Enhanced-Fluidity Liquid Chromatography-Mass Spectrometry for Intact Protein Separation and Characterization.

Recent advances in the analysis of proteins have increased the demand for more efficient techniques to separate intact proteins. Enhanced-fluidity liquid chromatography (EFLC) involves the addition of liquefied CO2 to conventional liquid mobile phases. The addition of liquefied CO2 increases diffusivity and decreases viscosity, which inherently leads to a more efficient separation. Herein, EFLC is applied to hydrophobic interaction chromatography (HIC) stationary phases for the first time to study the impact of liquefied CO2 to the chromatographic behavior of proteins. The effects of liquefied CO2 on chromatographic properties, charge state distributions (CSDs), and ionization efficiencies were evaluated. EFLC offered improved chromatographic performance compared to conventional liquid chromatography (LC) methods including a shorter analysis time, better peak shapes, and higher plate numbers. The addition of liquefied CO2 to the mobile phase provided an electrospray ionization (ESI)-friendly and "supercharging" reagent without sacrificing chromatographic performance, which can be used to improve peptide and protein identification in large-scale application.

Hybrid phospholipid bilayer coatings for separations of cationic proteins in capillary zone electrophoresis.

Protein separations in CZE suffer from nonspecific adsorption of analytes to the capillary surface. Semipermanent phospholipid bilayers have been used to minimize adsorption, but must be regenerated regularly to ensure reproducibility. We investigated the formation, characterization, and use of hybrid phospholipid bilayers (HPBs) as more stable biosurfactant capillary coatings for CZE protein separations. HPBs are formed by covalently modifying a support with a hydrophobic monolayer onto which a self-assembled lipid monolayer is deposited. Monolayers prepared in capillaries using 3-cyanopropyldimethylchlorosilane (CPDCS) or n-octyldimethylchlorosilane (ODCS) yielded hydrophobic surfaces with lowered surface free energies of 6.0 ± 0.3 or 0.2 ± 0.1 mJ m(-2) , respectively, compared to 17 ± 1 mJ m(-2) for bare silica capillaries. HPBs were formed by subsequently fusing vesicles comprised of 1,2-dilauroyl-sn-glycero-3-phosphocholine or 1,2-dioleoyl-sn-glycero-3-phosphocholine to CPDCS- or ODCS-modified capillaries. The resultant HPB coatings shielded the capillary surface and yielded reduced electroosmotic mobility (1.3-1.9 × 10(-4) cm(2) V(-1) s(-1) ) compared to CPDCS- and ODCS-modified or bare capillaries (3.6 ± 0.2 × 10(-4) cm(2) V(-1) s(-1) , 4.8 ± 0.4 × 10(-4) cm(2) V(-1) s(-1) , and 6.0 ± 0.2 × 10(-4) cm(2) V(-1) s(-1) , respectively), with increased stability compared to phospholipid bilayer coatings. HPB-coated capillaries yielded reproducible protein migration times (RSD ≤ 3.6%, n ≥ 6) with separation efficiencies as high as 200 000 plates/m.

Purification, cDNA cloning, and recombinant expression of chymotrypsin C from porcine pancreas.

Chymotrypsin C is a bifunctional secretory-type serine protease in pancreas; besides proteolytical activity, it also exhibits a calcium-decreasing activity in serum. In this study, we purified activated chymotrypsin C from porcine pancreas, and identified its three active forms. Active chymotrypsin C was found to be different in the length of its 13-residue activation peptide due to carboxydipeptidase (present in the pancreas) degradation or autolysis of the activated chymotrypsin C itself, resulting in the removal of several C-terminus residues from the activation peptide. After limited chymotrypsin C cleavage with endopeptidase Lys C, several purified peptides were partially sequenced, and the entire cDNA sequence for porcine chymotrypsin C was cloned. Recombinant chymotrypsinogen C was successfully expressed in Escherichia coli cells as inclusion bodies. After refolding and activation with trypsin, the comparison of the recombinant chymotrypsin C with the natural form showed that their proteolytic and calcium-decreasing activities were at the same level. The successful expression of chymotrypsin C gene paves the way to further mutagenic structure-function studies.

