A multispecific antibody against SARS-CoV-2 prevents immune escape in vitro and confers prophylactic protection in vivo.

Despite effective countermeasures, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) persists worldwide because of its ability to diversify and evade human immunity. This evasion stems from amino acid substitutions, particularly in the receptor binding domain (RBD) of the spike protein that confers resistance to vaccine-induced antibodies and antibody therapeutics. To constrain viral escape through resistance mutations, we combined antibody variable regions that recognize different RBD sites into multispecific antibodies.

Site-Specific Fluorescent Labeling of Hemagglutinin-Specific Antigen Binding Fragment through Amine Chemistry Revealed by Mass Spectrometry.

To capture the structure of assembled hemagglutinin (HA) nanoparticles at single-particle resolution, HA-specific antigen binding fragments (Fabs) were labeled by fluorescent (FLR) dyes as probes to highlight the HA trimers displayed on the assembled tetravalent HA nanoparticles for a qualitative localization microscopic study. The FLR dyes were conjugated to the Fabs through -hydroxysuccinimide (NHS) ester mediated amine coupling chemistry. The labeling profile, including labeling ratio, distribution, and site-specific labeling occupancy, can affect the imaging results and introduce inconsistency.

Large-Scale Interlaboratory DI-FT-ICR MS Comparability Study Employing Various Systems.

Ultrahigh resolution mass spectrometry (UHR-MS) coupled with direct infusion (DI) electrospray ionization offers a fast solution for accurate untargeted profiling. Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometers have been shown to produce a wealth of insights into complex chemical systems because they enable unambiguous molecular formula assignment even if the vast majority of signals is of unknown identity. Interlaboratory comparisons are required to apply this type of instrumentation in quality control (for food industry or pharmaceuticals), large-scale environmental studies, or clinical diagnostics.

A multispecific antibody prevents immune escape and confers pan-SARS-CoV-2 neutralization.

Despite effective countermeasures, SARS-CoV-2 persists worldwide due to its ability to diversify and evade human immunity. This evasion stems from amino-acid substitutions, particularly in the receptor-binding domain of the spike, that confer resistance to vaccines and antibodies . To constrain viral escape through resistance mutations, we combined antibody variable regions that recognize different receptor binding domain (RBD) sites into multispecific antibodies.

Spatially resolved isotope tracing reveals tissue metabolic activity.

Isotope tracing has helped to determine the metabolic activities of organs. Methods to probe metabolic heterogeneity within organs are less developed. We couple stable-isotope-labeled nutrient infusion to matrix-assisted laser desorption ionization imaging mass spectrometry (iso-imaging) to quantitate metabolic activity in mammalian tissues in a spatially resolved manner.

Cholesterol-binding translocator protein TSPO regulates steatosis and bile acid synthesis in nonalcoholic fatty liver disease.

Translocator protein (TSPO, 18 kDa) levels increase in parallel with the evolution of simple steatosis (SS) to nonalcoholic steatohepatitis (NASH) in nonalcoholic fatty liver disease (NAFLD). However, TSPO function in SS and NASH is unknown. Loss of TSPO in hepatocytes downregulated acetyl-CoA acetyltransferase 2 and increased free cholesterol (FC).

High Mass Analysis with a Fourier Transform Ion Cyclotron Resonance Mass Spectrometer: From Inorganic Salt Clusters to Antibody Conjugates and Beyond.

Analysis of proteins and complexes under native mass spectrometric (MS) and solution conditions was typically performed using time-of-flight (ToF) analyzers, due to their routine high / transmission and detection capabilities. However, over recent years, the ability of Orbitrap-based mass spectrometers to transmit and detect a range of high molecular weight species is well documented. Herein, we describe how a 15 Tesla Fourier transform ion cyclotron resonance mass spectrometer (15 T FT-ICR MS) is more than capable of analyzing a wide range of ions in the high / scale (>5000), in both positive and negative instrument polarities, ranging from the inorganic cesium iodide salt clusters; a humanized IgG1 monoclonal antibody (mAb; 148.

Best practices and benchmarks for intact protein analysis for top-down mass spectrometry.

One gene can give rise to many functionally distinct proteoforms, each of which has a characteristic molecular mass. Top-down mass spectrometry enables the analysis of intact proteins and proteoforms. Here members of the Consortium for Top-Down Proteomics provide a decision tree that guides researchers to robust protocols for mass analysis of intact proteins (antibodies, membrane proteins and others) from mixtures of varying complexity.

Using Tumor Explants for Imaging Mass Spectrometry Visualization of Unlabeled Peptides and Small Molecules.

