS: HG20 had an adjuvant therapeutic effect on arthritis in CIA rats, and its mechanism might be related to the inflammatory and apoptosis pathway. SIGNIFICANCE AND IMPACT OF STUDY: These results revealed that HG20 could be used as a functional probiotic in the field of food and medical, and which played a potential role in the prevention and treatment of arthritis.

Gated Recurrent Unit Network for Psychological Stress Classification Using Electrocardiograms from Wearable Devices.

In recent years, research on human psychological stress using wearable devices has gradually attracted attention. However, the physical and psychological differences among individuals and the high cost of data collection are the main challenges for further research on this problem. In this work, our aim is to build a model to detect subjects' psychological stress in different states through electrocardiogram (ECG) signals. Therefore, we design a VR high-altitude experiment to induce psychological stress for the subject to obtain the ECG signal dataset. In the experiment, participants wear smart ECG T-shirts with embedded sensors to complete different tasks so as to record their ECG signals synchronously. Considering the temporal continuity of individual psychological stress, a deep, gated recurrent unit (GRU) neural network is developed to capture the mapping relationship between subjects' ECG signals and stress in different states through heart rate variability features at different moments, so as to build a neural network model from the ECG signal to psychological stress detection. The experimental results show that compared with all comparison methods, our method has the best classification performance on the four stress states of resting, VR scene adaptation, VR task and recovery, and it can be a remote stress monitoring solution for some special industries.

Footprinting Mass Spectrometry of Membrane Proteins: Ferroportin Reconstituted in Saposin A Picodiscs.

Membrane proteins participate in a broad range of cellular processes and represent more than 60% of drug targets. One approach to their structural analyses is mass spectrometry (MS)-based footprinting including hydrogen/deuterium exchange (HDX), fast photochemical oxidation of proteins (FPOP), and residue-specific chemical modification. Studying membrane proteins usually requires their isolation from the native lipid environment, after which they often become unstable. To overcome this problem, we are pursuing a novel methodology of incorporating membrane proteins into saposin A picodiscs for MS footprinting. We apply different footprinting approaches to a model membrane protein, mouse ferroportin, in picodiscs and achieve high coverage that enables the analysis of the ferroportin structure. FPOP footprinting shows extensive labeling of the extramembrane regions of ferroportin and protection at its transmembrane regions, suggesting that the membrane folding of ferroportin is maintained throughout the labeling process. In contrast, an amphipathic reagent, N-ethylmaleimide (NEM), efficiently labels cysteine residues in both extramembrane and transmembrane regions, thereby affording complementary footprinting coverage. Finally, optimization of sample treatment gives a peptic-map of ferroportin in picodiscs with 92% sequence coverage, setting the stage for HDX. These results, taken together, show that picodiscs are a new platform broadly applicable to mass spectrometry studies of membrane proteins.

Membrane Protein Structure in Live Cells: Methodology for Studying Drug Interaction by Mass Spectrometry-Based Footprinting.

Mass spectrometry-based footprinting is an emerging approach for studying protein structure. Because integral membrane proteins are difficult targets for conventional structural biology, we recently developed a mass spectrometry (MS) footprinting method to probe membrane protein-drug interactions in live cells. This method can detect structural differences between apo and drug-bound states of membrane proteins, with the changes inferred from MS quantification of the cysteine modification pattern, generated by residue-specific chemical labeling. Here, we describe the experimental design, interpretation, advantages, and limitations of using cysteine footprinting by taking as an example the interaction of warfarin with vitamin K epoxide reductase, a human membrane protein. Compared with other structural methods, footprinting of proteins in live cells produces structural information for the near native state. Knowledge of cellular conformational states is a necessary complement to the high-resolution structures obtained from purified proteins in vitro. Thus, the MS footprinting method is broadly applicable in membrane protein biology. Future directions include probing flexible motions of membrane proteins and their interaction interface in live cells, which are often beyond the reach of conventional structural methods.

Human Metabolome-derived Cofactors Are Required for the Antibacterial Activity of Siderocalin in Urine.

