Assessment of differences in the conformational flexibility of hepatitis B virus core-antigen and e-antigen by hydrogen deuterium exchange-mass spectrometry.

Hepatitis B virus core-antigen (capsid protein) and e-antigen (an immune regulator) have almost complete sequence identity, yet the dimeric proteins (termed Cp149d and Cp(-10)149d , respectively) adopt quite distinct quaternary structures. Here we use hydrogen deuterium exchange-mass spectrometry (HDX-MS) to study their structural properties. We detect many regions that differ substantially in their HDX dynamics. Significantly, whilst all regions in Cp(-10)149d exchange by EX2-type kinetics, a number of regions in Cp149d were shown to exhibit a mixture of EX2- and EX1-type kinetics, hinting at conformational heterogeneity in these regions. Comparison of the HDX of the free Cp149d with that in assembled capsids (Cp149c ) indicated increased resistance to exchange at the C-terminus where the inter-dimer contacts occur. Furthermore, evidence of mixed exchange kinetics were not observed in Cp149c , implying a reduction in flexibility upon capsid formation. Cp(-10)149d undergoes a drastic structural change when the intermolecular disulphide bridge is reduced, adopting a Cp149d -like structure, as evidenced by the detected HDX dynamics being more consistent with Cp149d in many, albeit not all, regions. These results demonstrate the highly dynamic nature of these similar proteins. To probe the effect of these structural differences on the resulting antigenicity, we investigated binding of the antibody fragment (Fab E1) that is known to bind a conformational epitope on the four-helix bundle. Whilst Fab E1 binds to Cp149c and Cp149d , it does not bind non-reduced and reduced Cp(-10)149d , despite unhindered access to the epitope. These results imply a remarkable sensitivity of this epitope to its structural context.

Sizing up large protein complexes by electrospray ionisation-based electrophoretic mobility and native mass spectrometry: morphology selective binding of Fabs to hepatitis B virus capsids.

The capsid of hepatitis B virus (HBV) is a major viral antigen and important diagnostic indicator. HBV capsids have prominent protrusions ('spikes') on their surface and are unique in having either T = 3 or T = 4 icosahedral symmetry. Mouse monoclonal and also human polyclonal antibodies bind either near the spike apices (historically the 'α-determinant') or in the 'floor' regions between them (the 'ß-determinant'). Native mass spectrometry (MS) and gas-phase electrophoretic mobility molecular analysis (GEMMA) were used to monitor the titration of HBV capsids with the antigen-binding domain (Fab) of mAb 3120, which has long defined the ß-determinant. Both methods readily distinguished Fab binding to the two capsid morphologies and could provide accurate masses and dimensions for these large immune complexes, which range up to ~8 MDa. As such, native MS and GEMMA provide valuable alternatives to a more time-consuming cryo-electron microscopy analysis for preliminary characterisation of virus-antibody complexes.

Epitope-distal effects accompany the binding of two distinct antibodies to hepatitis B virus capsids.

Infection of humans by hepatitis B virus (HBV) induces the copious production of antibodies directed against the capsid protein (Cp). A large variety of anticapsid antibodies have been identified that differ in their epitopes. These data, and the status of the capsid as a major clinical antigen, motivate studies to achieve a more detailed understanding of their interactions. In this study, we focused on the Fab fragments of two monoclonal antibodies, E1 and 3120. E1 has been shown to bind to the side of outward-protruding spikes whereas 3120 binds to the "floor" region of the capsid, between spikes. We used hydrogen-deuterium exchange coupled to mass spectrometry (HDX-MS) to investigate the effects on HBV capsids of binding these antibodies. Conventionally, capsids loaded with saturating amounts of Fabs would be too massive to be readily amenable to HDX-MS. However, by focusing on the Cp protein, we were able to acquire deuterium uptake profiles covering the entire 149-residue sequence and reveal, in localized detail, changes in H/D exchange rates accompanying antibody binding. We find increased protection of the known E1 and 3120 epitopes on the capsid upon binding and show that regions distant from the epitopes are also affected. In particular, the α2a helix (residues 24-34) and the mobile C-terminus (residues 141-149) become substantially less solvent-exposed. Our data indicate that even at substoichiometric antibody binding an overall increase in the rigidity of the capsid is elicited, as well as a general dampening of its breathing motions.

Structure, stability and dynamics of norovirus P domain derived protein complexes studied by native mass spectrometry.