Evaluation of chemically selective displacer analogues for protein purification.

A library of molecular analogues to the selective displacer, N'1'-(4-methylquinolin-2-yl)ethane-1,2-diamine dinitrate, was employed to study the effects of changes in displacer chemistry on their efficacy for selective separations. High throughput screens were carried out using a robotic liquid handling system to examine the ability of these compounds to selectively displace proteins in batch adsorption systems. Experiments were conducted using the model protein pairs ribonuclease A/alpha-chymotrypsinogen A and cytochrome C/lysozyme on a strong cation exchanger. Selectivity pathway and DC-50 plots were constructed from the analogue screen data, and results indicated that minor changes in the molecular design of the displacer can have a significant impact on the separation behavior. Specifically, charge density and spacing of resin and protein interaction moieties were found to be important. The screen also identified a new displacer, 4-methyl-2-piperazin-1-yl-quinoline, which produced a more selective displacement than previously reported with the original compound. A steric mass action dynamic affinity plot was constructed to validate that this new displacer was acting as a chemically selective, rather than a steric mass action selective displacer. Finally, saturation transfer difference NMR experiments were conducted to examine protein-displacer interactions with these displacers and protein pairs. These results demonstrate how subtle changes in displacer design can be employed to fine-tune the separation performance of chemically selective displacers.

Noncovalent poly(1-vinylpyrrolidone)-based copolymer coating for the separation of basic proteins and lipoproteins by CE.

A new simple and fast noncovalent coating method based on poly(1-vinylpyrrolidone-co-2-dimethylaminoethyl methacrylate) copolymer was developed for CE. Merely 2 min flushing of the capillary with the poly(1-vinylpyrrolidone-co-2-dimethylaminoethyl methacrylate) copolymer was required. The copolymer is adsorbed onto the fused-silica surface by hydrogen bonding and electrostatic interactions. EOF was almost totally suppressed over a wide pH range. The coating conditions (flushing time, copolymer concentration, and the concentration and pH of background electrolyte solution) and the stability of the coating were optimized, and the coated capillary was successfully applied to the fast separation of four basic proteins: lysozyme, cytochrome c, ribonuclease A, and alpha-chymotrypsinogen A. Separation efficiencies were high, ranging from 386 000 to 738 000 plates/m at 40 mM pH 4.0 acetate buffer being comparable to values obtained on classical covalent PVP-coated capillary. The RSD of migration times of basic proteins for 200 times successive runs were all below 1.0% (n=200, 3 days). A successful capillary performance was demonstrated also to the separation of low- and high-density lipoproteins at acidic pH.

Immunocytochemical localization of amylase and chymotrypsinogen in the exocrine pancreatic cell with special attention to the Golgi complex.

Affinity-purified, monospecific rabbit antibodies against rat pancreatic alpha-amylase and bovine pancreatic alpha-chymotrypsinogen were used for immunoferritin observations of ultrathin frozen sections of mildly fixed exocrine pancreatic tissue from secretion-stimulated (pilocarpine) rats and from overnight-fasted rats and guinea pigs. The labeling patterns for both antibodies were qualitatively alike: Labeling occurred in (a) the cisternae of the rough endoplasmic reticulum (RER) including the perinuclear cisterna, in (b) the peripheral area between the RER and cis-Golgi face, and (c) all Golgi cisternae, condensing vacuoles, and secretory granules. Labeling of cytoplasmic matrix was negligible. Structures that appeared to correspond to rigid lamellae were unlabeled. Differences in labeling intensities indicated that concentration of the zymogens starts at the boundary of the RER and cis-side of the Golgi complex. These data support the view that the Golgi cisternae are involved in protein processing in both stimulated and unstimulated cells and that Golgi cisternae and condensing vacuoles constitute a functional unit.

Capillary electrophoresis of intact basic proteins using noncovalently triple-layer coated capillaries.

