Analysis by capillary electrophoresis of the kinetics of charge ladder formation for bovine carbonic anhydrase

A series of charge ladders of bovine carbonic anhydrase II were synthesized and the relative abundances of the rungs analyzed by capillary electrophoresis as a function of the quantity of acylating agent used. A simulation that models the kinetics of formation of the members of the charge ladders is described. The observed rate constants decreased as the extent of acylation increased. These rate constants correlated adequately with theoretical rate constants calculated using Debye-Hückel theory. The data are compatible with, but do not demand, a model for the formation of this charge ladder in which all unacetylated amino groups in each rung have indistinguishable reactivity and in which the reactivity of the amines in each rung decreases as the net charge on the protein increases; in this model, decreased reactivity is due to increased extent of protonation. This agreement between experiment and model suggests that the charge shielding that results from an ionic strength of 130 mM is not sufficient to suppress the influence of the increasingly negative charge of the protein with acetylation on the extent of protonation of Lys epsilon-NH2 groups.     

Influence of charge regulation in electrostatic interaction chromatography of proteins

Recently, the "slab model" was proposed to describe the interaction between a protein and the charged stationary phase surface in electrostatic interaction chromatography. The model is based on the solution of the linearized Poisson-Boltzmann equation for a system consisting of two charged planar surfaces in contact with an electrolyte solution. In the model it is assumed that the charge densities of both the protein and the stationary phase are constant during the adsorption process. However, as the protein comes close to the oppositely charged stationary phase surface, the protein net charge will change due to the electrical field from the stationary phase. In this paper, the theory for charge regulation is applied to the original slab model, and simple algebraic equations are developed in order to include the effect of charge regulation on the capacity factor. A large body of retention data are reanalyzed with the new model, and it is found that there is good agreement between the chromatographically and titrimetrically obtained protein net charge. An interesting consequence of charge regulation is that it gives a contribution to the retention of proteins with zero net charge and even to proteins with the same sign of charge as the stationary phase.     

Role of charge suppression and ionic strength in free zone electrophoresis of proteins.

The free zone electrophoretic mobility of proteins can be predicted from the protein's amino acid content by applying a model based on the Debye-Hückle-Henry theory and Henderson-Hasselbalch equation. Calculated mobilities are always greater than actual mobility but a pH-independent proportionality (described by the constant FZ) is found between the two. Thus, determination of a protein's mobility at one pH allows, with the use of the model and FZ, calculation of its mobility at other pH conditions. This leads directly to optimum conditions for the electrophoretic resolution of proteins in capillary zone electrophoresis. The fundamental nature of FZ is examined and found to be a function of a proteins molecular weight, charge, and solution ionic strength. This work aids in explaining the form of previously proposed empirically based equations for peptide and protein mobility.     

"One-pot" methylation in glycomics application: esterification of sialic acids and permanent charge construction

A simple and rapid "one-pot" methylation method to esterify sialic acids and construct a permanent charge was developed for N-linked glycan analysis, which combined complete nonspecific proteolytic digestion and methylation. A mixture of Asn-glycans prepared from Pronase E digestion of the glycoprotein was passed through a cation-exchange column to convert carboxylic acids to the Na+ form before being methylated with methyl iodide. Derivatives could be easily purified with a hydrophilic affinity chromatography cartridge. Mass spectrometry analysis was performed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) and MALDI-TOF/TOF. The mass spectrometric data indicated that carboxylic acids were methylated in addition to the formation of a quaternary ammonium in the amino group of asparagine residues. Three model glycoproteins, including ribonuclease B, ovalbumin, and transferrin, were employed to demonstrate the merits of this technique. Results showed that the stabilization of sialic acid was achieved in addition to the formation of a permanent charge. Compared to the analysis of underivatized N-glycans, detection sensitivity improved approximately 10-fold. The new technique was further evaluated with glycan profiling of serum transferrin and proved to be a sensitive method for the characterizing protein glycosylation.     