In human urinary tract infections, host cells release the antimicrobial protein siderocalin (SCN; also known as lipocalin-2, neutrophil gelatinase-associated lipocalin, or 24p3) into the urinary tract. By binding to ferric catechol complexes, SCN can sequester iron, a growth-limiting nutrient for most bacterial pathogens. Recent evidence links the antibacterial activity of SCN in human urine to iron sequestration and metabolomic variation between individuals. To determine whether these metabolomic associations correspond to functional Fe(III)-binding SCN ligands, we devised a biophysical protein binding screen to identify SCN ligands through direct analysis of human urine. This screen revealed a series of physiologic unconjugated urinary catechols that were able to function as SCN ligands of which pyrogallol in particular was positively associated with high urinary SCN activity. In a purified, defined culture system, these physiologic SCN ligands were sufficient to activate SCN antibacterial activity against Escherichia coli In the presence of multiple SCN ligands, native mass spectrometry demonstrated that SCN may preferentially combine different ligands to coordinate iron, suggesting that availability of specific ligand combinations affects in vivo SCN antibacterial activity. These results support a mechanistic link between the human urinary metabolome and innate immune function.

Laser-Initiated Radical Trifluoromethylation of Peptides and Proteins: Application to Mass-Spectrometry-Based Protein Footprinting.

Described is a novel, laser-initiated radical trifluoromethylation for protein footprinting and its broad residue coverage. . CF3 reacts with 18 of the 20 common amino acids, including Gly, Ala, Ser, Thr, Asp, and Glu, which are relatively silent with regard to . OH. This new approach to footprinting is a bridge between trifluoromethylation in materials and medicinal chemistry and structural biology and biotechnology. Its application to a membrane protein and to myoglobin show that the approach is sensitive to protein conformational change and solvent accessibility.

Paenibacillus solani sp. nov., isolated from potato rhizosphere soil.

A novel Gram-stain-positive, endospore-forming bacterium, designated FJAT-22460T, was isolated from a soil sample of a potato field in Xinjiang Autonomous Region, China. Cells were rods that were catalase-positive and motile by peritrichous flagella. The strain was found to grow at temperatures ranging from 10 to 40 °C (optimum 30 °C) and at pH 5.0-12.0 (optimum pH 7) with 0-5 % (w/v) NaCl (optimum 0 % NaCl). Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain FJAT-22460T belonged to the genus Paenibacillus and exhibited 16S rRNA gene sequence similarities of 97.3, 97.2, 97.2 and 97.0 % with Paenibacillus glucanolyticus DSM 5162T, Paenibacillus lautus DSM 3035T, Paenibacillus lactis MB 1871T and Paenibacillus chibensis JCM 9905T, respectively. DNA-DNA relatedness of strain FJAT-22460T with Paenibacillus glucanolyticusDSM 5162T and Paenibacillus lautus DSM 3035T was 62.6 % and 33.3 %, respectively, lower than the 70 % accepted for species delineation. The menaquinone was identified as MK-7. The major fatty acids detected were anteiso-C15 : 0 (51.4 %), iso-C15 : 0 (5.3 %), C16 : 0 (12.1 %), iso-C16 : 0 (10.7 %) and anteiso-C17 : 0 (6.9 %). The DNA G+C content was determined to be 50.9 mol%. Phenotypic, chemotaxonomic and genotypic properties clearly indicated that isolate FJAT-22460T represents a novel species within the genus Paenibacillus, for which the name Paenibacillus solani sp. nov. is proposed. The type strain is FJAT-22460T (=DSM 100999T=CCTCC AB 2015207T).

[Preventive Effect of Fangshuan Capsule on PCI-induced Myocardial Damage and Vascular Endo- thelial Injury in Patients with Unstable Angina Pectoris].

