Analysis of Intact Monoclonal Antibody IgG1 by Electron Transfer Dissociation Orbitrap FTMS

The primary structural information of proteins employed as biotherapeutics is essential if one wishes to understand their structure–function relationship, as well as in the rational design of new therapeutics and for quality control. Given both the large size (around 150 kDa) and the structural complexity of intact immunoglobulin G (IgG), which includes a variable number of disulfide bridges, its extensive fragmentation and subsequent sequence determination by means of tandem mass spectrometry (MS) are challenging. Here, we applied electron transfer dissociation (ETD), implemented on a hybrid Orbitrap Fourier transform mass spectrometer (FTMS), to analyze a commercial recombinant IgG in a liquid chromatography (LC)-tandem mass spectrometry (MS/MS) top-down experiment. The lack of sensitivity typically observed during the top-down MS of large proteins was addressed by averaging time-domain transients recorded in different LC-MS/MS experiments before performing Fourier transform signal processing. The results demonstrate that an improved signal-to-noise ratio, along with the higher resolution and mass accuracy provided by Orbitrap FTMS (relative to previous applications of top-down ETD-based proteomics on IgG), is essential for comprehensive analysis. Specifically, ETD on Orbitrap FTMS produced about 33% sequence coverage of an intact IgG, signifying an almost 2-fold increase in IgG sequence coverage relative to prior ETD-based analysis of intact monoclonal antibodies of a similar subclass. These results suggest the potential application of the developed methodology to other classes of large proteins and biomolecules.Top-down mass spectrometry (MS)¹ (1–3) has continued to demonstrate its particular advantages over traditionally employed bottom-up MS strategies (4). Specifically, top-down MS allows the characterization of specific protein isoforms originating from the alternative splicing of mRNA that code single nucleotide polymorphisms and/or post-translational modifications (PTMs) of protein species (5). Intact protein molecular weight (MW) determination and subsequent gas-phase fragmentation of selected multiply charged protein ions (referred to as tandem MS or MS/MS) theoretically might result in complete protein sequence coverage and precise assignment of the type and position of PTMs, amino acid substitutions, and C- or N-terminal truncations (6), whereas the bottom-up MS approach allows only the identification of a certain protein family when few or redundant peptides are found for a particular protein isoform. At a practical level, however, top-down MS-based proteomics struggles not only with the single- or multi-dimensional separation of undigested proteins, which demonstrates lower reproducibility and repeatability than for peptides, but also with technical limitations present in even state-of-the-art mass spectrometers. The outcome of a top-down MS experiment depends indeed on the balance between the applied resolution of the mass spectrometer and its sensitivity. The former is required for unambiguous assignment of ion isotopic clusters in both survey and MS/MS scans, whereas the latter is ultimately dependent on the scan speed of the mass analyzer, which determines the number of scans that can be accumulated for a given analyte ion on the liquid chromatography (LC) timescale to enhance the resulting signal-to-noise ratio (SNR). Until recently, the instrument of choice for top-down MS has been the Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer, primarily because of its superior resolving power and the availability of electron capture dissociation for the efficient MS/MS of large biomolecules (7, 8). However, this solution has been shown to have some limitations in the analysis of large proteins (9). The main issue, as described by Compton et al. (10), is that the SNR in Fourier transform mass spectrometry (FTMS) is inversely proportional to the width of the isotopic and charge state distributions (11), which both increase as a function of MW. Particularly, the SNR dramatically decreases with MW under standard on-line LC-MS/MS operating conditions if isotopic resolution is required. It is noteworthy that such SNR reduction can affect not only intact mass measurements, but also the subsequent MS/MS performance.The most widely employed solution for improving top-down analysis is thus a substantial reduction of the protein mixture complexity, for example, through off-line sample prefractionation (12). Furthermore, when the MW exceeds 100 kDa, proteins are often analyzed via direct infusion after off-line purification of the single isoform or species of interest (13). Overall, these strategies aim to improve the quality of mass spectra, specifically their SNR, by increasing the number of scans dedicated to each selected isoform or species. However, off-line intact protein analysis has limitations, including sample degradation and modification (e.g., oxidation during long off-line measurements and sample storage). The time required for multistep LC-based protein purification can also be substantial.Electron capture dissociation (ECD) (14, 15) and electron transfer dissociation (ETD) (16) are ion activation techniques that allow polypeptide fragmentation with reduced PTM losses (17, 18). Nevertheless, ECD and ETD generally provide larger sequence coverage for intact proteins than slow-heating activation methods such as collision induced dissociation (CID) and infrared multiple photon dissociation (19, 20). Furthermore, ECD and ETD are known to cleave disulfide bonds, a fundamental feature for the analysis of proteins in their native state (i.e., without cysteine reduction and alkylation) (21–23).The structural analysis of high MW intact proteins with MS has garnered much recent attention in the literature (24, 25), mainly because of the improved capabilities offered by rapidly developing sample preparation, protein separation, and mass spectrometric methods and techniques. Immunoglobulin G (IgG) proteins are antibodies with an MW of about 150 kDa that are composed of two identical sets of light and glycosylated heavy chains with both intra- and intermolecular disulfide bridges (Fig. 1) (26). IgGs represent an attractive target for structural analysis method development, given their high importance as biotherapeutics (27). A unit-mass resolution mass spectrum demonstrating an isotopic distribution of an isolated charge state of a 148 kDa IgG1 has been recently achieved with FT-ICR MS equipped with 9.4 T superconducting magnet and a statically harmonized ICR cell (24). However, further analytical improvements are needed to achieve routine and reproducible MS operation at the required level of resolution and sensitivity.Open in a separate windowFig. 1.Schematic representation of IgG1. Two identical light (blue) and two identical heavy (fucsia) chains form the intact IgG. The light chain is composed of a variable domain (V_L) and a constant domain (C_L), whereas the heavy chain comprises one variable domain (V_H) and three constant domains (C_H1–3). Each domain contains an intramolecular disulfide bridge (in red); intermolecular disulfide bridges link the heavy chains to each other (two bonds) and each heavy chain to one light chain (one bond). Each heavy chain includes an N-glycosylation site (located at Asn²⁹⁷; here, a G0F/G0F glycosylation is shown).Fragmentation of intact antibodies in the gas phase following the top-down MS approach has been previously attempted without precursor ion charge state isolation by means of nozzle-skimmer CID on a linear trap quadrupole (LTQ)-Orbitrap™ (28, 29) and with precursor ion isolation via ETD on a high resolution quadrupole time-of-flight (qTOF) mass spectrometer (25). Relative to the results previously obtained with slow-heating MS/MS methods, the ETD qTOF MS/MS demonstrated substantially higher sequence coverage, reaching 15% for human and 21% for murine IgGs. Important for future top-down proteomics development for complex protein mixtures, the ETD qTOF MS/MS results were obtained on the LC timescale. To increase the sequence coverage and confidence in product ion assignment, a substantial increase in SNR was achieved by averaging MS/MS data from up to 10 identical LC-MS/MS experiments. The high complexity of the product ion population reduced the effective resolution to about 30,000, presumably limiting the assignment of overlapping high charge state product ions in the 1000–2000 m/z range. Even higher peak complexity was observed in the region of charge reduced species and complementary heavy product ions, above 3000 m/z. Finally, numerous disulfide bonds drastically reduced MS/MS efficiency in the disulfide bond-protected regions.Here we demonstrate that ETD-enabled hybrid linear ion trap Orbitrap FTMS allows us to further improve the top-down ETD-based LC-MS/MS of monoclonal antibodies, introduced earlier for TOF-based MS. To fully take advantage of the high resolving power of Orbitrap MS/MS for increasing both the number of assigned product ions and the confidence of the assignments, maintaining an LC-MS/MS setup useful in a general proteomics workflow for protein desalting and separation, we averaged time-domain transients (derived from separated LC-MS/MS runs) before Fourier transform signal processing.     