Expression of the protruding (P) domain of the norovirus capsid protein, in vitro, results in the formation of P dimers and larger oligomers, 12-mer and 24-mer P particles. All these P complexes retain the authentic antigenicity and carbohydrate-binding function of the norovirus capsid. They have been used as tools to study norovirus-host interactions, and the 24-mer P particle has been proposed as a vaccine and vaccine platform against norovirus and other pathogens. In view of their pharmaceutical interest it is important to characterise the structure, stability and dynamics of these protein complexes. Here we use a native mass spectrometric approach. We analyse the P particles under both non-reducing and reducing conditions, as it is known that the macromolecular assemblies are stabilised by inter-subunit disulphide bonding. A novel 18-mer complex is identified, and we show that under reducing conditions the 24-mer dissociates into P dimers that reassemble into the 12-mer small P particle and another novel 36-mer complex. The collisional cross-sections of the 12-mer and 24-mer P particles determined by ion mobility MS are in good agreement with theoretical predictions and electron microscopy data. We propose a model structure for the 18-mer based on ion mobility experiments. Our results demonstrate the interchangeable nature and dynamic relationship of all P domain complexes and confirm their binding activity to the host receptors - human histo blood group antigens (HBGAs). These findings, together with the identification of the 18-mer and 36-mer P complexes add new information to the intriguing interactions of the norovirus P domain.

Offline and online liquid chromatography mass spectrometry in quantitative proteomics.

Hyphenation with liquid chromatography has become indispensable in mass spectrometry-based proteomics. Sample complexity together with the large variations in dynamic range can be only tackled using techniques that isolate and/or concentrate individual components prior to mass spectrometric analysis. In this review the most recent developments in micro/nanoliquid chromatography interfaced with MALDI and electrospray ionisation are discussed. Particular attention is focused on all applications related to quantitative proteomics.

Transcriptome and proteome analysis of osteocytes treated with nitrogen-containing bisphosphonates.

We combined high-throughput screening of differential mRNAs with mass spectrometric characterization of proteins obtained from osteocytes untreated and treated with Risedronate. Microarray analysis revealed, upon treatment, a marked upregulation of messengers encoding zinc-proteins. MS analysis identified 84 proteins in the osteocytes proteome map. Risedronate affected the expression of 10 proteins, associated with cytoskeleton, stress-response and metabolism. Data validated using gel imaging in combination with the GLaD post digestion isotopic labeling method provide the molecular basis for understanding the role of bisphosphonates as antiapoptotic drugs for osteocytes.

Development of novel guanidino-labelling derivatisation (GLaD) reagents for liquid chromatography/matrix-assisted laser desorption/ionisation analysis.

A new generation of guanidino-labelling (GLaD) reagents were developed for quantitative proteomics using offline microcapillary liquid chromatography (LC) matrix-assisted laser desorption/ionisation mass spectrometry (MALDI-MS). In order to reduce the unwanted overlapping between the isotopic envelopes of the two differentially labelled peptide ions, a novel synthetic route was described for production of both (13)C- and (15)N-containing isotopomers of N,O-dimethylisourea. The use of these types of isotopes has no deleterious effect on the retention times of both differentially labelled peptides during offline microbore reversed-phase LC. In addition, the possibility to incorporate a mass difference of 4 Da can be exploited during post-source decay analysis to generate product ion spectra in which fragment ions containing the modifications appear as doublets in the corresponding product ion spectra, thus facilitating identification of the C-terminal fragment ions.

Relative quantification of tau-related peptides using guanidino-labeling derivatization (GLaD) with online-LC on a hybrid ion trap (IT) time-of-flight (ToF) mass spectrometer.

Development of a quantification method based on isotopic variants of O-methyl isourea (OMIU) in conjunction with reversed-phase (RP) liquid chromatography (LC) electrospray mass spectrometry is described for determining the relative quantification of tau-related peptides Ac-VQIVXK-NH2. Extracted ion chromatograms of the mass spectrometric data derived from online microcapillary LC separation identifies the retention times of the isotopically derivatized peptides together with their ion abundances. Data-dependent MSMS analysis of both derivatized variants of the same peptide provides a complementary method for identification and resolution between isobaric species. In addition, with respect to offline LC MALDI a larger number of analogues are detected and formation of amyloid is also observed for the aspartic acid and histidine-containing peptides.

Comprehensive analysis of glycated human serum albumin tryptic peptides by off-line liquid chromatography followed by MALDI analysis on a time-of-flight/curved field reflectron tandem mass spectrometer.

Glycated peptides arising from in vivo digestion of glycated proteins, usually called advanced glycation end (AGE) product peptides, are biologically relevant compounds due to their reactivity towards circulating and tissue proteins. To investigate their structures, in vitro glycation of human serum albumin (HSA) has been performed and followed by enzymatic digestion. Using different MALDI based approaches the digestion products obtained have been compared with those arising from enzymatic digestion of the protein. Results obtained using 2,5-dihydroxybenzoic acid (DHB) indicate this as the most effective matrix, leading to an increase in the coverage of the glycated protein. Off-line microbore liquid chromatography prior to MALDI analysis reveals that 63% of the free amino groups amenable to glycation are modified. In addition, the same approach shows the co-presence of underivatised peptides. This indicates that, regardless of the high glucose concentration employed for HSA incubation, glycation does not go to completion. Tandem mass spectrometric data suggest that the collision induced dissociation of singly charged glycated peptides leads to specific fragmentation pathways related to the condensed glucose molecule. The specific neutral losses derived from the activated glycated peptides can be used as signature for establishing the occurrence of glycation processes.