Objective To observe the prevention of Fangshuan Capsule (FC) on percutaneous coronary intervention (PCI) induced myocardial damage and vascular endothelial injury in patients with un- stable angina pectoris (UAP). Methods Totally 100 UAP patients undergoing PCI were assigned to the control group and the treatment group by random digit table, 50 in each group. All patients received routine Western medicine therapy. Those in the treatment group additionally took FC, 6 pills each time, three times per day for at least 2 days before PCI operation. The therapeutic course for each group was 2 weeks. The clinical therapeutic effect was observed in the two groups. Heart rate (HR), systolic blood pressure (SBP) , changes of myocardial oxygen consumption ( HR x SBP, kPa/min) were compared. The levels of serum troponin I (cTn 1), creatinine kinase-MB (CK-MB) , myoglobin (MYO) , endothelin (ET), and nitric oxide (NO) were measured before PCI, and 6, 12, 24 h, 3 and 7 days after PCI. Results The markedly effective rate of Chinese medical syndromes was 54% (17/50) and the total effective rate was 94% (47/50) in the treatment group, obviously higher than those of the control group [26% (13/50) and 88% (44/50) ; P <0. 01]. Compared with before treatment in the same group, HR, SBP, myocardial oxygen consumption, and plasma ET level were reduced, plasma NO level was elevated in two groups after treatment (P <0.05, P <0. 01). cTnl concentration increased at 6, 12, 24 h, and day 3 (P <0. 05, P <0. 01 ) ; CK-MB concentration was elevated at 6, 12, and 24 h (P <0. 05, P <0. 01) ; MYO concentration increased at 6 and 12 h (P < 0. 01) in the control group after treatment. cTnl concentration increased at 12 and 24 h (P <0. 05, P <0. 01); CK-MB concentration was elevated at 6 and 12 h (P <0. 05) ; MYO concentration increased at 6 h (P <0. 01) in the treatment group after treatment. Compared with the control group at the same time point, HR, myocardial oxygen consumption, and plasma ET level decreased (P <0. 05); cTnl decreased at 6, 12, and 24 h (P <0. 05); CK-MB concentration decreased at 12 h (P <0. 05); MYO concentration decreased at 6 and 12 h (P <0. 05) in the treatment group after treatment. Conclusion FC could effectively improve scores of Chinese medical syndromes after PCI surgery, reduce myocardial oxygen consumption, attenuate myocar- dial damage and vascular endothelial injury in UAP patients after PCI.

Top-Down Mass Spectrometry Analysis of Membrane-Bound Light-Harvesting Complex 2 from Rhodobacter sphaeroides.

We report a top-down proteomic analysis of the membrane-bound peripheral light-harvesting complex LH2 isolated from the purple photosynthetic bacterium Rhodobacter sphaeroides. The LH2 complex is coded for by the puc operon. The Rb. sphaeroides genome contains two puc operons, designated puc1BAC and puc2BA. Although previous work has shown consistently that the LH2 ß polypeptide coded by the puc2B gene was assembled into LH2 complexes, there are contradictory reports as to whether the Puc2A polypeptides are incorporated into LH2 complexes. Furthermore, post-translational modifications of this protein offer the prospect that it could coordinate bacteriochlorophyll a (Bchl a) by a modified N-terminal residue. Here, we describe the components of the LH2 complex on the basis of electron-capture dissociation fragmentation to confirm the identity and sequence of the protein's subunits. We found that both gene products of the ß polypeptides are expressed and assembled in the mature LH2 complex, but only the Puc1A-encoded polypeptide α is observed here. The methionine of the Puc2B-encoded polypeptide is missing, and a carboxyl group is attached to the threonine at the N-terminus. Surprisingly, one amino acid encoded as an isoleucine in both the puc2B gene and the mRNA is found as valine in the mature LH2 complex, suggesting an unexpected and unusual post-translational modification or a specific tRNA recoding of this one amino acid.

Bacillus solani sp. nov., isolated from rhizosphere soil of a potato field.

