Conformational heterogeneity of Savinase from NMR, HDX-MS and X-ray diffraction analysis.

BACKGROUND: Several examples have emerged of enzymes where slow conformational changes are of key importance for function and where low populated conformations in the resting enzyme resemble the conformations of intermediate states in the catalytic process. Previous work on the subtilisin protease, Savinase, from Bacillus lentus by NMR spectroscopy suggested that this enzyme undergoes slow conformational dynamics around the substrate binding site. However, the functional importance of such dynamics is unknown. METHODS: Here we have probed the conformational heterogeneity in Savinase by following the temperature dependent chemical shift changes. In addition, we have measured changes in the local stability of the enzyme when the inhibitor phenylmethylsulfonyl fluoride is bound using hydrogen-deuterium exchange mass spectrometry (HDX-MS). Finally, we have used X-ray crystallography to compare electron densities collected at cryogenic and ambient temperatures and searched for possible low populated alternative conformations in the crystals. RESULTS: The NMR temperature titration shows that Savinase is most flexible around the active site, but no distinct alternative states could be identified. The HDX shows that modification of Savinase with inhibitor has very little impact on the stability of hydrogen bonds and solvent accessibility of the backbone. The most pronounced structural heterogeneities detected in the diffraction data are limited to alternative side-chain rotamers and a short peptide segment that has an alternative main-chain conformation in the crystal at cryo conditions. Collectively, our data show that there is very little structural heterogeneity in the resting state of Savinase and hence that Savinase does not rely on conformational selection to drive the catalytic process.

Investigating surrogate cerebrospinal fluid matrix compositions for use in quantitative LC-MS analysis of therapeutic antibodies in the cerebrospinal fluid.

As quantitative analysis of biotherapeutics in cerebrospinal fluid (CSF) with LC-MS becomes increasingly widespread, there is a need for method developments towards higher sensitivity. By using artificial CSF (aCSF) in the development phase, the consumption of costly and sparsely available CSF can be limited. The aCSF compositions tested here were made from various dilutions of bovine serum albumin (BSA) or rat plasma to mimic the total protein concentration found in CSF. Focusing on monoclonal antibodies, the aCSF was spiked with human immunoglobulin (hIgG) and prepared with the bottom-up analysis technique using LC-MS. Assuming that the composition of the aCSF would affect the digest, the response from aCSF matrices was compared with CSF from rat, monkey, and dog in terms of estimated sample concentration and matrix effects. The samples were spiked with hIgG in the range of 10 to 1000 ng/mL and volumes of 10 µL were transferred to sample preparation. The results indicate that BSA dilutions from 300 to 2000 µg/mL and rat plasma dilutions of 0.5-2% provide the most accurate concentration estimates when compared with rat CSF. 1000 µg/mL BSA did not produce significantly different concentration estimates for 500 ng/mL samples when compared with CSF from rat, monkey, and dog, and can therefore be used as aCSF for several different species.

Molecular Basis of the Mechanisms Controlling MASTL.

The human MASTL (Microtubule-associated serine/threonine kinase-like) gene encodes an essential protein in the cell cycle. MASTL is a key factor preventing early dephosphorylation of M-phase targets of Cdk1/CycB. Little is known about the mechanism of MASTL activation and regulation. MASTL contains a non-conserved insertion of 550 residues within its activation loop, splitting the kinase domain, and making it unique. Here, we show that this non-conserved middle region (NCMR) of the protein is crucial for target specificity and activity. We performed a phosphoproteomic assay with different MASTL constructs identifying key phosphorylation sites for its activation and determining whether they arise from autophosphorylation or exogenous kinases, thus generating an activation model. Hydrogen/deuterium exchange data complements this analysis revealing that the C-lobe in full-length MASTL forms a stable structure, whereas the N-lobe is dynamic and the NCMR and C-tail contain few localized regions with higher-order structure. Our results indicate that truncated versions of MASTL conserving a cryptic C-Lobe in the NCMR, display catalytic activity and different targets, thus establishing a possible link with truncated mutations observed in cancer-related databases.

Molecular architecture of the Jumonji C family histone demethylase KDM5B.

