Journal of Chromatography B (v.808, #1)

Preface by M.A Vijayalakshmi (1).

Monoliths for microfluidic devices in proteomics by Séverine Le Gac; Julien Carlier; Jean-Christophe Camart; Cécile Cren-Olivé; Christian Rolando (3-14).
We report here on the preparation of monolithic capillary columns in view to their integration in a microsystem for on-chip sample preparation before their on-line analysis by electrospray and mass spectrometry (ESI–MS). These monolithic columns are based on polymer materials and consist of reverse phases for peptide separation and/or desalting. They were prepared using lauryl methacrylate (LMA), ethylene dimethacrylate (EDMA) as well as a suitable porogenic mixture composed of cyclohexanol and ethylene glycol. The resulting stationary phases present thus a C12-functionality. The LMA-based columns were first prepared in a capillary format using capillary tubing of 75 μm i.d. and tested in nanoLC–MS experiments for the separation of a commercial Cytochrome C digest composed of 12 peptidic fragments whose isoelectric point values and hydrophobic character cover a wide range. The LMA-based columns were capable of separating the peptidic fragments and their performances were seen to be similar as those of standard commercial columns dedicated to proteomic purposes with calculated separation efficiencies up to 145×103 plates/m. Monolithic LMA-based phases were then successfully polymerized in microchannels fabricated using the negative photoresist SU-8. After the polymerization, the systems were seen to withstand the pressures applied during the nanoLC–MS separation tests that were carried out in the same conditions as for the monolithic capillary columns. The pressure drop during these tests of the in-microchannel monoliths was as high as 50 bar; however, the separation was not as good as for a capillary format which could be accounted for by the monolith dimensions.
Keywords: Polymer monoliths; Microfluidic devices; Miniaturization; Proteomics;

Utilization of newly developed immobilized enzyme reactors for preparation and study of immunoglobulin G fragments by Lucie Korecká; Zuzana Bı́lková; Michal Holèapek; Josef Královský; Milan Beneš; Jiøı́ Lenfeld; Nicolas Minc; Roxana Cecal; Jean-Louis Viovy; Michael Przybylski (15-24).
The newly developed immobilized enzyme reactors (IMERs) with proteolytic enzymes chymotrypsin, trypsin or papain were used for specific fragmentation of high molecular-mass and heterogeneous glycoproteins immunoglobulin G (IgG) and crystallizable fragment of IgG (Fc). The efficiency of splitting or digestion were controlled by RP-HPLC. The specificity of digestion by trypsin reactor was controlled by MS. IMERs (trypsin immobilized on magnetic microparticles focused in a channel of magnetically active microfluidic device) was used for digestion of the whole IgG molecule. The sufficient conditions for IgG digestion in microfluidic device (flow rate, ratio S:E, pH, temperature) were optimized. It was confirmed that the combination of IMERs with microfluidic device enables efficient digestion of highly heterogeneous glycoproteins such as IgG in extremely short time and minimal reaction volume.
Keywords: Immobilized enzyme reactors; Immunoglobulin G;

2-Mercapto-5-benzimidazolesulfonic acid: an effective multimodal ligand for the separation of antibodies by Pierre Girot; Emmanuelle Averty; Isabelle Flayeux; E Boschetti (25-33).
The report describes the use of 2-mercapto-5-benzimidazolesulfonic acid (MBISA) as a ligand for the separation of antibodies by chromatography. The ligand shows a relatively specific adsorption property for antibodies from very crude biologicals at pH 5.0–5.5. At this pH range most of other proteins do not interact with the resin especially when the ionic strength is similar to physiological conditions. Several characterization studies are described such as antibody adsorption in different conditions of ionic strength, pH and temperature. These properties are advantageously used to selectively capture antibodies from very crude feed stocks without dilution or addition of lyotropic salts. Demonstration was made that the adsorption mechanism is neither based on ion exchange nor on hydrophobic associations, but rather as an assembly of a variety of properties of the ligand itself. Binding capacity in the described conditions ranges between 25 and 30 mg/mL of resin. The sorbent does not co-adsorb albumin (Alb) and seems compatible with a large variety of feedstocks. Quantitative antibody desorption occurs when the pH is raised above 8.5. The final purity of the antibody depends on the nature of the feedstock, and can reach levels of purity as high as 98%. Even with very crude biological liquids such as ascites fluids, cell culture supernatants and Chon fraction II + III from human plasma fractionation where the number of protein impurities is particularly large, immunoglobumins G (IgG) were separated at high purity level in a single step.
Keywords: Heterocyclic ligands; 2-Mercapto-5-benzimidazolesulfonic acid; Antibodies;