A novel Gram-stain-positive, endospore-forming bacterium, designated strain FJAT-18043T, was isolated from a soil sample of a potato field in Xinjiang Autonomous Region, China. Cells were rods that were catalase-positive and motile by peritrichous flagella. The strain grew at 20-45 °C (optimum 35 °C), at pH 6.0-10.0 (optimum pH 9) and with 0-10 % (w/v) NaCl (optimum 0 %). Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain FJAT-18043T belonged to the genus Bacillus and exhibited similarities of 97.7, 97.6, 97.2 and 97.2 % with Bacillus eiseniae A1-2T, Bacillus horneckiae DSM 23495T, Bacillus gottheilii WCC 4585T and Bacillus purgationiresistens DS22T, respectively. DNA-DNA relatedness between strain FJAT-18043T and B. eiseniae A1-2 T was lower than 70 % (36.1 %). The menaquinone was identified as MK-7 and the major polar lipids were diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine. The major fatty acids detected were anteiso-C15 : 0, iso-C15 : 0, iso-C16 : 0, anteiso-C17 : 0, C16 : 0 and iso-C14 : 0. The DNA G+C content was 48.8 mol%. Phenotypic, chemotaxonomic and genotypic properties clearly indicated that isolate FJAT-18043T represents a novel species within the genus Bacillus, for which the name Bacillus solani sp. nov. is proposed. The type strain is FJAT-18043T ( = DSM 29501T = CCTCC AB 2014277T).

Mapping conformational changes on bispecific antigen-binding biotherapeutic by covalent labeling and mass spectrometry.

Biotherapeutic's higher order structure (HOS) is a critical determinant of its functional properties and conformational relevance. Here, we evaluated two covalent labeling methods: diethylpyrocarbonate (DEPC)-labeling and fast photooxidation of proteins (FPOP), in conjunction with mass spectrometry (MS), to investigate structural modifications for the new class of immuno-oncological therapy known as bispecific antigen-binding biotherapeutics (BABB). The evaluated techniques unveiled subtle structural changes occurring at the amino acid residue level within the antigen-binding domain under both native and thermal stress conditions, which cannot be detected by conventional biophysical techniques, e.g., near-ultraviolet circular dichroism (NUV-CD). The determined variations in labeling uptake under native and stress conditions, corroborated by binding assays, shed light on the binding effect, and highlighted the potential of covalent-labeling methods to effectively monitor conformational changes that ultimately influence the product quality. Our study provides a foundation for implementing the developed techniques in elucidating the inherent structural characteristics of novel therapeutics and their conformational stability.

Intelligence Sparse Sensor Network for Automatic Early Evaluation of General Movements in Infants.

General movements (GMs) have been widely used for the early clinical evaluation of infant brain development, allowing immediate evaluation of potential development disorders and timely rehabilitation. The infants' general movements can be captured digitally, but the lack of quantitative assessment and well-trained clinical pediatricians presents an obstacle for many years to achieve wider deployment, especially in low-resource settings. There is a high potential to explore wearable sensors for movement analysis due to outstanding privacy, low cost, and easy-to-use features. This work presents a sparse sensor network with soft wireless IMU devices (SWDs) for automatic early evaluation of general movements in infants. The sparse network consisting of only five sensor nodes (SWDs) with robust mechanical properties and excellent biocompatibility continuously and stably captures full-body motion data. The proof-of-the-concept clinical testing with 23 infants showcases outstanding performance in recognizing neonatal activities, confirming the reliability of the system. Taken together with a tiny machine learning algorithm, the system can automatically identify risky infants based on the GMs, with an accuracy of up to 100% (99.9%). The wearable sparse sensor network with an artificial intelligence-based algorithm facilitates intelligent evaluation of infant brain development and early diagnosis of development disorders.

Highly efficient ionization of phosphopeptides at low pH by desorption electrospray ionization mass spectrometry.

A fast and novel strategy for efficient ionization of phosphopeptides in mixtures is reported, in which the sample is acidified to low pH to suppress the deprotonation of phosphate groups and then followed by direct analysis using liquid sample desorption electrospray ionization mass spectrometry (DESI-MS).

Native electrospray ionization and electron-capture dissociation for comparison of protein structure in solution and the gas phase.