The full length human histone 3 lysine 4 demethylase KDM5B (PLU-1/Jarid1B) has been studied using Hydrogen/Deuterium exchange mass spectrometry, homology modelling, sequence analysis, small angle X-ray scattering and electron microscopy. This first structure on an intact multi-domain Jumonji histone demethylase reveal that the so-called PLU region, in the central region of KDM5B, has a curved α-helical three-dimensional structure, that acts as a rigid linker between the catalytic core and a region comprising four α-helices, a loop comprising the PHD2 domain, two large intrinsically disordered loops and the PHD3 domain in close proximity. The dumbbell shaped and curved KDM5B architecture observed by electron microscopy is complementary to the nucleosome surface and has a striking overall similarity to that of the functionally related KDM1A/CoREST complex. This could suggest that there are similarities between the demethylation mechanisms employed by the two histone 3 lysine 4 demethylases at the molecular level.

Circularized and solubility-enhanced MSPs facilitate simple and high-yield production of stable nanodiscs for studies of membrane proteins in solution.

Recently, an enzymatic reaction was utilized to covalently link the N and C termini of membrane scaffold proteins to produce circularized nanodiscs that were more homogeneous and stable than standard nanodiscs. We continue this development and aim for obtaining high yields of stable and monodisperse nanodiscs for structural studies of membrane proteins by solution small-angle scattering techniques. Based on the template MSP1E3D1, we designed an optimized membrane scaffold protein (His-lsMSP1E3D1) with a sortase recognition motif and high abundance of solubility-enhancing negative charges. With these modifications, we show that high protein expression is maintained and that the circularization reaction is efficient, such that we obtain a high yield of circularized membrane scaffold protein (csMSP1E3D1) and downstream circularized nanodiscs. We characterize the circularized protein and corresponding nanodiscs biophysically by small-angle X-ray scattering, size-exclusion chromatography, circular dichroism spectroscopy, and light scattering and compare to noncircularized samples. First, we show that circularized and noncircularized (lsMSP1E3D1) nanodiscs are structurally similar and have the expected nanodisc structure. Second, we show that lsMSP1E3D1 nanodiscs are more stable compared to the template MSP1E3D1 nanodiscs as an effect of the extra negative charges and that csMSP1E3D1 nanodiscs have further improved stability as an effect of circularization. Finally, we show that a membrane protein can be efficiently incorporated in csMSP1E3D1 nanodiscs. Large-scale production methods for circularized nanodiscs with improved thermal and temporal stability will facilitate better access to the nanodisc technology and enable applications at physiologically relevant temperatures.

Investigating the utility of minimized sample preparation and high-resolution mass spectrometry for quantification of monoclonal antibody drugs.

Determination of the pharmacokinetic (PK) properties of therapeutic monoclonal antibodies (mAbs) is essential for their successful development as drugs. For this purpose, besides the traditional ligand binding assay (LBA), LC-MS/MS method using low resolution mass spectrometers (e.g. triple quadrupole (QqQ)) has become routinely used, however, complicated and lengthy sample pre-treatment (employing immuno-affinity) is often necessary for obtaining sufficient sensitivity and selectivity. In this study, we investigate the capabilities of high-resolution MS instruments for circumventing the complex sample preparation currently needed for sensitive LC-MS/MS-based quantification of mAbs. Employing a simple one-step sample pre-treatment workflow, we compare the ability of three different LC-MS platforms for absolute quantification of a representative monoclonal antibody Rendomab-B1 in serum and plasma. The samples are subjected to protein precipitation with methanol, followed by pellet digestion with trypsin prior to LC-MS analysis. AQUA peptides based on two surrogate mAb peptides selected from an extensive in-silico and experimental screening are used as internal standards. MS/MS acquisitions are developed and systematically examined for 1) a low-resolution QqQ operated in selected reaction monitoring (SRM) acquisition mode, 2) a high-resolution hybrid Quadrupole-Orbitrap (Q-Orbitrap) operated in parallel reaction monitoring (PRM) acquisition mode and 3) a high-resolution hybrid Quadrupole-Time-of-flight (Q-TOF) operated in SRM acquisition mode with enhanced duty cycle (EDC) function. The sensitivity of the high-resolution Q-Orbitrap and Q-TOF methods was significantly higher (LOD of 80 ng/mL) in serum/plasma samples than the low-resolution QqQ method. Finally, the real-world utility of the developed high-resolution MS method with minimized sample handling was demonstrated and validated by determining the PK profile of Rendomab-B1 in mice by a 10-point in vivo study over 15 days.