Silica beads are coated with dextran carrying a calculated amount of positively charged diethylassminoethyl groups (DEAE) in order to neutralize negative charged silanol groups at the silica surface and in this way to minimize non specific interactions between silica surface and proteins in solution. Dextran-coated silica supports are potentially excellent stationary phases for high-performance liquid chromatography of proteins. These supports combine the advantages of polysaccharide phases with the excellent mechanical characteristics of silica. These supports (silica–dextran–DEAE = SID) are easily functionalized by grafting N-acetylglucosamine (GlcNAc) using conventional coupling methods. The performances of the support bearing GlcNAc are studied by high-performance liquid affinity chromatography (HPLAC) of insulin, the hypoglycemic peptide hormone of the human organism. The study shows that these supports exhibit a reversible and specific affinity towards insulin and allow separations with high purification yields. Moreover, the influence of different physico-chemical parameters (pH, NaCl and insulin concentration) on insulin retention on the support was analysed. This allowed us to optimize the conditions of adsorption and to better understand the interaction mechanisms between insulin and GlcNAc as biospecific ligand.
Keywords: N-Acetylglucosamine; Insulin;

The binding of l-Boc-phenylalanine anilide (BFA) and l-Boc-phenylalanine (phe) to molecularly imprinted and non-imprinted polymer nanoparticles consisting of poly[(ethylene glycol dimethacrylate)-co-(methacrylic acid)] has been investigated by adsorption experiments and mathematical modeling. The experimental isotherms have been mathematically adapted following the models of Freundlich, Langmuir, Langmuir–Freundlich, Bi-Langmuir, and extended Langmuir. The extended Langmuir model differentiated between specific and nonspecific binding of the ligand to the receptor nanoparticles and rendered excellent fitting of the experimental data. It delivered a thermodynamic and kinetic parameter set on the experimental association curves of l-BFA by l-BFA-imprinted nanospheres in suspension experiments with the equilibrium constant K D=4.09±0.69 μmol L−1 and the kinetic association rate constant k a=5.60 mL μmol−1  min−1.
Keywords: Molecular recognition; Adsorption isotherm; Amino acids; Molecularly imprinted nanospheres; Poly[(ethylene glycol dimethacrylate)-co-(methacrylic acid)];

In this study, we attempted a limited combinatorial approach for designing affinity ligands based on mercaptoheterocyclic components. The template, divinyl sulfone structure (DVS), which was grafted on poly(ethylene vinyl alcohol) (PEVA) hollow fiber membrane, has served for the tethering of different heterocyclic compounds as pyridine, imidazole, purine and pyrimidine rings. Their ability to adsorb specifically IgG in a salt independent manner out of pure IgG solution, mixture of IgG/albumin and human plasma was demonstrated. Mercapto methyl imidazole (MMI) has shown the best adsorption of IgG in terms of binding capacity. No subclass discrimination was observed on all tested ligands except for mercapto methyl pyrimidine where the major IgG subclass adsorbed was IgG3. MMI gave an IgG binding capacity of 100 μg/cm2 of hollow fiber membrane surface area.
Keywords: Salt-independent thiophilic chromatography; Mercaptoheterocyclic ligands; Poly(ethylene vinyl alcohol); Immunoglobulin G;