The usefulness of a noncovalent, positively charged capillary coating for the efficient analysis of intact basic proteins with CE was studied. Capillaries were coated by subsequent flushing with solutions of 10% w/v Polybrene (PB), 3% w/v dextran sulfate (DS), and again 10% w/v PB. Coating characterization studies showed that stable coatings could be produced which exhibited a pH-independent and highly reproducible EOF. The PB-DS-PB coating was evaluated with Tris phosphate BGEs of various pH using the four basic model proteins: alpha-chymotrypsinogen A, ribonuclease A, cytochrome c, and lysozyme. Typical migration time RSDs for the proteins were less than 0.85%, and apparent plate numbers were above 125,000 using a capillary length of 40 cm. The high separation efficiency allowed detection of several minor impurities in the model proteins. Using a BGE of medium pH, the CE system with triple-layer coating appeared to be useful for the repeatable profiling of recombinant humanized mouse monoclonal immunoglobulin G(1) showing a characteristic pattern of glycoforms. The CE system was also applied to the characterization of two llama antibodies, which were produced in Saccharomyces cerevisiae, revealing the presence of a side product in one of the antibodies. The high migration time stability allowed the reliable determination of antibody-antigen binding by monitoring migration time shifts. Finally, the feasibility of using the PB-DS-PB coated capillaries for CE with mass spectrometric detection was shown by the characterization of the impure llama antibody sample.

Reaction of fluorogenic reagents with proteins II: capillary electrophoresis and laser-induced fluorescence properties of proteins labeled with Chromeo P465.

The fluorogenic reagent Chromeo P465 is considered for the analysis of proteins by capillary electrophoresis with laser-induced fluorescence detection. The reagent was first used to label alpha-lactalbumin; the product was analyzed by capillary zone electrophoresis in a sub-micellar sodium dodecyl sulfate (SDS) buffer. The product generated a set of equally spaced but poorly resolved peaks that formed a broad envelope with a net mobility of 4 x 10(-4)cm(2) V(-1) s(-1). The components of the envelope were presumably protein that had reacted with different numbers of labels. The mobility of these components decreased by roughly 1% with the addition of each label. The signal increased linearly from 1.0 nM to 100 nM alpha-lactalbumin (r(2)=0.99), with a 3sigma detection limit of 70 pM. We then considered the separation of a mixture of ovalbumin, alpha-chymotrypsinogen A, and alpha-lactalbumin labeled with Chromeo P465; unfortunately, baseline resolution was not achieved with a borax/SDS buffer. Better resolution was achieved with N-cyclohexyl-2-aminoethanesulfonic acid/Tris/SDS/dextran capillary sieving electrophoresis; however, dye interactions with this buffer system produced a less than ideal blank.

Single cell analysis in full body quartz glass chips with native UV laser-induced fluorescence detection.

In order to investigate the individual and inhomogenous cellular response, e.g. to external stimuli, single cell analysis is mandatory and may provide new cognitions in proteomics as well as in other fields of systems biology in the future. Here, we report on novel chip architectures for single cell analysis based on full body quartz glass microfluidic chips (QG chips) that extend our previous studies in polydimethylsiloxane (PDMS) chips, and enhance the detection sensitivity of native UV laser-induced fluorescence (UV-LIF) detection. Detection of a 10nM tryptophan solution with an S/N ratio of 11.9, which gives a theoretical limit of detection of 2.5 nM (with S/N=3), was possible. With these optimizations the three proteins alpha-chymotrypsinogen A, ovalbumin and catalase each at a concentration of 100 microg/mL (equal to 4 microM, 0.4 microM and 2.2 microM) were injected electrokinetically and could be separated with nearly baseline resolution. Furthermore, fluorescence spectra (excitation wavelength, lambda(ex) = 266 nm) clearly demonstrate the favourable properties like the very high UV transparency and the nearly vanishing background fluorescence of the QG chips as compared to PDMS chips and to PDMS quartz window (PQW) chips. Finally we exploit the improved sensitivity for single cell electropherograms of Spodoptera frugiperda (Sf9) insect cells.

