CD5L is a canonical component of circulatory IgM.

Immunoglobulin M (IgM) is an evolutionary conserved key component of humoral immunity, and the first antibody isotype to emerge during an immune response. IgM is a large (1 MDa), multimeric protein, for which both hexameric and pentameric structures have been described, the latter additionally containing a joining (J) chain. Using a combination of single-particle mass spectrometry and mass photometry, proteomics, and immunochemical assays, we here demonstrate that circulatory (serum) IgM exclusively exists as a complex of J-chain-containing pentamers covalently bound to the small (36 kDa) protein CD5 antigen-like (CD5L, also called apoptosis inhibitor of macrophage). In sharp contrast, secretory IgM in saliva and milk is principally devoid of CD5L. Unlike IgM itself, CD5L is not produced by B cells, implying that it associates with IgM in the extracellular space. We demonstrate that CD5L integration has functional implications, i.e., it diminishes IgM binding to two of its receptors, the FcαµR and the polymeric Immunoglobulin receptor. On the other hand, binding to FcµR as well as complement activation via C1q seem unaffected by CD5L integration. Taken together, we redefine the composition of circulatory IgM as a J-chain containing pentamer, always in complex with CD5L.

Local structural flexibility drives oligomorphism in computationally designed protein assemblies.

Many naturally occurring protein assemblies have dynamic structures that allow them to perform specialized functions. For example, clathrin coats adopt a wide variety of architectures to adapt to vesicular cargos of various sizes. Although computational methods for designing novel self-assembling proteins have advanced substantially over the past decade, most existing methods focus on designing static structures with high accuracy. Here we characterize the structures of three distinct computationally designed protein assemblies that each form multiple unanticipated architectures, and identify flexibility in specific regions of the subunits of each assembly as the source of structural diversity. Cryo-EM single-particle reconstructions and native mass spectrometry showed that only two distinct architectures were observed in two of the three cases, while we obtained six cryo-EM reconstructions that likely represent a subset of the architectures present in solution in the third case. Structural modeling and molecular dynamics simulations indicated that the surprising observation of a defined range of architectures, instead of non-specific aggregation, can be explained by constrained flexibility within the building blocks. Our results suggest that deliberate use of structural flexibility as a design principle will allow exploration of previously inaccessible structural and functional space in designed protein assemblies.

Comparative Analysis of Antibodies and Heavily Glycosylated Macromolecular Immune Complexes by Size-Exclusion Chromatography Multi-Angle Light Scattering, Native Charge Detection Mass Spectrometry, and Mass Photometry.

Qualitative and quantitative mass analysis of antibodies and related macromolecular immune complexes is a prerequisite for determining their identity, binding partners, stoichiometries, and affinities. A plethora of bioanalytical technologies exist to determine such characteristics, typically based on size, interaction with functionalized surfaces, light scattering, or direct mass measurements. While these methods are highly complementary, they also exhibit unique strengths and weaknesses. Here, we benchmark mass photometry (MP), a recently introduced technology for mass measurement, against native mass spectrometry (MS) and size exclusion chromatography multi-angle light scattering (SEC-MALS). We examine samples of variable complexity, namely, IgG4Δhinge dimerizing half-bodies, IgG-RGY hexamers, heterogeneously glycosylated IgG:sEGFR antibody-antigen complexes, and finally megadalton assemblies involved in complement activation. We thereby assess the ability to determine (1) binding affinities and stoichiometries, (2) accurate masses, for extensively glycosylated species, and (3) assembly pathways of large heterogeneous immune complexes. We find that MP provides a sensitive approach for characterizing antibodies and stable assemblies, with dissociation correction enabling us to expand the measurable affinity range. In terms of mass resolution and accuracy, native MS performs the best but is occasionally hampered by artifacts induced by electrospray ionization, and its resolving power diminishes when analyzing extensively glycosylated proteins. In the latter cases, MP performs well, but single-particle charge detection MS can also be useful in this respect, measuring masses of heterogeneous assemblies even more accurately. Both methods perform well compared to SEC-MALS, still being the most established method in biopharma. Together, our data highlight the complementarity of these approaches, each having its unique strengths and weaknesses.

Probing Affinity, Avidity, Anticooperativity, and Competition in Antibody and Receptor Binding to the SARS-CoV-2 Spike by Single Particle Mass Analyses.

Determining how antibodies interact with the spike (S) protein of the SARS-CoV-2 virus is critical for combating COVID-19. Structural studies typically employ simplified, truncated constructs that may not fully recapitulate the behavior of the original complexes. Here, we combine two single particle mass analysis techniques (mass photometry and charge-detection mass spectrometry) to enable the measurement of full IgG binding to the trimeric SARS-CoV-2 S ectodomain. Our experiments reveal that antibodies targeting the S-trimer typically prefer stoichiometries lower than the symmetry-predicted 3:1 binding. We determine that this behavior arises from the interplay of steric clashes and avidity effects that are not reflected in common antibody constructs (i.e., Fabs). Surprisingly, these substoichiometric complexes are fully effective at blocking ACE2 binding despite containing free receptor binding sites. Our results highlight the importance of studying antibody/antigen interactions using complete, multimeric constructs and showcase the utility of single particle mass analyses in unraveling these complex interactions.