Immobilized metal-ion affinity chromatography of human antibodies and their proteolytic fragments by Daniela Todorova-Balvay; Olivier Pitiot; Mustapha Bourhim; Thamarapu Srikrishnan; Mookambeswaran Vijayalakshmi (57-62).
Immobilized metal-ion affinity chromatography (IMAC) performed with four different transition metal ions: copper(II), nickel(II), zinc(II) and cobalt(II), was used to study the adsorption properties of human polyclonal γ-globulines (IgG), Cohn II–III fractions, and their pepsin cleaved fragments: F(ab′)2 and F′c. In each case, digested products showed lower affinity for metal ions, as well by decreasing pH elution as by competition with imidazole. An explanation was proposed by the presence of a histidine (His) cluster in the F′c domain of IgGs, identified by computer calculation (accessible surface area (ASA) determination) as the more probable His 433-x-His 435 sequence presented in the CH3 domain of human IgG heavy chain. As shown by IMAC and electrophoresis, F′c and undigested IgG have higher affinity for transition metal ions than F(ab′)2 fragments and could be then separated in one step by IMAC. When chelated Zn(II) or Co(II) are used as ligands, the F(ab′)2 fragment could be easily recovered under mild conditions (pH 7) in the non-retained fraction. This approach could be used as a powerful alternative to conventional protein A/G methods for the commercial preparation of non immunogen active F(ab′)2 fragments.
Keywords: Immobilized metal-ion affinity chromatography; Immunoglobulins G; F(ab′)2 fragment; F′c fragment; Histidine;

Immobilization of a (dextran-adamantane-COOH) polymer onto β-cyclodextrin-modified silica by Carole Karakasyan; Marie-Claude Millot; Claire Vidal-Madjar (63-67).
Adamantane-modified compounds are known to form stable complexes with β-cyclodextrins (β-CD) by host–guest interactions. In this study, the inclusion complex formed between β-CD cavities and the adamantane group was evaluated for the elaboration of a cation-exchange support. The synthesis of the chromatographic supports involved three steps: (i) a polymer of β-CD was grafted to diol-modified silica, (ii) a dextran polymer was modified by both adamantane groups and ionizable COOH functions, (iii) the dextran derivative (Ad-Dex-COOH) was bound to the chromatographic support by complexation between the adamantane groups of the dextran and β-CD cavities of the support. The polymer immobilization on the β-CD support was successful as the resulting support exhibited weak cation-exchange properties. The stationary phase was easy to prepare under mild conditions (aqueous media, room temperature) and was quite stable when using aqueous mobile phases. The chromatographic behaviour of model proteins was studied in isocratic elution by examining the effect of salt concentration in the buffer on retention. A mixed retention mode was found for lysozyme, revealing both electrostatic and hydrophobic interactions with the stationary phase.
Keywords: Immobilization; (Dextran-adamantane-COOH) polymer; Basic proteins; Poly-β-cyclodextrin;

Mycelium of the mold Aspergillus niger was used as a raw material for the preparation of microbial chitosan. Aspergillus niger, the mold used for the production of citric acid, contains approx. 15% of chitin, which can be separated, transformed into chitosan, and used as a sorbent for chromatography. The main advantage of this material in comparison with krill chitosan is the uniformity of particle size leading to the low back-pressure in the column. The other advantage is the fact, that original fibrous structure of mycelial pellets could be stabilized before chitosan preparation by cross-linking with glutaraldehyde. The product prepared by this way – crosslinked chitosan of uniform particle size, is highly porous, with high water regain and, as a result, low sedimentation velocity. Low sedimentation velocity is not disadvantage in chromatographic application, but may form some problems in batchwise operation. Chitosan as a polymer of glucosamine is anion exchanger in nature and the chromatographic properties of this anion exchanger was demonstrated by the chromatography of bovine blood plasma, glucose oxidase, and chicken pepsinogen. In all cases, the course of chromatography on crosslinked chitosan was compared with the chromatography on MONO Q (bovine blood plasma) or DEAE-cellulose (glucose oxidase, chicken pepsinogen) under the same protocol.
Keywords: Fungal mycelium; Chitosan;

Capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC) were used for the analysis of new synthetic derivatives of hypophysis neurohormones—vasopressin and oxytocin, and pancreatic hormone—human insulin (HI) and its octapeptide fragment, derivatized by fluorescent probe, 4-chloro-7-nitrobenzo[1,2,5]oxadiazol (NBD). The suitable composition of background electrolytes (BGEs) was selected on the basis of calculated pH dependence of effective charge of analyzed peptides. Basic ionogenic peptides were analyzed by CZE in the acidic BGE composed of 100 mM H3PO4, 50 mM Tris, pH 2.25. The ionogenic peptides with fluorescent label, NBD, were analyzed in 0.5 M acetic acid, pH 2.5. The best MEKC separation of non-ionogenic peptides was achieved in alkaline BGE, 20 mM Tris, 5 mM H3PO4, with micellar pseudophase formed by 50 mM sodium dodecylsulfate (SDS), pH 8.8. Selected characteristics (noise, detectability of substance, sensitivity of detector) of the UV-absorption detectors (single wavelength detector, multiple-wavelength photodiode array detector (PDA), both of them operating at constant wavelength 206 nm) and laser-induced fluorescence (LIF) detector (excitation/emission wavelength 488/520 nm) were determined. The detectability of peptides in the single wavelength detector was 1.3–6.0 μmol dm−3 and in the PDA detector 1.6–3.1 μmol dm−3. The LIF detection was more sensitive, the applied concentration of NBD derivative of insulin fragment in CZE analysis with LIF detection was three orders lower than in CZE with UV-absorption detector, and the detectability of this peptide was improved to 15.8 nmol dm−3.
Keywords: Derivatization, CE; Derivatization, MEKC; Insulin; Peptides;

Chromatographic purification of an insoluble histidine tag recombinant Ykt6p SNARE from Arabidopsis thaliana over-expressed in E. coli by Patrick Vincent; Wilfrid Dieryck; Lilly Maneta-Peyret; Patrick Moreau; Claude Cassagne; Xavier Santarelli (83-89).
In order to undertake in plant cell the study of the endoplasmic reticulum (ER)-Golgi apparatus (GA) protein and/or lipid vesicular transport pathway, expressed sequence tag (EST) coding for a homologue to the yeast soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) Ykt6p has been cloned in Arabidopsis thaliana by reverse transcription polymerase chain reaction (RT-PCR). The corresponding protein was over-expressed as a recombinant histidine-tag (his-tag) protein in E. coli. Starting from one litter of culture, an ultrasonic homogenization was performed for cell disruption and after centrifugation the Arabidopsis Ykt6p SNARE present in inclusion bodies in the pellet was solubilized. After centrifugation, the clarified feedstock obtained was injected onto an immobilized metal affinity chromatography (IMAC) in presence of 6 M guanidine and on-column refolding was performed. Folded and subsequently purified (94% purity) recombinant protein was obtained with 82% of recovery.
Keywords: Protein purification; Histidine tag; Arabidopsis SNARE Ykt6p;

Three anion exchanger expanded bed adsorption (EBA) matrices: Streamline DEAE, Streamline Q XL and Q Hyper Z were evaluated with the aid of EFGP from an ultrasonic homogenate of Escherichia coli. Two pH of buffer were tested. Capture was done in an expanded mode whereas elution was done in a packed mode. The same conditions were chosen for evaluation of the three matrices. We observed a loss of EGFP (8–15%) in the through flow fraction especially with the Streamline Q XL matrix, probably due to an aggregation of beads during sample application. The beads of this matrix possess tentacles which probably retain a lot of cellular and molecular debris. The two other matrices gave a good purification of the EGFP (7–15-fold) but the Q Hyper Z matrix appeared to give the best results. It is composed of little size and density beads which lead to a higher exchange surface and then a better mass transfer.
Keywords: Escherichia coli; Expanded-bed adsorption anion exchange matrices; Enhanced green fluorescent protein;