Enhanced stability of surfactant-based semipermanent wall coatings in capillary electrophoresis using oppositely charged surfactant.

Semipermanent surfactant coatings are effective for the prevention of wall adsorption of proteins in CE. However, they often suffer from their unsatisfactory coating stability as they essentially degrade from the capillary walls after the surfactants are removed from the buffer. In this paper, we proposed a facile and universal method to improve the stability of semipermanent surfactant coatings based on addition of an oppositely charged surfactant into the coating. Didodecyldimethylammonium bromide (DDAB) and a gemini surfactant, 18-6-18, were used as the model semipermanent coatings, and sodium dodecyl sulfate (SDS) was chosen as their oppositely charged surfactant. SDS can strongly alter the packing parameter P of the cationic surfactants, and consequently mediates the coating stability. With the increase of SDS concentration in coating, the coating stability first dramatically increases due to the enlarged P, and then decreases due to the weakness of electrostatic interaction between the capillary wall and surfactant coating. At the proper SDS concentration, very stable coatings can be obtained that, even after rinsing under 138 kPa for 60 min, the reversed electroosmotic flow (EOF) only decreases by 3.6%. These SDS-enhanced coatings show excellent stability and reproducibility in protein separation (RSD of migration time <1.1% for run-to-run assay, n=9). Also, the high separation efficiency (>500,000 plates/m) and fine recovery of tested proteins indicate that these coatings are powerful in wall adsorption suppression. Finally, we found that the separation efficiency of protein was a more exact indicator for the coating stability than the traditional EOF magnitude.

Features of partitioning pattern of two pancreatic enzymatic precursors: trypsinogen and chymotrypsinogen in polyethyleneglycol-sodium citrate aqueous biphasic systems.

The partitioning behaviour of bovine trypsinogen and alpha-chymotrypsinogen, enzymatic precursors with similar physicochemical properties, was investigated in different polyethyleneglycol/sodium citrate aqueous two-phase systems. The effect of different factors such as polyethyleneglycol molecular weight, pH, tie line length and temperature was also examined. The increase of pH and the decrease of polyethyleneglycol molecular weight displaced the partitioning equilibrium of both proteins to the top phase. An enthalpy-entropy compensation pattern was observed, indicating the participation of water molecules in the partitioning mechanism. TRPz phase equilibrium showed to be more displaced to the citrate-rich phase than ChTRPz for most of the assayed systems. From a practical view, the aqueous two-phase system formed by polyethylenglycol of molecular weight 1450 and sodium citrate pH 8.20 showed the best capability for separating both proteins. When a mixture formed by equal quantities of both zymogens was partitioned in this system, significant recoveries (about 60%) were obtained. Purity values were improved significantly (84-89%) by either developing a second extractive step or increasing the top-bottom volume ratio.

Control of the adsorption properties of alginate - guar gum matrix functionalized with epichlorohydrin through the addition of different flexible chain polymers as toll for the chymotrypsinogen isolation.

This work addresses the obtaining and characterization of alginate-guar gum matrix, cross-linked with epichlorohydrin in the presence of different flexible chain polymers: polyvinyl alcohol, polyvinyl pyrrolidine and Pluronic® F68. These matrixes were used for the adsorption of chymotrypsinogen and showed an increasing uptake in presence of the flexible chain polymer in the sense: none < Pluronic 68 < polyvinyl pyrrolidine < polyvinyl alcohol. The adsorption process was found to follow a first order kinetics model and was not influenced by the polymer type. It was found that Freundlich model was more suitable for our data. Polyvinyl alcohol and polyvinyl pyrrolidine addition increase the adsorption capacity of the original bed due to an increment in the rigidity of the gel caused by the formation of hydrogen bound between the polysaccharides and synthetics polymers.

Displacement chromatography of proteins on hydrophobic charge induction adsorbent column.