The extraordinary thermal stability of EstA from S. islandicus is independent of post translational modifications.

Enzymes from thermophilic and hyper-thermophilic organisms have an intrinsic high stability. Understanding the mechanisms behind their high stability will be important knowledge for the engineering of novel enzymes with high stability. Lysine methylation of proteins is prevalent in Sulfolobus, a genus of hyperthermophilic and acidophilic archaea. Both unspecific and temperature dependent lysine methylations are seen, but the significance of this post-translational modification has not been investigated. Here, we test the effect of eliminating in vivo lysine methylation on the stability of an esterase (EstA). The enzyme was purified from the native host S. islandicus as well as expressed as a recombinant protein in E. coli, a mesophilic host that does not code for any machinery for in vivo lysine methylation. We find that lysine mono methylation indeed has a positive effect on the stability of EstA, but the effect is small. The effect of the lysine methylation on protein stability is secondary to that of protein expression in E. coli, as the E. coli recombinant enzyme is compromised both on stability and activity. We conclude that these differences are not attributed to any covalent difference between the protein expressed in hyperthermophilic versus mesophilic hosts.

Expression, Purification and Characterization of GMZ2'.10C, a Complex Disulphide-Bonded Fusion Protein Vaccine Candidate against the Asexual and Sexual Life-Stages of the Malaria-Causing Plasmodium falciparum Parasite.

PURPOSE: Production and characterization of a chimeric fusion protein (GMZ2'.10C) which combines epitopes of key malaria parasite antigens: glutamate-rich protein (GLURP), merozoite surface protein 3 (MSP3), and the highly disulphide bonded Pfs48/45 (10C). GMZ2'.10C is a potential candidate for a multi-stage malaria vaccine that targets both transmission and asexual life-cycle stages of the parasite. METHODS: GMZ2'.10C was produced in Lactococcus lactis and purified using either an immunoaffinity purification (IP) or a conventional purification (CP) method. Protein purity and stability was analysed by RP-HPLC, SEC-HPLC, 2-site ELISA, gel-electrophoresis and Western blotting. Structural characterization (mass analysis, peptide mapping and cysteine connectivity mapping) was performed by LC-MS/MS. RESULTS: CP-GMZ2'.10C resulted in similar purity, yield, structure and stability as compared to IP-GMZ2'.10C. CP-GMZ2'.10C and IP-GMZ2'.10C both elicited a high titer of transmission blocking (TB) antibodies in rodents. The intricate disulphide-bond connectivity of C-terminus Pfs48/45 was analysed by tandem mass spectrometry and was established for GMZ2'.10C and two reference fusion proteins encompassing similar parts of Pfs48/45. CONCLUSION: GMZ2'.10C, combining GMZ2' and correctly-folded Pfs48/45 can be produced by the Lactoccus lactis P170 based expression system in purity and quality for pharmaceutical development and elicit high level of TB antibodies. The cysteine connectivity for the 10C region of Pfs48/45 was revealed experimentally, providing an important guideline for employing the Pfs48/45 antigen in vaccine design.

Thiol-ene Monolithic Pepsin Microreactor with a 3D-Printed Interface for Efficient UPLC-MS Peptide Mapping Analyses.

To improve the sample handling, and reduce cost and preparation time, of peptide mapping LC-MS workflows in protein analytical research, we here investigate the possibility of replacing conventional enzymatic digestion methods with a polymer microfluidic chip based enzyme reactor. Off-stoichiometric thiol-ene is utilized as both bulk material and as a monolithic stationary phase for immobilization of the proteolytic enzyme pepsin. The digestion efficiency of the, thiol-ene based, immobilized enzyme reactor (IMER) is compared to that of a conventional, agarose packed bed, pepsin IMER column commonly used in LC-MS based protein analyses. The chip IMER is found to rival the conventional column in terms of digestion efficiency at comparable residence time and, using a 3D-printed interface, be directly interfaceable with LC-MS.

A simple sheathless CE-MS interface with a sub-micrometer electrical contact fracture for sensitive analysis of peptide and protein samples.