Cobalt binding proteins from mouse liver, which were expressed in response to CoCl2 poisoning, were separated using gel permeation chromatography and then immobilised metal ion affinity chromatography (IMAC) with immobilized cobalt ions. Conditions used in IMAC-Co2+ were optimised. The fractions eluted with 60 mM imidazole were analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS–PAGE). Differences between the samples were also evaluated by a two-dimensional electrophoresis. Samples from the Co2+-treated mice provided higher number of electrophoretic spots than those from the untreated mice. Relative molecular masses of these proteins are appropriately 37,000; 32,000 and 26,000 and their isoelectric points (pI) are 6.5–7.5.
Keywords: Immobilization; Cobalt-binding proteins;

Purification of human galectin-1 produced in high-cell density cultures of recombinant Escherichia coli: a comparison with classic shake flask cultivation by Didier Lutomski; Naima Imam-Sghiouar; Karine Blondeau; Michel Caron; Raymonde Joubert-Caron (105-109).
The aim of the present work was to develop a highly productive and simplified process for active human galectin-1 (Gal1) production. Gal1 is a β-galactoside binding lectin that differentially affects biological and cellular functions such as immune surveillance and apoptosis. These effects have attracted the attention of researchers in cell biology, biochemistry and immunology. However, the production of sufficient amounts of recombinant human Gal1 (rhGal1) is needed to study of the effects of Gal1 during cell treatments. To this end, an high-yield expression of rhGal1 was achieved by high-cell density fed-batch cultivation using an exponential glycerol feeding strategy and rhGal1 was purified by a one-step purification scheme using affinity chromatography.
Keywords: Escherichia coli; Galectin-1;

Penicillin acylase purification with the aid of hydrophobic charge induction chromatography by D Coulon; C Cabanne; V Fitton; A.M Noubhani; E Saint-Christophe; X Santarelli (111-115).
The aim of this work was to test a chromatographic support, 4-mercaptoethyl pyridine (4-MEP) Hypercel, for penicillin acylase purification by using pure penicillin acylase and crude extract. Two equilibration buffers with various salt concentrations and different flow rates were tested. The relationships between electrostatic and hydrophobic interactions and proteins are demonstrated. (NH4)2SO4 proved preferable because no salting-in occurred, contrary to NaCl. The recovery and purification fold were similar to those obtained in pseudo-affinity chromatography with a three-fold reduction of the (NH4)2SO4 concentration.
Keywords: Purification; Penicillinacylase;

Analysis of liquid extracts from tree and grass pollens by capillary electromigration methods by Petra Sázelová; Václav Kašička; Dušan Koval; Gabriel Peltre (117-123).
Capillary electromigration methods, zone electrophoresis (CZE), micellar electrokinetic chromatography (CMEKC) and isotachophoresis (CITP), have been used for analysis of water and water–buffer extracts from tree—common birch (Betula verrucosa) and grass—orchardgrass (Dactylis glomerata) pollen samples. Water extracts were analyzed by CZE using acetic acid as background electrolyte (BGE), by CMEKC in tris-phosphate BGE with anionic detergent sodium dodecyl sulfate (SDS) micellar pseudophase (TP-SDS) and by CITP in cationic mode with leading/terminating cations K+/BALA+ (β-alanine (BALA)) and in anionic mode with leading/terminating anions Cl/MES (2-(N-morpholino)ethanesulphonic acid (MES)). Moreover, acetic acid extracts were analyzed by CZE using acetic acid as BGE, and alkaline water–SDS-buffer extracts were analyzed by CMEKC using TP-SDS as BGE. Extracted amounts of pollen allergens and other UV-absorbing compounds and the number of resolved components were evaluated from CZE, CMEKC and CITP analyses of the liquid extracts. Larger amounts of UV-absorbing material were found in the water–buffer pollen extracts than in the water extracts. More UV-absorbing material was found in all extracts from D. glomerata pollen than in relevant extracts from B. verrucosa pollen. It was found by CITP that the extracted amounts of anionic components and their number were much higher than those of cationic components. Concentrations of some inorganic ions (e.g. Cl, K+, Na+, Ca2+) in pollen samples were also determined by CITP.
Keywords: Betula verrucosa; Dactylis glomerata; Pollen allergens;