Displacement chromatography of protein mixtures is proposed on hydrophobic charge induction chromatography (HCIC). We have used an HCIC medium, MEP-Hypercel as the stationary phase and a quaternary ammonium salt, benzethonium chloride, as the displacer. It was found that the multiple interactions between proteins/displacer and the HCIC sorbent, i.e. hydrophobic interaction and charge repulsion, enabled a greater flexibility for the design of displacement processes and ease of column regeneration by adjustment of pH. The capacity factors of proteins and displacers were used to predict their performances in column displacement, and the experimental results agreed well with the prediction. An isotachic displacement train of lysozyme and alpha-chymotrypsinogen A was formed with benzethonium chloride as the displacer at pH 5.0 with good yields and purities of the two proteins. Column regeneration was efficiently achieved by charge repulsion between the displacer and the adsorbent at lower pH values (pH 3 and 4). The results indicate that the displacement chromatography on HCIC is a good alternative to traditional hydrophobic displacement chromatography.

A molecular modeling based method to predict elution behavior and binding patches of proteins in multimodal chromatography.

Multimodal (MM) chromatography provides a powerful means to enhance the selectivity of protein separations by taking advantage of multiple weak interactions that include electrostatic, hydrophobic and van der Waals interactions. In order to increase our understanding of such phenomena, a computationally efficient approach was developed that combines short molecular dynamics simulations and continuum solvent based coarse-grained free energy calculations in order to study the binding of proteins to Self Assembled Monolayers (SAM) presenting MM ligands. Using this method, the free energies of protein-MM SAM binding over a range of incident orientations of the protein can be determined. The resulting free energies were then examined to identify the more "strongly bound" orientations of different proteins with two multimodal surfaces. The overall free energy of protein-MM surface binding was then determined and correlated to retention factors from isocratic chromatography. This correlation, combined with analytical expressions from the literature, was then employed to predict protein gradient elution salt concentrations as well as selectivity reversals with different MM resin systems. Patches on protein surfaces that interacted strongly with MM surfaces were also identified by determining the frequency of heavy atom contacts with the atoms of the MM SAMs. A comparison of these patches to Electrostatic Potential and hydrophobicity maps indicated that while all of these patches contained significant positive charge, only the highest frequency sites also possessed hydrophobicity. The ability to identify key binding patches on proteins may have significant impact on process development for the separation of bioproduct related impurities.

Cationic lipid vesicles as coating precursors in capillary electrochromatography: separation of basic proteins and neutral steroids.

1,2-Dioleyl-3-trymethylammoniumpropane (DOTAP) lipid vesicles were employed as coating precursors to obtain a semipermanent cationic lipid bilayer in silica capillary. The coating procedure was relatively fast and simple. Reliable results for the separation of four basic proteins (alpha-chymotrypsinogen A, ribonuclease A, cytochrome C, lysozyme) were obtained by using an acetate buffer under acidic conditions. The RSDs of the migration times were not higher than 0.5% run-to-run and about 1% day-to-day (3 days), while the RSDs of the peak areas were within 7% day-to-day (3 days). The day-to-day RSD of the EOF mobility of about 1%, confirmed that the DOTAP coating was stable for the separation of basic proteins, under acidic buffers. In addition to basic proteins the DOTAP coating was found suitable under acidic conditions for the repeatable separation of neutral steroids. The potential of DOTAP as a carrier in background electrolyte solution was studied.

Purification and properties of multiple forms of mouse trypsinogen.

Three trypsinogens and one chymotrypsinogen were found in and purified from the pancreas of a mouse strain (CFO). The molecular weights of the trypsinogens and the chymotrypsinogen were all estimated as 25 000. The enzyme properties of the three trypsinogens were studied and they showed very low Km values (3.2-6.5 microM) for the substrates, Bz-Arg-OEt and Tos-Arg-OMe, and the same pH optimum profile between pH 8.0-10.0. However, the ratios of catalytic rate constants, kcat (s-1), with Bz-Arg-OEt as substrates compared to that with Tos-Arg-OMe were very different. The values of Try-III were similar with the two substrates, Try-I was slightly higher value with Tos-Arg-OMe than with Bz-Arg-OEt, and the values of Try-II were much higher with Tos-Arg-OMe than with Bz-Arg-OEt. Also, the trypsinogens and the chymotrypsinogen were purified from pancreas of Mol-A strain mice. When the enzyme properties of the three trypsinogens were examined, one form of trypsinogen (Try-I) was shown to have different properties in kcat (s-1) for the two substrates, compared to the trypsinogen of CFO mice.