Negative electrospray droplet exposure to gaseous bases for the manipulation of protein charge state distributions

The exposure of electrospray droplets to vapors of reagents of various base strengths affects protein negative charge state distributions independent of initial solution conditions. Volatile bases are introduced into the counter-current nitrogen drying gas of an electrospray interface to interact with charged droplets as they undergo desolvation/disintegration, shifting charge state distributions of proteins to higher, more negative, charge states. Alterations of charge state distributions can implicate protein folding/unfolding phenomena. Species bound by relatively weak interactions can be preserved, at least to some extent, allowing for the observation of high charge states of protein-ligand complexes, such as high negative charge states of holomyoglobin. The binding of carbonic anhydrase with its Zn(2+) cofactor is apparently preserved when the holo-form of the protein is exposed to basic vapors (i.e., the Zn(2+) ion remains associated with the protein), but this prevents the appearance of charge states higher than -17. Charge state distributions of proteins containing disulfide bonds shift slightly with the leak-in of basic vapors, but when these disulfide bonds are reduced with dithiothreitol in solution, charge states higher than the number of acidic sites (Asp, Glu, and C-terminus) are observed. Since there is no observed change in the distributions of buffered proteins exposed to these reagent vapors, the charge state changes are attributed largely to a pH affect. High pK(a) and highly volatile reagents have been found to be the most effective in terms of observing the maximum negative charge state of the biomolecule of interest.     

Proteolysis in mixed organic-aqueous solvent systems: applications for peptide mass mapping using mass spectrometry

The rate of protein digestion imposes significant limitations on high-throughput protein identification using mass spectrometry. In this report, we demonstrate that proteins are readily digested by trypsin in the presence of organic solvents such as methanol, acetone, 2-propanol, and acetonitrile. The rates of protein digestion in organic solvents, as indicated by the abundances of digest fragment ions in the mass spectrum, are increased relative to aqueous solution. In addition, amino acid coverage for the analyzed proteins increases in the presence of the organic solvents, and proteins that are resistant to proteolysis are readily digested. For example, a 68% amino acid sequence coverage was attained from a tryptic digest of myoglobin in < 5 min from an 80% acetonitrile solution, whereas no digest fragments were detected from a 5 min digestion in an aqueous solution. Moreover, the tryptic digestion of a complex protein mixture in an organic-aqueous solvent system showed significantly enhanced digestion for nearly all of the protein components. Enzymatic digestion in an organic-aqueous solvent system is a rapid, simple, and effective peptide mass-mapping technique.     

Manipulation of protein charge states through continuous flow-extractive desorption electrospray ionization: a new ambient ionization technique

Manipulation of protein charge states in electrospray ionization-mass spectrometry (ESI-MS) has implications for the study of intact proteins, protein-protein interactions, post-translational modifications, and protein sequencing. Control of these protein charge states is often difficult to achieve with conventional methods of analysis. A novel ambient ionization configuration, continuous flow-extractive desorption electrospray ionization (CF-EDESI), is presented as a means to control the charge state distribution of proteins. A key feature of the CF-EDESI technique is the continuous flow needle, which is a hypodermic needle presented orthogonal to the electrospray source and delivers a solvent flow containing analytes for extractive desorption ionization. With this source design, the successful manipulation of cytochrome c and lysozyme charge states with the use of different additives, such as acetic acid and sulfolane, was demonstrated. Results were compared to data obtained with conventional electrospray ionization. Good agreement with previously reported studies of cytochrome c unfolding/folding studies, performed by conventional ESI-MS, is evident. In addition to the protein analysis presented, the CF-EDESI-MS technique should be applicable for analyzing atypical analyte and solvent systems by mass spectrometry while maintaining optimal electrospray source conditions.     

Use of vapor-phase acid hydrolysis for mass spectrometric peptide mapping and protein identification

A method for mass spectrometric peptide mapping was developed, based on hydrolysis of a solid protein by acid vapor followed by mass spectrometric analysis of the cleavage products. The method is applicable to lyophilized samples as well as proteins present in gels after separation by SDS-PAGE. The cleavage specificity was established using a number of standard proteins. Three different types of cleavages were observed: specific internal backbone cleavages at Asp, Ser, Thr, and Gly and N- and C-terminal sequence ladders. On the basis of the observed cleavage characteristics, a strategy for protein identification based on the peptide mass maps was developed. The identification strategy utilizes the specific internal backbone cleavages as well as the partial sequence information, obtained from the sequence ladders.     

Refrigerant charge in refrigerating systems and strategies of charge reduction

For some years, European regulation concerning refrigerating systems has become more and more severe. Because of their design and technology, refrigeration units cannot avoid refrigerant leaks, and can thus have a harmful impact on the environment. Many studies aimed at minimizing the charge in a refrigerating machine were thus developed. On a global level, reduction of refrigerant charges must not affect energy aspects while respecting environmental constraints [Montreal 1987, Kyoto 1997]. The architecture and refrigerant charge distribution in the systems are of particular importance; their analyses permit the determination of the target elements and thus define those in which the refrigerant charge could be reduced (heat exchangers, liquid pipes, receivers, …). This paper presents a review of the refrigerant charge studies in a refrigerating plant (methodologies of charge measurement, void fraction correlations and characteristic values for various technologies). It also evaluates the influence of the refrigerant charge on the coefficient of performance (COP) and on the cooling capacity. Finally, it inventories the different means that allow the charge in a refrigerating system to be minimized.     