A novel type of allosteric regulation: functional cooperativity in monomeric proteins

Cooperative functional properties and allosteric regulation in cytochromes P450 play an important role in xenobiotic metabolism and define one of the main mechanisms of drug-drug interactions. Recent experimental results suggest that ability to bind simultaneously two or more small organic molecules can be the essential feature of cytochrome P450 fold, and often results in rich and complex pattern of allosteric behavior. Manifestations of non-Michaelis kinetics include homotropic and heterotropic activation and inhibition effects depending on the stoichiometric ratios of substrate and effector, changes in the regio- and stereospecificity of catalytic transformations, and often give rise to the clinically important drug-drug interactions. In addition, functional response of P450 systems is modulated by the presence of specific and non-specific effector molecules, metal ions, membrane incorporation, formation of homo- and hetero-oligomers, and interactions with the protein redox partners. In this article we briefly overview the main factors contributing to the allosteric effects in cytochromes P450 with the main focus on the sources of cooperative behavior in xenobiotic metabolizing monomeric heme enzymes with their conformational flexibility and extremely broad substrate specificity. The novel mechanism of functional cooperativity in P450 enzymes does not require substantial binding cooperativity, rather it implies the presence of one or more binding sites with higher affinity than the single catalytically active site in the vicinity of the heme iron.     