The two human chymotrypsinogens. Purification and characterization.

The two chymotrypsinogens present in human pancreatic juice have been purified and characterized. The zymogens are two immunologically and electrophoretically different proteins. Chymotrypsinogen A, the major chymotryptic component (90% of the total potential N-acetyl-L-tyrosine ethylester activity) is stable in acidic medium. By its molecular weight (approx. 24 000), specific activity (530) and amino acid composition, human chymotrypsinogen A resembles chymotrypsinogens A and B form bovine and porcine pancreas. Chymotrypsinogen B is a minor chymotryptic component (7% of the total potential N-acetyl-L-tyrosine ethylester activity) unstable in acidic medium with a molecular weight slightly higher (approx. 27 000) and a specific activity slightly lower (300) than chymotrypsinogen A. The last 3% of the total potential N-acetyl-L-tyrosine ethylester activity corresponds to a proelastase that we have partially characterized.

Free-flow electrophoresis of proteins in phenol-containing solvents at various pH values.

1. Several proteins were found to migrate when subjected to free-flow electrophoresis in buffered phenol-ethanediol-water (3:2:3, w/v/v) solvent mixtures. Mobility of these proteins changed with changing pH (apparent) values of this medium. A pH value of zero mobility for each individual protein could be estimated. 2. Founded on these observations, a high-voltage electrophoresis method in free-flowing buffer films was worked out. The method as presented here was particularly suitable for the separation of proteins on a preparative scale. Application of this and other protein fractionation techniques in dissociating media for the investigation of structural and other insoluble proteins was discussed.

Preparative parallel protein purification (P4).

In state of the art drug discovery, it is essential to gain structural information of pharmacologically relevant proteins. Increasing the output of novel protein structures requires improved preparative methods for high throughput (HT) protein purification. Currently, most HT platforms are limited to small-scale and available technology for increasing throughput at larger scales is scarce. We have adapted a 10-channel parallel flash chromatography system for protein purification applications. The system enables us to perform 10 different purifications in parallel with individual gradients and UV monitoring. Typical protein purification applications were set up. Methods for ion exchange chromatography were developed for different sample proteins and columns. Affinity chromatography was optimized for His-tagged proteins using metal chelating media and buffer exchange by gel filtration was also tested. The results from the present system were comparable, with respect to resolution and reproducibility, with those from control experiments on an AKTA purifier system. Finally, lysates from 10 E. coli cultures expressing different His-tagged proteins were subjected to a three-step parallel purification procedure, combining the above-mentioned procedures. Nine proteins were successfully purified whereas one failed probably due to lack of expression.

Displacement chromatography of proteins using a retained pH front in a hydrophobic charge induction chromatography column.

The chromatographic separation of two proteins into a displacement train of two adjoined rectangular bands was accomplished using a novel method for hydrophobic charge induction chromatography (HCIC) which employs a self-sharpening pH front as the displacer. This method exploits the fact that protein elution in HCIC is promoted by a pH change, but is relatively independent of salt effects, so that a retained pH front can be used in place of a traditional displacer in displacement chromatography. The retained pH front was produced using the two adsorbed buffering species tricine and acetic acid. The separation of lysozyme and α-chymotrypsinogen A into adjoined, rectangular bands was accomplished with overall recoveries based on the total mass injected greater than 90 and 70%, respectively. The addition of urea to the buffer system increased the sharpness of the pH front by 36% while the yields of lysozyme and α-chymotrypsinogen A based on the total mass eluted increased from 76% to 99% and from 37% to 85%, respectively, when the purities of both proteins in their product fractions were fixed at 85%. The results demonstrate that the method developed in this study is a useful variant of HCIC and is also a useful alternative to other displacement chromatography methods.