Bacterial adhesion to phosphorylcholine-based polymers with varying cationic charge and the effect of heparin pre-adsorption

The steady increase in the use of medical implants and the associated rise of medical device infections has fuelled the need for the production of biomaterials with improved biocompatibility. 2-(methacryloyloxyethyl phosphorylcholine) (MPC) based coatings have been used to improve the biocompatibility of a number of different medical devices. Recent studies have investigated the use of a phosphorylcholine modified with cationic charge to encourage specific bio-interaction. Until now the affect of cationic charge incorporation in MPC copolymers on bacterial adhesion has not been investigated. This study attempts to address this by investigating the affect of charge on four different strains of bacteria commonly associated with medical device infections. In addition, the affect of pre-incubating these MPC-copolymers in heparin is also evaluated as this has previously been shown to improve biocompatibility and reduce bacterial adhesion. Bacterial adhesion was assessed by ATP bioluminescence and Scanning Electron Microscopy (SEM). Results suggest that bacterial adhesion generally increased with increasing cationic charge. When samples were however, pre-incubated with heparin a significant reduction in bacterial adhesion to the MPC-based samples was observed. The heparin remained bound and effective at reducing bacterial adhesion to the cationic MPC-based samples even after three weeks incubation in PBS. To conclude, the MPC-based cationic polymer coatings complexed with heparin may provide a promising solution to reduce medical device related infections.     

Identification of bacteriophage MS2 coat protein from E. coli lysates via ion trap collisional activation of intact protein ions

Collisional activation of the intact MS2 viral capsid protein with subsequent ion/ion reactions has been used to identify the presence of this virus in E. coli lysates. Tandem ion trap mass spectrometry experiments on the +7, +8, and +9 charge states, followed by ion/ion reactions, provided the necessary sequence tag information (and molecular weight data) needed for protein identification via database searching. The most directly informative structural information is obtained from those charge states that produce a series of product ions arising from fragmentation at adjacent residues. The formation of these product ions via dissociation at adjacent amino acid residues depends greatly on the charge state of the parent ion. Database searching of the charge-state-specific sequence tags was performed by two different search engines: the ProteinInfo program from the Protein information Retrieval On-line World Wide Web Lab or PROWL and the TagIdent program from the ExPASy molecular biology server. These search engines were used in conjunction with the sequence tag information generated via collisional activation of the intact viral coat protein. These programs were used to evaluate the feasibility of generating sequence tags from collisional activation of intact multiply charged protein ions in a quadrupole ion trap.     

Dissociation of multiple protein ion charge states following a single gas-phase purification and concentration procedure

The formation of a range of precursor ion charge states from a single concentrated and purified charge state, followed by activation of each charge state, is introduced as a means to obtain more protein structural information than is available from dissociation of a single charge state alone. This approach is illustrated using off-resonance collisional activation of the [M + 8H]8+ to [M + 6H]6+ precursor ions of the bacteriophage MS2 viral coat protein following concentration and purification of the [M + 8H]8+ charge state. This range of charge states was selected on the basis of an ion trap collisional activation study of the effects of precursor ion charge state on the dissociation of the [M + 12H]12+ to [M + 5H]5+ ions. Gas-phase ion/ion proton-transfer reactions and the ion parking technique were applied to purify and concentrate selected precursor ion charge states as well as to simplify the product ion spectra. The high-charge-state ions fragment preferentially at the N-terminal side of proline residues while the product ion spectra of the lowest charge states investigated are dominated by C-terminal aspartic acid cleavages. Maximum structural information is obtained by fragmentation of the intermediate-charge states.     

Effects of peptide hydrophobicity and charge state on molecular ion yields in plasma desorption mass spectrometry

Plasma desorption mass spectra were obtained for a series of peptides, grouped in four mass ranges having approximately 9, 20, 30, and 40 amino acid residues. Within each group, the individual peptides differed in hydrophobicity, charge state, and retention time, as measured on a reversed-phase HPLC column. Comparison of the molecular ion intensities in the positive ion mass spectra of peptides from each group showed a strong dependence upon hydrophobicity and no correlation with charge state. Plasma desorption mass spectra of mixtures of all the peptides within each mass range generally resulted in the desorption of a single residue and suppression of the ion signal from other components. In most cases, this could be correlated with hydrophobicity, as calculated from the Bull and Breese index; however, a better correlation existed when the results were compared with reversed-phase retention times. In general the spectra of mixtures were not influenced by charge state (except in the absence of hydrophobic peptides), as the same component in each peptide mixture produced the most abundant ions in both positive and negative ion spectra.     