Extending Native Top-Down Electron Capture Dissociation to MDa Immunoglobulin Complexes Provides Useful Sequence Tags Covering Their Critical Variable Complementarity-Determining Regions.

Native top-down mass spectrometry (MS) is gaining traction for the analysis and sequencing of intact proteins and protein assemblies, giving access to their mass and composition, as well as sequence information useful for identification. Herein, we extend and apply native top-down MS, using electron capture dissociation, to two submillion Da IgM- and IgG-based oligomeric immunoglobulins. Despite structural similarities, these two systems are quite different. The ∼895 kDa noncovalent IgG hexamer consists of six IgG subunits hexamerizing in solution due to three specifically engineered mutations in the Fc region, whereas the ∼935 kDa IgM oligomer results from the covalent assembly of one joining (J) chain and 5 IgM subunits into an asymmetric "pentamer" stabilized by interchain disulfide bridges. Notwithstanding their size, structural differences, and complexity, we observe that their top-down electron capture dissociation spectra are quite similar and straightforward to interpret, specifically providing informative sequence tags covering the highly variable CDR3s and FR4s of the Ig subunits they contain. Moreover, we show that the electron capture dissociation fragmentation spectra of immunoglobulin oligomers are essentially identical to those obtained for their respective monomers. Demonstrated for recombinantly produced systems, the approach described here opens up new prospects for the characterization and identification of IgMs circulating in plasma, which is important since IgMs play a critical role in the early immune response to pathogens such as viruses and bacteria.

Single-particle mass analysis of intact ribosomes by mass photometry and Orbitrap-based charge detection mass spectrometry.

Standard methods for mass analysis measure ensembles of thousand to millions of molecules. This approach enables analysis of monodisperse recombinant proteins, whereas some heterogeneous protein assemblies pose a significant challenge, whereby co-occurring stoichiometries, sub-complexes, and modifications hamper analysis using native mass spectrometry. To tackle the challenges posed by mass heterogeneity, single-particle methods may come to the rescue. Recently, two such approaches have been introduced, namely, mass photometry (MP) and Orbitrap-based charge detection mass spectrometry (CDMS). Both methods assess masses of individual molecules, albeit adhering to distinct physical principles. To evaluate these methods side by side, we analyzed a set of ribosomal particles, representing polydisperse ribonucleoprotein assemblies in the MDa range. MP and CDMS provide accurate masses for intact ribosomes and enable quantitative analysis of concomitant distinct particles within each ribosome sample. Here, we discuss pros and cons of these single-molecule techniques, also in the context of other techniques used for mass analysis.

Requirements and attributes of nano-resonator mass spectrometry for the analysis of intact viral particles.

When studying viruses, the most prevalent aspects that come to mind are their structural and functional features, but this leaves in the shadows a quite universal characteristic: their mass. Even if approximations can be derived from size and density measurements, the multi MDa to GDa mass range, featuring a majority of viruses, has so far remained largely unexplored. Recently, nano-electromechanical resonator-based mass spectrometry (NEMS-MS) has demonstrated the ability to measure the mass of intact DNA filled viral capsids in excess of 100 MDa. However, multiple factors have to be taken in consideration when performing NEMS-MS measurements. In this article, phenomena influencing NEMS-MS mass estimates are listed and discussed, including some particle's extraneous physical properties (size, aspect ratio, stiffness), and the influence of frequency noise and device fabrication defects. These factors being accounted for, we could begin to notice subtler effects linked with (e.g.) particle desolvation as a function of operating parameters. Graphical abstract.

Development of a focused high-energy macromolecular ion beam.

In this work, we report the development of a focused macromolecular ion beam with kinetic energy of up to 110 keV. The system consists of a quadrupole ion trap (QIT), einzel lens and linear accelerator (LINAC). Based on the combination of matrix-assisted laser desorption ionization (MALDI) and quadrupole ion trapping (QIT), ions were desorbed from the surface and trapped with an ion trap to form biomolecular ion packets. Positive- and negative-pulsed voltages were applied on each end-cap electrode of the QIT to extract the ion packets and form an ion beam that was subsequently focused via an einzel lens and accelerated by stepwise pulsed voltages. The tabletop instrument was designed and successfully demonstrated via measurements of molecular ions of insulin, cytochrome c and bovine serum albumin (BSA) with mass-to-charge ratios (m/z) ranging from ∼5.8 to 66.5 k. This is the first report of both a focused and high-kinetic-energy protein ion beam. In addition, both secondary ions and electrons were observed from the surface by hypervelocity ion beam bombardment. This focused macromolecular ion beam has demonstrated its potential in the study of interactions between large molecular ions with other molecules either in the gas phase or upon a surface.