Online coupling of capillary electrophoresis (CE) to electrospray ionization mass spectrometry (MS) has shown considerable potential, however, technical challenges have limited its use. In this study, we have developed a simple and sensitive sheathless CE-MS interface based on the novel concept of forming a sub-micrometer fracture directly in the capillary. The simple interface design allowed the generation of a stable ESI spray capable of ionization at low nanoliter flow-rates (45-90 nL/min) for high sensitivity MS analysis of challenging samples like those containing proteins and peptides. By analysis of a model peptide (leucine enkephalin), a limit of detection (LOD) of 0.045 pmol/µL (corresponding to 67 attomol in a sample volume of ∼15 nL) was obtained. The merit of the CE-MS approach was demonstrated by analysis of bovine serum albumin (BSA) tryptic peptides. A well-resolved separation profile was achieved and comparable sequence coverage was obtained by the CE-MS method (73%) compared to a representative UPLC-MS method (77%). The CE-MS interface was subsequently used to analyse a more complex sample of pharmaceutically relevant human proteins including insulin, tissue factor and α-synuclein. Efficient separation and protein ESI mass spectra of adequate quality could be achieved using only a small amount of sample (30 fmol). In addition, analysis of ubiquitin samples under both native and denatured conditions, indicate that the CE-MS setup can facilitate native MS applications to probe the conformational properties of proteins. Thus, the described CE-MS setup should be useful for a wide range of high-sensitivity applications in protein research.

A method for studies on interactions between a gold-based drug and plasma proteins based on capillary electrophoresis with inductively coupled plasma mass spectrometry detection.

An analytical method based on capillary electrophoresis (CE) and inductively coupled plasma mass spectrometry (ICP-MS) detection was developed for studies on the interaction of gold-containing drugs and plasma proteins using auranofin as example. A detection limit of 18 ng/mL of auranofin corresponding to 5.2 ng/mL Au and a precision of 1.5 % were obtained. Kinetic studies of the interaction between auranofin and protein were performed by incubation in aqueous solutions as well as 20 % human plasma at 37 °C. The reaction of auranofin with human serum albumin (HSA) and plasma proceeded fast; 50 % of un-bound auranofin disappeared within 2 and 3 min, respectively. By blocking the free cysteine (Cys-34) by iodoacetamide on HSA, it was shown that Cys-34 was the main reaction site for auranofin. By selective labeling of HSA present in 20 % human plasma with iophenoxate, it was demonstrated that HSA was the major auranofin-interacting protein in plasma. The CE-ICP-MS method is proposed as a novel approach for kinetic studies of the interactions between gold-based drugs and plasma proteins. Graphical Abstract Development of a CE-ICP-MS based method allows for studies on interaction of the gold containing drug auranofin with plasma proteins.

Selective analysis of human serum albumin based on SEC-ICP-MS after labelling with iophenoxic acid.

Human serum albumin (HSA) is the most abundant protein in the human plasma. HSA has several physiological roles in the human body, including storage and transport. Owing to the predominance of albumin in plasma, HSA is often involved in the protein binding of drugs. The aim of this work was to develop a selective, quantitative method for determining albumin in plasma with the purpose of clarifying the fate of metal-based drugs in biological systems. The method can also be applied for determination of urine albumin, which is of relevance in diagnostics of kidney disease. A selective method for quantification of HSA based on labelling the protein with iophenoxic acid (IPA) was developed. Samples were subjected to size exclusion chromatography (SEC) and detection by inductively coupled plasma mass spectrometry (ICP-MS) monitoring iodine and platinum. The iodine signal for the HSA-IPA complex showed linearity in the range 1 to 250 mg L(-1). The precision was 3.7% and the accuracy 100.7% determined by analysis of a certified HSA reference material. The limit of detection (LOD) and limit of quantification (LOQ) were 0.23 and 9.79 mg L(-1), respectively. The method was applied for analysis of HSA in human plasma and urine samples and for studying the binding of cisplatin to proteins in the human plasma.

Determination of platinum drug release and liposome stability in human plasma by CE-ICP-MS.