Fundamental design principles that guide induction of helix upon formation of stable peptide-nanoparticle complexes

We have shown that it is possible to design a peptide that has a very low helical content when free in solution but that adopts a well-defined helix when interacting with silica nanoparticles. From a systematic variation of the amino acid composition and distribution in designed peptides, it has been shown that the ability to form helical structure upon binding to the silica surface is dominated by two factors. First, the helical content is strongly correlated with the net positive charge on the side of the helix that interacts with the silica, and arginine residues are strongly favored over lysine residues in these positions. The second important factor is to have a high net negative charge on the side of the helix that faces the solution. Apparently, both attractive and repulsive electrostatic forces dominate the induction and stabilization of a bound helix. It is also evident that using amino acids that have high propensity to form helix in solution are also advantageous for the formation of helix on surfaces.     

Enhancement of MALDI-MS spectra of C-terminal peptides by the modification of proteins via an active ester generated in situ from an oxazolone

For selective C-terminal derivatization of peptides and proteins, we have devised a method for activating the C-terminal carboxyl group by extending the oxazolone chemistry. A mixture of formic acid and acetic anhydride was found to be effective for the formation of an oxazolone, which was converted to an active ester in situ in the presence of a phenol or an N-hydroxide. In particular, the resulting active ester with pentafluorophenol facilitated the subsequent reaction with an amine and the hydrazine derivative to yield the C-terminal amide and hydrazide, respectively. The peptides thus coupled with arginine methyl ester or 2-hydrazino-2-imidazoline containing the guanidino moiety exhibited the positive-ion peaks in matrix-assisted laser desorption/ionization (MALDI) mass spectra with appreciably enhanced intensities. As expected from the reaction mechanism, the carboxyl groups of aspartic and glutamic acid residues were not modified, while the amino groups that could react with the activated peptides were concomitantly protected by formylation. The MALDI peaks corresponding to the C-terminal peptide fragments of proteins were specifically enhanced, discriminating against those from internal peptides that were not tagged with a positive charge. In favorable cases, the C-terminal peptide fragments were clearly discerned by MALDI-MS after chymotryptic digestion and were identified by their MALDI postsource decay analysis. Based on these results, we suggest a method for C-terminal sequencing of a protein.     

Revealing a positive charge patch on a recombinant monoclonal antibody by chemical labeling and mass spectrometry

During purification process development and analytical characterization, a recombinant human monoclonal antibody, referred to as rmAb1, showed an anomalous charge heterogeneity profile by cation-exchange chromatography (CIEC), characterized by extremely high retention and poor resolution between charge variants. Mass spectrometry-based footprinting methodologies that include selective labeling of lysine with sulfosuccinimidyl acetate and arginie with p-hydroxyphenylglyoxal were developed to map the positive charges on the rmAb1 surface. On the basis of the average percentages of labeling obtained for the lysine and arginine residues by peptide mapping analysis, the positive charges were more distributed on the surface in the Fab region than in the Fc region of rmAb1. By a comparative study of in-solution and on-resin labeling reaction dynamics, seven positively charged residues were identified to bind to the cation-exchange resin and they were located in the variable domains. Among them, three lysine and one arginine residues appeared to cluster together on the surface to form a positive charge patch. When the charge patch residues were neutralized by chemical labeling, rmAb1 exhibited a more typical CIEC retention time, confirming that the charge patch was responsible for the atypical CIEC profile of rmAb1. To our knowledge, this work is the first report revealing the amino acid composition of a surface charge patch on therapeutic monoclonal antibodies.     

Surface charge sensitivity of silicon nanowires: size dependence

Silicon nanowires of different widths were fabricated in silicon on insulator (SOI) material using conventional process technology combined with electron-beam lithography. The aim was to analyze the size dependence of the sensitivity of such nanowires for biomolecule detection and for other sensor applications. Results from electrical characterization of the nanowires show a threshold voltage increasing with decreasing width. When immersed in an acidic buffer solution, smaller nanowires exhibit large conductance changes while larger wires remain unaffected. This behavior is also reflected in detected threshold shifts between buffer solutions of different pH, and we find that nanowires of width >150 nm are virtually insensitive to the buffer pH. The increased sensitivity for smaller sizes is ascribed to the larger surface/volume ratio for smaller wires exposing the channel to a more effective control by the local environment, similar to a surrounded gate transistor structure. Computer simulations confirm this behavior and show that sensing can be extended even down to the single charge level.     