Matrix assisted laser desorption ionization time-of-flight (MALDI-TOF) imaging mass spectrometry has emerged as a powerful, label-free technique to visualize penetration of small molecules and , including in 3D cell culture spheroids; however, some spheroids do not grow sufficiently large to provide enough area for imaging mass spectrometry. Here, we describe an method for visualizing unlabeled peptides and small molecules in tumor explants, which can be divided into pieces of desired size, thus circumventing the size limitations of many spheroids. As proof-of-concept, a small molecule drug (4-hydroxytamoxifen), as well as a peptide drug (cyclosporin A) and peptide chemical probe, can be visualized after incubation with tumor explants so that this technique may provide a solution to robing cell penetration by unlabeled peptides.

Surface-Induced Dissociation of Protein Complexes in a Hybrid Fourier Transform Ion Cyclotron Resonance Mass Spectrometer.

Mass spectrometry continues to develop as a valuable tool in the analysis of proteins and protein complexes. In protein complex mass spectrometry studies, surface-induced dissociation (SID) has been successfully applied in quadrupole time-of-flight (Q-TOF) instruments. SID provides structural information on noncovalent protein complexes that is complementary to other techniques.

Towards the Analysis of High Molecular Weight Proteins and Protein complexes using TIMS-MS.

In the present work, we demonstrate the potential and versatility of TIMS for the analysis of proteins, DNA-protein complexes and protein-protein complexes in their native and denatured states. In addition, we show that accurate CCS measurement are possible and in good agreement with previously reported CCS values using other IMS analyzers (<5% difference). The main challenges for the analysis of high mass proteins and protein complexes in the mobility and domain are described.

Gated Trapped Ion Mobility Spectrometry Coupled to Fourier Transform Ion Cyclotron Resonance Mass Spectrometry.

Analysis of molecules by ion mobility spectrometry coupled with mass spectrometry (IMS-MS) provides chemical information on the three dimensional structure and mass of the molecules. The coupling of ion mobility to trapping mass spectrometers has historically been challenging due to the large differences in analysis time between the two devices. In this paper we present a modification of the trapped ion mobility (TIMS) analysis scheme termed "Gated TIMS" that allows efficient coupling to a Fourier Transform Ion Cyclotron Resonance (FT-ICR) analyzer.

Comprehensive assessment of chamber-specific and transmural heterogeneity in myofilament protein phosphorylation by top-down mass spectrometry.

The heart is characterized by a remarkable degree of heterogeneity, the basis of which is a subject of active investigation. Myofilament protein post-translational modifications (PTMs) represent a critical mechanism regulating cardiac contractility, and emerging evidence shows that pathological cardiac conditions induce contractile heterogeneity that correlates with transmural variations in the modification status of myofilament proteins. Nevertheless, whether there exists basal heterogeneity in myofilament protein PTMs in the heart remains unclear.

Site-specific Labeling of a Protein Lysine Residue By Novel Kinetic Labeling Combinatorial Libraries.

The first example of a kinetic labeling library designed to enable the discovery of affinity labels is presented. Each library component (1) consists of a variable peptidyl component linked to a biotinyl moiety by a 4-mercaptobenzoyl linker in thioester format. We demonstrate that an affinity label can be uncovered by measuring reaction rates between library pools and the protein target, human serum albumin (HSA) and identifying significant outliers.

The first pilot project of the consortium for top-down proteomics: a status report.

Pilot Project #1--the identification and characterization of human histone H4 proteoforms by top-down MS--is the first project launched by the Consortium for Top-Down Proteomics (CTDP) to refine and validate top-down MS. Within the initial results from seven participating laboratories, all reported the probability-based identification of human histone H4 (UniProt accession P62805) with expectation values ranging from 10(-13) to 10(-105). Regarding characterization, a total of 74 proteoforms were reported, with 21 done so unambiguously; one new PTM, K79ac, was identified.

Top-down protein identification of proteasome proteins with nanoLC-FT-ICR-MS employing data-independent fragmentation methods.

A comparison of different data-independent fragmentation methods combined with LC coupled to high-resolution FT-ICR-MS/MS is presented for top-down MS of protein mixtures. Proteins composing the 20S and 19S proteasome complexes and their PTMs were identified using a 15 T FT-ICR mass spectrometer. The data-independent fragmentation modes with LC timescales allowed for higher duty-cycle measurements that better suit online LC-FT-ICR-MS.

Native top-down electrospray ionization-mass spectrometry of 158 kDa protein complex by high-resolution Fourier transform ion cyclotron resonance mass spectrometry.

Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS) delivers high resolving power, mass measurement accuracy, and the capabilities for unambiguously sequencing by a top-down MS approach. Here, we report isotopic resolution of a 158 kDa protein complex, tetrameric aldolase with an average absolute deviation of 0.36 ppm and an average resolving power of ~520 000 at m/z 6033 for the 26+ charge state in magnitude mode.

Hexuronic acid stereochemistry determination in chondroitin sulfate glycosaminoglycan oligosaccharides by electron detachment dissociation.

Electron detachment dissociation (EDD) has previously provided stereo-specific product ions that allow for the assignment of the acidic C-5stereochemistry in heparan sulfate glycosaminoglycans (GAGs), but application of the same methodology to an epimer pair in the chondroitin sulfate glycoform class does not provide the same result. A series of experiments have been conducted in which glycosaminoglycan precursor ions are independently activated by electron detachment dissociation (EDD), electron induced dissociation (EID), and negative electron transfer dissociation (NETD) to assign the stereochemistry in chondroitin sulfate (CS) epimers and investigate the mechanisms for product ion formation during EDD in CS glycoforms. This approach allows for the assignment of electronic excitation products formed by EID and detachment products to radical pathways in NETD, both of which occur simultaneously during EDD.

Meta-omic characterization of the marine invertebrate microbial consortium that produces the chemotherapeutic natural product ET-743.

In many macroorganisms, the ultimate source of potent biologically active natural products has remained elusive due to an inability to identify and culture the producing symbiotic microorganisms. As a model system for developing a meta-omic approach to identify and characterize natural product pathways from invertebrate-derived microbial consortia, we chose to investigate the ET-743 (Yondelis) biosynthetic pathway. This molecule is an approved anticancer agent obtained in low abundance (10(-4)-10(-5) % w/w) from the tunicate Ecteinascidia turbinata and is generated in suitable quantities for clinical use by a lengthy semisynthetic process.

Negative electron transfer dissociation Fourier transform mass spectrometry of glycosaminoglycan carbohydrates.

Electron transfer through gas phase ion-ion reactions has led to the widespread application of electron- based techniques once only capable in ion trapping mass spectrometers. Although any mass analyzer can in theory be coupled to an ion-ion reaction device (typically a 3-D ion trap), some systems of interest exceed the capabilities of most mass spectrometers. This case is particularly true in the structural characterization of glycosaminoglycan (GAG) oligosaccharides.

Negative electron transfer dissociation of glycosaminoglycans.

Structural characterization of glycosaminoglycans (GAGs) has been a challenge in the field of mass spectrometry, and the application of electron detachment dissociation (EDD) Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) has shown great promise to GAG oligosaccharide characterization in a single tandem mass spectrometry experiment. In this work, we apply the technique of negative electron transfer dissociation (NETD) to GAGs on a commercial ion trap mass spectrometer. NETD of GAGs, using fluoranthene or xenon as the reagent gas, produces fragmentation very similar to previously observed EDD fragmentation.

Synthesis of 7-(15)N-Oroidin and evaluation of utility for biosynthetic studies of pyrrole-imidazole alkaloids by microscale (1)H-(15)N HSQC and FTMS.

Numerous marine-derived pyrrole-imidazole alkaloids (PIAs), ostensibly derived from the simple precursor oroidin, 1a, have been reported and have garnered intense synthetic interest due to their complex structures and in some cases biological activity; however very little is known regarding their biosynthesis. We describe a concise synthesis of 7-(15)N-oroidin (1d) from urocanic acid and a direct method for measurement of (15)N incorporation by pulse labeling and analysis by 1D (1)H-(15)N HSQC NMR and FTMS. Using a mock pulse labeling experiment, we estimate the limit of detection (LOD) for incorporation of newly biosynthesized PIA by 1D (1)H-(15)N HSQC to be 0.

EVALUATION OF THE EXPERIMENTAL PARAMETERS WHICH CONTROL ELECTRON DETACHMENT DISSOCIATION, AND THEIR EFFECT ON THE FRAGMENTATION EFFICIENCY OF GLYCOSAMINOGLYCAN CARBOHYDRATES.

The efficiency of conversion of precursor ions to observable products for electron detachment dissociation (EDD) was measured as a function of the key experimental parameters to determine their optimal values for the Fourier transform mass spectrometry analysis of anionic glycosaminoglycan carbohydrates. These parameters include electron current, electron energy, dispenser cathode heater current, electron beam duration, charge state of the precursor ion, oligomer length, and precursor ion number accumulated in an external radio frequency multipole trap. Precursor conversion is most efficient at an electron current of 15 µA, and decreases at higher and lower values.