Cees Versluis

High-resolution mass spectrometry of viral assemblies: molecular composition and stability of dimorphic hepatitis B virus capsids.

Hepatitis B virus (HBV) is a major human pathogen. In addition to its importance in human health, there is growing interest in adapting HBV and other viruses for drug delivery and other nanotechnological applications. In both contexts, precise biophysical characterization of these large macromolecular particles is fundamental. HBV capsids are unusual in that they exhibit two distinct icosahedral geometries, nominally composed of 90 and 120 dimers with masses of ≈3 and ≈4 MDa, respectively. Here, a mass spectrometric approach was used to determine the masses of both capsids to within 0.1%. It follows that both lattices are complete, consisting of exactly 180 and 240 subunits. Nanoindentation experiments by atomic-force microscopy indicate that both capsids have similar stabilities. The data yielded a Youngs modulus of ≈0.4 GPa. This experimental approach, anchored on very precise and accurate mass measurements, appears to hold considerable potential for elucidating the assembly of viruses and other macromolecular particles.

Chaperonin complexes monitored by ion mobility mass spectrometry.

The structural analysis of macromolecular functional protein assemblies by contemporary high resolution structural biology techniques (such as nuclear magnetic resonance, X-ray crystallography, and electron microscopy) is often still challenging. The potential of a rather new method to generate structural information, native mass spectrometry, in combination with ion mobility mass spectrometry (IM-MS), is highlighted here. IM-MS allows the assessment of gas phase ion collision cross sections of protein complex ions, which can be related to overall shapes/volumes of protein assemblies, and thus be used to monitor changes in structure. Here we applied IM-MS to study several (intermediate) chaperonin complexes that can be present during substrate folding. Our results reveal that the protein assemblies retain their solution phase structural properties in the gas phase, addressing a long-standing issue in mass spectrometry. All IM-MS data on the chaperonins point toward the burial of genuine substrates inside the GroEL cavity being retained in the gas phase. Additionally, the overall dimensions of the ternary complexes between GroEL, a substrate, and cochaperonin were found to be similar to the dimensions of the empty GroEL-GroES complex. We also investigated the effect of reducing the charge, obtained in the electrospray process, of the protein complex on the global shape of the chaperonin. At decreased charge, the protein complex was found to be more compact, possibly occupying a lower number of conformational states, enabling an improved ion mobility separation. Charge state reduction was found not to affect the relative differences observed in collision cross sections for the chaperonin assemblies.

Stability and Shape of Hepatitis B Virus Capsids In Vacuo

The hepatitis B virus (HBV) is a major cause of liver disease in humans[1] and its non-infectious capsid is of interest for nanotechnology, including for drug-delivery applications. A precise biophysical characterization of these particles is of importance not only for these applications, but also because it may provide further insight into the replication cycle and assembly pathway of the virus, and thus contribute to the future development of drugs.[2,3] The HBV capsid protein (cp) forms icosahedral capsids of two sizes in vivo and in vitro (with triangulation numbers of T = 3 and T = 4[4] that contain 180 and 240 subunits, respectively[5–8]).The capsid protein has two domains—a core domain (amino acids 1–140) and a “protamine domain” (amino acids 150–183)—connected by a 10-residue linker;[9] of these, the core domain is necessary and sufficient for assembly of the capsid. The length of the linker and the conditions under which assembly take place determine the ratio of the T = 3 and T = 4 capsids obtained.[10] Capsid protein dimers are stabilized by an intermolecular four-helix bundle[11–13] and a disulfide bond within the bundle (Cys61); these dimers represent the building blocks for the formation of the capsid. However, the disulfide bond is not required for dimerization or assembly, as Cys61 can be replaced with Ala[9, 10] without affecting either process. The interfaces of the dimers display protruding spikes that result in an uneven surface.[6, 13] Although extensive structural studies of the HBV capsid structure have been performed by electron microscopy (EM) and X-ray crystallography,[14] knowledge of its biophysical properties is limited.[15, 16] Here, we present data from macromolecular tandem and ion mobility mass spectrometry (MS)[17–19] that have a bearing on the stability and conformational diversity of HBV capsids in vacuo.

A new chemical probe for proteomics of carbohydrate-binding proteins.

Selective capture of galectins, while leaving other proteins untouched, was achieved by activating photoaffinity labels that were precisely positioned on noncovalently bound carbohydrate ligands. The labelled proteins were visualised in-gel by “clicking-on” a rhodamine moiety afterwards.

Rolling Loop Scan: An Approach Featuring Ring-Closing Metathesis for Generating Libraries of Peptides with Molecular Shapes Mimicking Bioactive Conformations or Local Folding of Peptides and Proteins.

Libraries of loop-containing peptides (such as the one shown schematically) can be prepared from bis-N-alkylated peptides by ring-closing metathesis. In a general solid-phase procedure the peptides are accessible by site-specific N-alkylation. Since the amino acid side chains are not involved in cyclization, they remain available for interaction with, for example, a receptor.