The importance of protein and protein-complex structure motivates improvements in speed and sensitivity of structure determination in the gas phase and comparison with that in solution or solid state. An opportunity for the gas phase measurement is mass spectrometry (MS) combined with native electrospray ionization (ESI), which delivers large proteins and protein complexes in their near-native states to the gas phase. In this communication, we describe the combination of native ESI, electron-capture dissociation (ECD), and top-down MS for exploring the structures of ubiquitin and cytochrome c in the gas phase and their relation to those in the solid-state and solution. We probe structure by comparing the protein's flexible regions, as predicted by the B-factor in X-ray crystallography, with the ECD fragments. The underlying hypothesis is that maintenance of structure gives fragments that can be predicted from B-factors. This strategy may be applicable in general when X-ray structures are available and extendable to the study of intrinsically disordered proteins.

Structural model and spectroscopic characteristics of the FMO antenna protein from the aerobic chlorophototroph, Candidatus Chloracidobacterium thermophilum.

The Fenna-Matthews-Olson protein (FMO) binds seven or eight bacteriochlorophyll a (BChl a) molecules and is an important model antenna system for understanding pigment-protein interactions and mechanistic aspects of photosynthetic light harvesting. FMO proteins of green sulfur bacteria (Chlorobiales) have been extensively studied using a wide range of spectroscopic and theoretical approaches because of their stability, the spectral resolution of their pigments, their water-soluble nature, and the availability of high-resolution structural data. We obtained new structural and spectroscopic insights by studying the FMO protein from the recently discovered, aerobic phototrophic acidobacterium, Candidatus Chloracidobacterium thermophilum. Native C. thermophilum FMO is a trimer according to both analytical gel filtration and native-electrospray mass spectrometry. Furthermore, the mass of intact FMO trimer is consistent with the presence of 21-24 BChl a in each. Homology modeling of the C. thermophilum FMO was performed by using the structure of the FMO protein from Chlorobaculum tepidum as a template. C. thermophilum FMO differs from C. tepidum FMO in two distinct regions: the baseplate, CsmA-binding region and a region that is proposed to bind the reaction center subunit, PscA. C. thermophilum FMO has two fluorescence emission peaks at room temperature but only one at 77K. Temperature-dependent fluorescence spectroscopy showed that the two room-temperature emission peaks result from two excited-state BChl a populations that have identical fluorescence lifetimes. Modeling of the data suggests that the two populations contain 1-2 BChl and 5-6 BChl a molecules and that thermal equilibrium effects modulate the relative population of the two emitting states.

Electrochemistry-assisted top-down characterization of disulfide-containing proteins.

Covalent disulfide bond linkage in a protein represents an important challenge for mass spectrometry (MS)-based top-down protein structure analysis as it reduces the backbone cleavage efficiency for MS/MS dissociation. This study presents a strategy for solving this critical issue via integrating electrochemistry (EC) online with a top-down MS approach. In this approach, proteins undergo electrolytic reduction in an electrochemical cell to break disulfide bonds and then undergo online ionization into gaseous ions for analysis by electron-capture dissociation (ECD) and collision-induced dissociation (CID). The electrochemical reduction of proteins allows one to remove disulfide bond constraints and also leads to increased charge numbers of the resulting protein ions. As a result, sequence coverage was significantly enhanced, as exemplified by ß-lactoglobulin A (24 vs 75 backbone cleavages before and after electrolytic reduction, respectively) and lysozyme (5 vs 66 backbone cleavages before and after electrolytic reduction, respectively). This methodology is fast and does not need chemical reductants, which would have an important impact in high-throughput proteomics research.

Native mass spectrometry characterization of intact nanodisc lipoprotein complexes.

We describe here the analysis of nanodisc complexes by using native mass spectrometry (MS) to characterize their molecular weight (MW) and polydispersity. Nanodiscs are nanoscale lipid bilayers that offer a platform for solubilizing membrane proteins. Unlike detergent micelles, nanodiscs are native-like lipid bilayers that are well-defined and potentially monodisperse. Their mass spectra allow peak assignment based on differences in the mass of a single lipid per complex. Resultant masses agree closely with predicted values and demonstrate conclusively the narrow dispersity of lipid molecules in the nanodisc. Fragmentation with collisionally activated dissociation (CAD) or electron-capture dissociation (ECD) shows loss of a small number of lipids and eventual collapse of the nanodisc with release of the scaffold protein. These results provide a foundation for future studies utilizing nanodiscs as a platform for launching membrane proteins into the gas phase.