Biomolecular Clusters Distribution up to Mega Dalton Region Using MALDI-Quadrupole Ion Trap Mass Spectrometer.

We present the first report on complete cluster distributions of cytochrome c (molecular weight of 12.4 kDa) and bovine serum albumin ((BSA), molecular weight of 66.4 kDa) with mass-to-charge ratio (m/z) reaching 350,000 and 1,400,000, respectively, by matrix-assisted laser desorption/ionization (MALDI). Large cluster distributions of the analytes were measured by our homemade frequency-scanned quadrupole ion trap (QIT) mass spectrometer with a charge detector. To our knowledge, we report the highest m/z clusters of these two biomolecules. The quantitative results indicate that large clusters ions of cytochrome c and BSA follow the power law (r² > 0.99) with cluster size distribution, which provides experimental evidence for the laser ablation studies of MALDI.

ESI MS for Microsized Bioparticles.

An ESI ion trap mass spectrometer was designed for high-throughput and rapid mass analysis of large bioparticles. Mass calibration of the instrument was performed using commercially available polystyrene (PS) microparticles with a size comparable to cancer cells. Different sizes of MCF-7 breast cancer cells (8 to 15 µm) were used in this study. The masses of different cancer cells were measured. This system allows for the analysis of all types of particles.

Macromolecular ion accelerator.

Presented herein are the development of macromolecular ion accelerator (MIA) and the results obtained by MIA. This new instrument utilizes a consecutive series of planar electrodes for the purpose of facilitating stepwise acceleration. Matrix-assisted laser desorption/ionization (MALDI) is employed to generate singly charged macromolecular ions. A regular Z-gap microchannel plate (MCP) detector is mounted at the end of the accelerator to record the ion signals. In this work, we demonstrated the detection of ions with the mass-to-charge (m/z) ratio reaching 30,000,000. Moreover, we showed that singly charged biomolecular ions can be accelerated with the voltage approaching 1 MV, offering the evidence that macromolecular ions can possess much higher kinetic energy than ever before.

Frequency-scanning MALDI linear ion trap mass spectrometer for large biomolecular ion detection.

This study presents the first report on the development of a matrix-assisted laser desorption ionization (MALDI) linear ion trap mass spectrometer for large biomolecular ion detection by frequency scan. We designed, installed, and tested this radio frequency (RF) scan linear ion trap mass spectrometer and its associated electronics to dramatically extend the mass region to be detected. The RF circuit can be adjusted from 300 to 10 kHz with a set of operation amplifiers. To trap the ions produced by MALDI, a high pressure of helium buffer gas was employed to quench extra kinetic energy of the heavy ions produced by MALDI. The successful detection of the singly charged secretory immunoglobulin A ions indicates that the detectable mass-to-charge ratio (m/z) of this system can reach ~385 000 or beyond.

Disentangling intrinsic ultrafast excited-state dynamics of cytosine tautomers.

Gas-phase ultrafast excited-state dynamics of cytosine, 1-methylcytosine, and 5-fluorocytosine were investigated in molecular beams using femtosecond pump-probe photoionization spectroscopy to identify the intrinsic dynamics of the major cytosine tautomers. The results indicate that, upon photoexcitation in the first absorption band, the cytosine enol tautomer exhibits a significantly longer excited-state lifetime than its keto and imino counterparts. The initially excited states of the cytosine keto and imino tautomers decay with sub-picosecond dynamics for excitation wavelengths shorter than 300 nm, whereas that of the cytosine enol tautomer decays with time constants ranging from 3 to 45 ps for excitation between 260 and 285 nm.

Discrimination of mononuclear and dinuclear dinitrosyl iron complexes (DNICs) by S K-edge X-ray absorption spectroscopy: insight into the electronic structure and reactivity of DNICs.