Epitope mapping of antigenic determinants of hepatitis C virus proteins by phage display

Study of individual hepatitis C (HCV) proteins could help to find a molecular structure and conformation, localization of antigenic and immunogenic determinants, to reveal of protective epitopes. It is necessary for practical medicine - development of diagnostic test-systems, vaccines and therapeutics. Linear and conformation dependent epitopes of HCV proteins was localized in this work and immunogenic properties of phage displayed peptides screened on monoclonal antibodies to HCV proteins have been investigated. Eleven epitopes of four HCV proteins have been studied. Three epitopes was found as linear, two epitopes were dependent on secondary structure of proteins and one epitope was dependent on tertiary structure of NS3 protein. Aminoacid sequences of other determinants have been determined and the distinct localization of these determinants will be continued after discovering of tertiary structure of HCV proteins. It was shown, that phage mimotope 3f4 is immunogenic and could induce specific hu- moral immune response to NS5A HCV protein. The data obtained could be useful for improving of HCV diagnostic test-systems, studying of amino acid substitutions and its influence on antigenic properties of the HCV proteins. The results could help to study an immune response in patients infected with different genotypes of HCV. Phage displayed peptides mimicking the antigenic epitopes of HCV proteins could be applied to development of HCV vaccine.     

Generation of uniform low-temperature plasma in a pulsed non-self-sustained glow discharge with a large-area hollow cathode

Generation of plasma in a pulsed non-self-sustained glow discharge with a hollow cathode with an area of ≥2 m² at gas pressures of 0.4–1 Pa was studied experimentally. At an auxiliary arc-discharge current of 100 A and a main discharge voltage of 240 V, a pulse-periodic glow discharge with a current amplitude of 370 A, pulse duration of 340 μs, and repetition rate of 1 kHz was obtained. The possibility of creating a uniform gas-discharge plasma with a density of up to 10¹² cm^?3 and an electron temperature of 1 eV in a volume of >0.2 m³ was demonstrated. Such plasma can be efficiently used to treat material surfaces and generate pulsed ion beams with a current density of up to 15 mA/cm².     

Correlated Evolution of Nucleotide Positions within Splice Sites in Mammals

Splice sites (SSs)—short nucleotide sequences flanking introns—are under selection for spliceosome binding, and adhere to consensus sequences. However, non-consensus nucleotides, many of which probably reduce SS performance, are frequent. Little is known about the mechanisms maintaining such apparently suboptimal SSs. Here, we study the correlations between strengths of nucleotides occupying different positions of the same SS. Such correlations may arise due to epistatic interactions between positions (i.e., a situation when the fitness effect of a nucleotide in one position depends on the nucleotide in another position), their evolutionary history, or to other reasons. Within both the intronic and the exonic parts of donor SSs, nucleotides that increase (decrease) SS strength tend to co-occur with other nucleotides increasing (respectively, decreasing) it, consistent with positive epistasis. Between the intronic and exonic parts of donor SSs, the correlations of nucleotide strengths tend to be negative, consistent with negative epistasis. In the course of evolution, substitutions at a donor SS tend to decrease the strength of its exonic part, and either increase or do not change the strength of its intronic part. In acceptor SSs, the situation is more complicated; the correlations between adjacent positions appear to be driven mainly by avoidance of the AG dinucleotide which may cause aberrant splicing. In summary, both the content and the evolution of SSs is shaped by a complex network of interdependences between adjacent nucleotides that respond to a range of sometimes conflicting selective constraints.     

Effect of structural factors on the stability of duplexes formed by oligonucleotide conjugates with minor groove binders