High-resolution mapping of carbene-based protein footprints

Carbene chemistry has been used recently in structural mass spectrometry as a labeling method for mapping protein surfaces. The current study presents a method for quantitating label distribution at the amino acid level and explores the nature and basis for an earlier observation of labeling bias. With the use of a method based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) applied to digests of holo-calmodulin, we developed a quantitation strategy to map site-specific incorporation of carbene, generated from photolysis of ionic label precursors 2-amino-4,4-azipentanoic acid and 4,4-azipentanoic acid. The approach provides reliable incorporation data for fragments generated by electron-transfer dissociation, whereas high-energy collisional dissociation leads to energy and sequence-dependent loss of the label as a neutral. However, both can produce data suitable for mapping residues in the interaction of holo-calmodulin with M13 peptide ligand. Site-specific labeling was monitored as a function of reagent, ionic strength, and temperature, demonstrating that electrostatic interactions at the protein surface can "steer" the distribution of label precursors to sites of surface charge and favor label insertion into residues in the vicinity of the surface charge. A further preference for insertion into carboxylates was observed, based on chemical reactivity. We suggest that decoupling surface partitioning from the chemistry of insertion offers a flexible, tunable labeling strategy for structural mass spectrometry that can be applied to a broad range of protein surface compositions and promotes the design of reagents to simplify the workflow.     

Image charge detection mass spectrometry: pushing the envelope with sensitivity and accuracy

A novel image charge detection mass spectrometer (CDMS) with improved sensitivity and mass accuracy is described. The improved detector design and method of data analysis allow us to measure a reliable mass for a single macroion that is an order of magnitude smaller than previously achieved with CDMS. The apparatus employs an image charge detector array consisting of 22 detectors. The detectors are divided into two groups that can be floated at different potentials. The signals from the detector array are analyzed using a correlation approach to yield the velocities in the two groups of detectors and the charge. These quantities, together with the voltage difference between the two groups of detectors, provide a value for the mass. The mass, m/z, and charge distributions recorded for 300 kDa poly(ethylene oxide) (PEG) are presented. The mass distribution shows a peak at around 300 kDa with a width close to that expected from the polymer size distribution. In addition, there are broad peaks in the mass distribution at around 100 and 500 MDa. The 300 kDa ions have m/z ratios of ～2 kDa/e, and the 100 and 500 MDa ions have m/z ratios of ～40 kDa/e. The 100 and 500 MDa ions probably result from PEG aggregates that are either present in solution or the residue of large electrospray droplets.     

Charger: combination of signal processing and statistical learning algorithms for precursor charge-state determination from electron-transfer dissociation spectra

Tandem mass spectrometry in combination with liquid chromatography has emerged as a powerful tool for characterization of complex protein mixtures in a high-throughput manner. One of the bioinformatics challenges posed by the mass spectral data analysis is the determination of precursor charge when unit mass resolution is used for detecting fragment ions. The charge-state information is used to filter database sequences before they are correlated to experimental data. In the absence of the accurate charge state, several charge states are assumed. This dramatically increases database search times. To address this problem, we have developed an approach for charge-state determination of peptides from their tandem mass spectra obtained in fragmentations via electron-transfer dissociation (ETD) reactions. Protein analysis by ETD is thought to enhance the range of amino acid sequences that can be analyzed by mass spectrometry-based proteomics. One example is the improved capability to characterize phosphorylated peptides. Our approach to charge-state determination uses a combination of signal processing and statistical machine learning. The signal processing employs correlation and convolution analyses to determine precursor masses and charge states of peptides. We discuss applicability of these methods to spectra of different charge states. We note that in our applications correlation analysis outperforms the convolution in determining peptide charge states. The correlation analysis is best suited for spectra with prevalence of complementary ions. It is highly specific but is dependent on quality of spectra. The linear discriminant analysis (LDA) approach uses a number of other spectral features to predict charge states. We train LDA classifier on a set of manually curated spectral data from a mixture of proteins of known identity. There are over 5000 spectra in the training set. A number of features, pertinent to spectra of peptides obtained via ETD reactions, have been used in the training. The loading coefficients of LDA indicate the relative importance of different features for charge-state determination. We have applied our model to a test data set generated from a mixture of 49 proteins. We search the spectra with and without use of the charge-state determination. The charge-state determination helps to significantly save the database search times. We discuss the cost associated with the possible misclassification of charge states.