Metastable ion formation and disparate charge separation in the gas-phase dissection of protein assemblies studied by orthogonal time-of-flight mass spectrometry

The dissection of specific and nonspecific protein complexes in the gas phase is studied by collisionally activated decomposition. In particular, the gas phase dissection of multiple protonated homodimeric Human Galectin I, E. Coli Glyoxalase I, horse heart cytochrome c, and Hen egg Lysozyme have been investigated. Both the Human Galectin I and E. Coli Glyoxalase I enzymes are biologically active as a dimer, exhibiting molecular weights of approximately 30 kDa. Cytochrome c and Lysozyme are monomers, but may aggregate to some extent at high protein concentrations. The gas phase dissociation of these multiple protonated dimer assemblies does lead to the formation of monomers. The charge distribution over the two concomitant monomers following the dissociation of these multiple protonated dimers is found to be highly dissimilar. There is no evident correlation between the solution phase stability of the dimeric proteins and their gas-phase dissociation pattern. Additionally, in the collisionally activated decomposition spectra diffuse ion signals are observed, which are attributed to monomer ions formed via slow decay of the collisionally activated dimer ions inside the reflectron time-of-flight. Although, the formation of these diffuse metastable ions may complicate the interpretation of collisionally activated decomposition mass spectra, especially when studying noncovalent protein complexes, a simple mathematical equation may be used to reveal their origin and pathway of formation.

Capillary electrophoretic bioanalysis of therapeutically active peptides with UV and mass spectrometric detection after on-capillary preconcentration

An earlier developed capillary electrophoresis (CE) system with an on‐capillary adsorptive phase is investigated for its suitability to quantitate low concentrations of angiotensin II and gonadorelin in plasma. An off‐line solid‐phase extraction is used for sample preparation. The on‐line preconcentration CE system allows multiple capillary volumes of sample solution to be injected, increasing the concentration sensitivity of CE with 3–4 orders of magnitude. Furthermore, possible influence of matrix salts can be ruled out by employing a rinsing step after sample application. Using short‐wavelength UV detection, reproducibility and linearity in the low nanomolar range were satisfactory. The capillary could be efficiently regenerated using a programmed between‐run rinsing procedure, allowing 20–30 large injections of sample extracts. Coating of the capillary improved the robustness of the method. Mass spectrometric detection via a previously reported sheathless interface increased the selectivity and sensitivity substantially. Recommendations are provided for the sample preparation process, the most critical part of the system. Further purification of the sample is required to allow the loading of larger sample volumes and to optimize the systems robustness.

Robust and cost-effective capillary electrophoresis-mass spectrometry interfaces suitable for combination with on-line analyte preconcentration

This paper describes several successful cost‐effective attempts to couple capillary electrophoresis (CE) and mass spectrometry (MS) without make‐up flow or nebulizing gas. An in‐depth analysis of several interfaces using conductive spray tips was performed as well as an easy‐to‐prepare T‐junction with direct electrode contact, the latter being the most robust interface. No coating is necessary and the spray voltage is applied through a gold wire positioned at the gap between the separation and spray capillaries. The T‐junction interface is made by puncturing a small piece of transparent rubber. The on‐line preconcentration CE‐MS system allows immunoassay sensitivity, as is demonstrated by a calibration plot in the picomolar range for angiotensin II and gonadorelin. It also shows good reproducibility and has the ability of excellent automation. The secure electrical contact gives a constant spray quality, even with 100% aqueous separation buffers. The described setup has a wide applicability as is demonstrated by the analysis of larger peptides, such as insulin and cytochrome c. Detailed information is given on critical factors in the preparation of the described interfaces.

Structural reorganization of the antigen-binding groove of human CD1b for presentation of mycobacterial sulfoglycolipids

The mechanisms permitting nonpolymorphic CD1 molecules to present lipid antigens that differ considerably in polar head and aliphatic tails remain elusive. It is also unclear why hydrophobic motifs in the aliphatic tails of some antigens, which presumably embed inside CD1 pockets, contribute to determinants for T-cell recognition. The 1.9-Å crystal structure of an active complex of CD1b and a mycobacterial diacylsulfoglycolipid presented here provides some clues. Upon antigen binding, endogenous spacers of CD1b, which consist of a mixture of diradylglycerols, moved considerably within the lipid-binding groove. Spacer displacement was accompanied by F’ pocket closure and an extensive rearrangement of residues exposed to T-cell receptors. Such structural reorganization resulted in reduction of the A’ pocket capacity and led to incomplete embedding of the methyl-ramified portion of the phthioceranoyl chain of the antigen, explaining why such hydrophobic motifs are critical for T-cell receptor recognition. Mutagenesis experiments supported the functional importance of the observed structural alterations for T-cell stimulation. Overall, our data delineate a complex molecular mechanism combining spacer repositioning and ligand-induced conformational changes that, together with pocket intricacy, endows CD1b with the required molecular plasticity to present a broad range of structurally diverse antigens.

Gas-phase dissociation of hemoglobin

Abstract The collisionally induced gas-phase dissociation of protein assemblies of hemoglobin has been investigated. In particular, the gas-phase disassembly of the holo-hetero-dimers and the holo-tetramers of bovine, porcine, and human hemoglobin have been studied. The holo-hetero-dimer ions dissociate primarily in apo-α-chains and holo-β-chains, whereby the apo-α-chain ions accommodate more than a fair share of the initial charges present on the precursor ions. Comparing the collision-activated dissociation tandem mass spectrometry of three holo-tetramer assemblies one of the most important findings is that also these ions dissociate predominantly into the α-chain monomer concomitant with the αβ 2 trimer, whereby the α-chain monomer retains primarily 0 or 1, and the trimer 2–4 heme groups. Again the apo-α-chain ions accommodate more than a fair share of the initial charges, sometimes even more than the much larger αβ 2 trimer. The disparate charge distribution following the dissociation is in agreement with previous observations on other protein assemblies, and may to some extent be explained by assuming a charge-droplet fission model. Some significant differences are observed in the gas-phase disassembly of the hemoglobin complexes originating from the three different species. It is thought that these differences originate from differences in the gas-phase structures of these assemblies. The observed gas-phase dissociation patterns are in sharp contrast to the known solution-phase behaviour, providing a further indication that caution has to be taken when relating gas-phase data to solution-phase properties.