The effect of structural factors on the stability of duplexes formed by DNA minor groove binders conjugated with oligonucleotide mono- or diphosphoramidates of the general formula Oligo-MGB_m (where Oligo is an oligonucleotide; m = 1 or 2; MGB is -L(Py)₂R, -L(Py)₄R, -L(Im)₄R, or -L(Py)₄NH(CH₂)₃CO(Py)₄R; Py is a 4-aminopyrrole-2-carboxylic acid residue; L is a -aminobutyric acid or an -aminocaproic acid residue, R = OEt, NH(CH₂)₆NEt₂, or NH(CH₂)₆N⁺Me₃) was studied by the method of thermal denaturation. The mode of binder interaction with the minor groove depends on the conjugate structure; it may be of the parallel head to head type for bisphosphoramidates and of the antiparallel head to tail type for monophosphoramidates of a hairpin structure. The effects of the duplexes with parallel orientation (bisphosphoramidates, MGB is L(Py)₄R, m = 2) and those of the hairpin structure with the antiparallel orientation (monophosphoramidates, MGB is L(Py)₄(CH₂)₃CO(Py)₄R, m = 1) on T _m values were close. The influence of the linker (L) and substituent (R) structures upon T _m was more pronounced for monophosphoramidate (MGB is L(Py)_nR, m = 1) than for bisphosphoramidate (MGB is L(Py)_nR, m = 2). No more than two oligopyrrolecarboxamide residues (either in parallel or antiparallel orientations) can be incorporated into the duplex minor groove. Moreover, it was shown by the example of monophosphoramidates (Oligo-L(Py)₄R and Oligo-L(Py)₄NH(CH₂)₃CO(Py)₄R) that the addition of a second ligand capable of incorporation into the minor groove increased T _m of the corresponding duplex in comparison with the duplex formed by the starting monophosphoramidate. At the same time, the introduction of a ligand incapable of incorporating decreased the T _m value. The mode of interaction of the conjugated binder with the oligonucleotide duplex is determined by its structure. For example, dipyrrolecarboxamide containing an ethoxy group at the binder C-end stabilizes the duplex due to stacking interaction with the terminal A · T pair, whereas tetrapyrrolecarboxamides stabilize the duplex by incorporation into the minor groove.__________Translated from Bioorganicheskaya Khimiya, Vol. 31, No. 2, 2005, pp. 159–166.Original Russian Text Copyright © 2005 by Ryabinin, Butorin, Elen, Denisov, Pyshnyi, Sinyakov.     

Disposable luciferase‐based microfluidic chip for rapid assay of water pollution

In the present study, we demonstrate the use of a disposable luciferase‐based microfluidic bioassay chip for environmental monitoring and methods for fabrication. The designed microfluidic system includes a chamber with immobilized enzymes of bioluminescent bacteria Photobacterium leiognathi and Vibrio fischeri and their substrates, which dissolve after the introduction of the water sample and thus activate bioluminescent reactions. Limits of detection for copper (II) sulfate, 1,3‐dihydroxybenzene and 1,4‐benzoquinone for the proposed microfluidic biosensor measured 3 μM, 15 mM, and 2 μM respectively, and these values are higher or close to the level of conventional environmental biosensors based on lyophilized bacteria. Approaches for entrapment of enzymes on poly(methyl methacrylate) (PMMA) plates using a gelatin scaffold and solvent bonding of PMMA chip plates under room temperature were suggested. The proposed microfluidic system may be used with some available luminometers and future portable luminescence readers.     

A new view of water dynamics in immobilized proteins.

The inflection frequency of the deuteron magnetic relaxation dispersion from water in rotationally immobilized protein samples has recently been found to be essentially independent of temperature and protein structure. This remarkable invariance has been interpreted in terms of a universal residence time of 1 microseconds for protein-associated water molecules. We demonstrate here that this interpretation is an artifact of the conventional perturbation theory of spin relaxation, which is not valid for rotationally immobile proteins. Using a newly developed non-perturbative, stochastic theory of spin relaxation, we identify the apparent correlation time of 1 microseconds with the inverse of the nuclear quadrupole frequency, thus explaining its invariance. The observed dispersion profiles are consistent with a broad distribution of residence times, spanning the microseconds range. Furthermore, we argue that the deuteron dispersion is due to buried water molecules rather than to the traditional surface hydration previously invoked, and that the contribution from rapidly exchanging protein hydrogens cannot be neglected. The conclusions of the present work are also relevant to proton relaxation in immobilized protein samples and to magnetic resonance imaging of soft tissue.     

Linear growth of sparsely rakered whitefish <Emphasis Type="Italic">Coregonus lavaretus</Emphasis> (Coregonidae) of the Imandra Lake (Murmansk oblast)

This paper studies the properties of linear growth in sparsely rakered whitefish Coregonus lavaretus in the stretches of the large subarctic Imandra Lake, which are characterized by the diversity of habitat conditions and anthropogenic stress levels. The groups of sparsely rakered whitefish that are confined to different areas of the lake are marked out based on the properties of growth, trophic status, and anthropogenic stress intensity. A direct relationship of the linear growth in whitefish with the age of sexual development and trophic level of its habitats is found.