An in vitro method for simultaneous assessment of platinum release and liposome stability of liposomal formulations in human plasma is demonstrated. The development and assessment of the method was performed on a PEGylated liposomal formulation containing cisplatin. Complete separation of free cisplatin, encapsulated cisplatin and cisplatin bound to plasma components was achieved by capillary electrophoresis (CE) separation and simultaneous monitoring of phosphorous (phospholipid) and platinum (cisplatin) by inductively coupled plasma mass spectrometry (ICP-MS). The method allows assessment of the encapsulation efficiency of the formulation, the physical stability of liposomes as well as cisplatin leakage in human plasma. The method was applied for studying the disintegration of liposomes and the interactions of leaked cisplatin with plasma components. Triggered release of the drug into plasma by sonication was also demonstrated. Analysis of liposomal formulations with alternative phospholipid compositions containing oxaliplatin showed similar results. Thus, the present in vitro method is suitable for mimicking the in vivo drug release profile in human plasma after administration of liposomal platinum formulations to patients. This approach may be of use in early drug development as well as in quality control.

Metallomics in drug development: characterization of a liposomal cisplatin drug formulation in human plasma by CE-ICP-MS.

A capillary electrophoresis inductively coupled plasma mass spectrometry method for separation of free cisplatin from liposome-encapsulated cisplatin and protein-bound cisplatin was developed. A liposomal formulation of cisplatin based on PEGylated liposomes was used as model drug formulation. The effect of human plasma matrix on the analysis of liposome-encapsulated cisplatin and intact cisplatin was studied. The presence of 1 % of dextran and 4 mM of sodium dodecyl sulfate in HEPES buffer was demonstrated to be effective in improving the separation of liposomes and cisplatin bound to proteins in plasma. A detection limit of 41 ng/mL of platinum and a precision of 2.1 % (for 10 µg/mL of cisplatin standard) were obtained. Simultaneous measurements of phosphorous and platinum allows the simultaneous monitoring of the liposomes, liposome-encapsulated cisplatin, free cisplatin and cisplatin bound to plasma constituents in plasma samples. It was demonstrated that this approach is suitable for studies of the stability of liposome formulations as leakage of active drug from the liposomes and subsequent binding to biomolecules in plasma can be monitored. This methodology has not been reported before and will improve characterization of liposomal drugs during drug development and in studies on kinetics.

Investigation of a liposomal oxaliplatin drug formulation by capillary electrophoresis hyphenated to inductively coupled plasma mass spectrometry (CE-ICP-MS).

A capillary electrophoresis-inductively coupled plasma mass spectrometry (CE-ICP-MS) method was developed for separation of the free oxaliplatin drug substance from liposome-entrapped oxaliplatin. Simultaneous determination of phosphorous and platinum opened the possibility to simultaneously monitor the liposomes (phospholipids) and platinum-based drug. In order to suppress the interferences, argon gas was used as a collision gas in ICP-MS. A detection limit of 29 ng/mL of platinum and a precision of 2.9% (for 10 µg/mL of oxaliplatin standard) were obtained. Measurement of the total concentration of free and encapsulated oxaliplatin by CE-ICP-MS was compared with total determination by ICP-MS after microwave digestion and showed a good agreement. A liposomal formulation of oxaliplatin based on PEGylated liposomes was used as a model drug formulation. Studies of accelerated drug release induced by sonication and phospholipase A(2) catalyzed hydrolysis were performed. It was demonstrated that the CE-ICP-MS was an efficient in vitro characterization method in the development and quality assurance purposes of lipsome-based formulation of metallodrugs.

Characterization of a liposome-based formulation of oxaliplatin using capillary electrophoresis: encapsulation and leakage.

A capillary electrophoresis-based method to characterize a PEGylated liposomal drug formulation of the anti-cancer agent oxaliplatin was developed. Pharmaceutical characterization in terms of determination of the free and total oxaliplatin concentrations in the liposomal formulation was successfully performed allowing calculation of the percentage of encapsulated drug and encapsulation efficiency. The trapping efficiency was likewise calculated. The capillary electrophoresis method allowed liposome characterization in the intended formulation media (sucrose solution with low electrolyte concentration), and the attained results were consistent with inductively coupled plasma mass spectrometry measurements. Accelerated drug leakage studies were initiated by the sonication of the PEGylated formulation, using an ultrasound probe, subsequently the drug leakage was determined by capillary electrophoresis. The results obtained with the PEGylated liposomes demonstrate that capillary electrophoresis may be a useful tool for the characterization of liposomal drug formulations.