In addition to probing the formation of dinitrosyl iron complexes (DNICs) by the characteristic Fe K-edge pre-edge absorption energy ranging from 7113.4 to 7113.8 eV, the distinct S K-edge pre-edge absorption energy and pattern can serve as an efficient tool to unambiguously characterize and discriminate mononuclear DNICs and dinuclear DNICs containing bridged-thiolate and bridged-sulfide ligands. The higher Fe-S bond covalency modulated by the stronger electron-donating thiolates promotes the Fe → NO π-electron back-donation to strengthen the Fe-NO bond and weaken the NO-release ability of the mononuclear DNICs, which is supported by the Raman ν(Fe-NO) stretching frequency. The Fe-S bond covalency of DNICs further rationalizes the binding preference of the {Fe(NO)(2)} motif toward thiolates following the trend of [SEt](-) > [SPh](-) > [SC(7)H(4)SN](-). The relative d-manifold energy derived from S K-edge XAS as well as the Fe K-edge pre-edge energy reveals that the electronic structure of the {Fe(NO)(2)}(9) core of the mononuclear DNICs [(NO)(2)Fe(SR)(2)](-) is best described as {Fe(III)(NO(-))(2)}(9) compared to [{Fe(III)(NO(-))(2)}(9)-{Fe(III)(NO(-))(2)}(9)] for the dinuclear DNICs [Fe(2)(µ-SEt)(µ-S)(NO)(4)](-) and [Fe(2)(µ-S)(2)(NO)(4)](2-).

Dynamics of the excited states of [Ir(ppy)2bpy]+ with triple phosphorescence.

We investigated the relaxation dynamics of bis(2-phenylpyridinato-)(2,2'-bipyridine)iridium(III), [Ir(ppy)(2)bpy](+) using the technique of time-resolved spectroscopy. In the visible emission spectra this molecule exhibits triple phosphorescence: displaying blue, green, and orange bands. From the dependence of spectral shifts with polarity of solvent, decay lifetimes, and the results of calculations using time-dependent density functional theory, we assigned these three emitting states to be triplet interligand charge-transfer ((3)LLCT), metal-to-ligand ppy charge transfer ((3)MLCT(ppy)), and metal-to-ligand bpy charge transfer ((3)MLCT(bpy)) states. The blue states were formed promptly after excitation at wavelength 355 nm; the one lying at higher energy decaying with a time coefficient 0.79-2.56 ns is assigned to be a triplet MLCT, and the other at lower energy decaying in 1.5-2.8 µs is assigned to (3)LLCT(A), A symmetry. This decay time coefficient of (3)LLCT(A) decreases with increasing dielectric constant of the solvent indicating this state mixing of some MLCT character. The green state (3)MLCT(ppy) decays in 0.13-4.8 ns to a nearby intermediate state either (3)MLCT(ppy) or (3)MLCT(bpy). The orange state (3)MLCT(bpy) is coupled to the intermediate state to have a rise time about 0.36-0.84 ns and decays in 425-617 ns. Although many triplet states exist in a small energy range, they couple weakly to display triple emission. All (3)LLCT and (3)MCLT states are coupled to the singlet (1)LLCT manifold directly and/or indirectly and contribute to the emission in the visible range.

Metal-metal bonding and structures of metal string complexes Cr3(dpa)4Cl2, Cr3(dpa)4(NCS)2, and [Cr3(dpa)4Cl2](PF6) from IR, Raman, and surface-enhanced Raman spectra.

We recorded infrared, Raman, and surface-enhanced Raman scattering (SERS) spectra of metal-string complexes Cr(3)(dpa)(4)X(2) (dpa = di(2-pyridyl)amido, X = Cl, NCS) and [Cr(3)(dpa)(4)Cl(2)](PF(6)) and dipyridylamine (Hdpa) to determine their vibrational frequencies and to study their structures. For the SERS measurements these complexes were adsorbed on silver nanoparticles in aqueous solution to eliminate the constraints of a crystal lattice. From the results of analysis of the vibrational normal modes we assign the infrared band at 346 cm(-1) to the Cr(3) asymmetric stretching vibration of the symmetric form and the Raman line at 570 cm(-1) to the Cr-Cr stretching mode for the unsymmetric form of Cr(3)(dpa)(4)Cl(2). Complex Cr(3)(dpa)(4)Cl(2) exhibits both symmetric (s-) and unsymmetric (u-) forms in solution but Cr(3)(dpa)(4)(NCS)(2) only the s-form. The structures for both complexes in their ground states have the s-form. The oxidized complex [Cr(3)(dpa)(4)Cl(2)](PF(6)) has only a u-form for which the Cr-Cr stretching mode is assigned to the band at 570 cm(-1). From the variation with temperature from 23 to 60 degrees C of the intensity of this line, we obtained the proportion of the u-form Cr(3)(dpa)(4)Cl(2); the enthalpy change is thus obtained to be DeltaH = 46.2 +/- 3.3 kJ mol(-1) and the entropy change is DeltaS = 138 +/- 10.3 J K(-1) mol(-1) for the reaction u-Cr(3)(dpa)(4)Cl(2) <--> s-Cr(3)(dpa)(4)Cl(2). From the spectral intensities and band frequencies in SERS spectra, Hdpa is expected to adsorb on a silver nanoparticle with the amido nitrogen and pyridyl rings tilted from the silver surface, whereas the trichromium complex with the chromium ion line is orthogonal to the silver surface normal in aqueous silver solution.