Construction of polymer monolithic columns in polypropylene ink-pen tubes for separation of proteins by cation-exchange chromatography.

We describe the synthesis of polymer monoliths inside polypropylene tubes from ink pens. These tubes are cheap, chemically stable, and resistant to pressure. UV-initiated grafting with 5 wt% benzophenone in methanol for 20 min activated the internal surface, thus enabling the covalent binding of ethylene glycol dimethacrylate, also via photografting. The pendant vinyl groups attached a poly(glycidyl methacrylate-co-ethylene glycol dimethacrylate) monolith prepared via photopolymerization. These tubes measured 100-110 mm long, with 2 mm of internal diameter. The parent monoliths were functionalized with Na2 SO3 or iminodiacetate to produce strong and weak cation exchangers, respectively. The columns exhibited permeabilities varying from 2.7 to 3.3 × 10-13  m2 , which enabled the separation of proteins at 500 µL/min and back pressures <2.8 MPa. Neither structure collapse nor monolith detachment occurred at flow rates as high as 2.0 mL/min, which produced back pressures between 6.9 and 9.0 MPa. The retention times of ovalbumin, ribonuclease A, cytochrome C, and lysozyme in salt gradient at pH 7.0 followed the order of increasing isoelectric points, confirming the cation exchange mechanism. Separation and determination of lysozyme in egg white proved the applicability of the columns to the analysis of complex samples.

Fast construction of polymer monolithic columns inside fluorinated ethylene propylene (FEP) tubes for separation of proteins by reversed-phase liquid chromatography.

This paper describes the preparation of polymer monolithic columns in the confines of fluorinated ethylene propylene (FEP) tubes. These tubes are cheap, chemically stable, and widely used in flow analysis laboratories. UV-initiated grafting with 5 wt% benzophenone in methanol for 1 h activated the internal surface walls, thus enabling the further covalent binding of ethylene glycol dimethacrylate (EDMA) from a 15 wt% solution in methanol, also via photografting. Both steps used 254 nm radiation under a potency of 120 mJ cm2. ATR-FTIR measurements revealed the presence of carbonyl, alkyl and vinyl groups in the functionalized FEP. The density of vinyl groups was high enough to firmly attach a poly(lauryl methacrylate-co-ethylene glycol dimethacrylate) monolith in 120 × 1.57 mm i.d. tubes, prepared via photopolymerization. The total preparation lasts less than 2-h. The columns were permeable, (1.58 ± 0.06) × 10-13 m2, providing reproducible chromatographic parameters of retention times, retention factor, selectivity, and resolution. The monoliths were stable at flow rates of 500 µL min-1, collapsing only at flow rates >700 µL min-1, a condition that increased the backpressure over 1000 psi (experiments at the room temperature). The separation of proteins by reversed-phase liquid chromatography demonstrated the efficiency of the columns. Determination of egg white proteins (ovalbumin and lysozyme) and myoglobin in spiked urine proved the applicability to the analysis of real samples.

Synthesis of hydrophilic and conductive molecularly imprinted polyaniline particles for the sensitive and selective protein detection.

In this work, a novel kind of water-dispersible molecular imprinted conductive polyaniline particles was prepared through a facile and efficient macromolecular co-assembly of polyaniline with amphiphilic copolymer, and applied as the molecular recognition element to construct protein electrochemical sensor. In our strategy, an amphiphilic copolymer P(AMPS-co-St) was first synthesized using 2-acrylamido-2-methyl-1-propanesulfonic acid (AMPS) and styrene (St) as monomer, which could co-assemble with PANI in aqueous solution to generate PANI particles driven by the electrostatic interaction. During this process, ovalbumin (OVA) as template protein was added and trapped into the PANI NPs particles owing to their interactions, resulting in the formation of molecular imprinted polyaniline (MIP-PANI) particles. When utilizing the MIP-PANI particles as recognition element, the resultant imprinted PANI sensor not only exhibited good selectivity toward template protein (the imprinting factor α is 5.31), but also a wide linear range over OVA concentration from 10-11 to 10-6mgmL-1 with a significantly lower detection limit of 10-12mgmL-1, which outperformed most of reported OVA detecting methods. In addition, an ultrafast response time of less than 3min has also been demonstrated. The superior performance is ascribed to the water compatibility, large specific surface area of PANI particles and the electrical conductivity of PANI which provides a direct path for the conduction of electrons from the imprinting sites to the electrode surface. The outstanding sensing performance combined with its facile, quick, green preparation procedure as well as low production cost makes the MIP-PANI particles attractive in specific protein recognition and sensing.

Carboxyl modified magnetic nanoparticles coated open tubular column for capillary electrochromatographic separation of biomolecules.

Carboxyl modified magnetic nanoparticles (Fe3O4-COOH MNPs) coated open tubular (OT) columns were prepared for capillary electrochromatography. The Fe3O4-COOH MNPs coatings were constructed on the surface of positively charged poly(diallydimethylammonium chloride) (PDDA) modified capillaries through electrostatic self-assembly approach. The as-prepared PDDA@Fe3O4-COOH MNPs coated OT columns were characterized by scanning electron microscopy and electroosmotic flow measurement. The electrochromatographic characterization of the OT columns was evaluated by separation of amino acids, dipeptides and proteins. The influences of background solution pH, concentration, and organic modifier content on separation were investigated. The separation of these analytes was primarily based on the electrophoretic mechanism in combination with chromatographic mechanism. The Fe3O4-COOH MNPs coatings improved the separation resolution of these analytes due to their large surface area. Three variants of bovine serum albumin, two variants of ß-lactoglobulin and nine glycoisoforms of ovalbumin were successfully separated. The relative standard deviations of migration times of analytes representing run-to-run, day-to-day and column-to-column were less than 4.3%. Furthermore, the feasibility of the PDDA@Fe3O4-COOH MNPs coated OT column was verified by successful separation of acidic proteins in egg white.

Different components of chicken ovalbumin extract promote different cell proliferation.

The chicken ovalbumin extracts could promote cell survival and proliferation. In the present study, the different components in chicken ovalbumin extracts were further separated to find the component primarily responsible for promoting cell proliferation. Components of differing molecular weight were separated from chicken ovalbumin extracts by ultrafiltration. Different components were co—cultured with different cells at different final concentrations, and the effects on cell proliferation were subsequently determined by a CellTiter 96 Aqueous One Solution Cell Proliferation Assay kit (Promega). Components from chicken ovalbumin extracts less than 3 kD in size can promote 293T cell and 293T—GFP cell proliferation. The components from chicken ovalbumin extract more than 3 kD in size can promote bone marrow or umbilical cord mesenchymal stem cell (MSC) proliferation. The separation of components from chicken ovalbumin less than and more than 3 kD in size that are able to function as active components for the promotion of different cellular proliferation. This discovery may identify a new and convenient additive for cell culture media, promoting cell growth and proliferation.

A unique boronic acid functionalized monolithic capillary for specific capture, separation and immobilization of cis-diol biomolecules.

We report a sulfonyl substituted phenylboronic acid containing monolith capillary that exhibited not only a strong boronate affinity at neutral pH but also secondary separation capability to cis-diol biomolecules.

Influence of a poly-ethylene glycol spacer on antigen capture by immobilized antibodies.

The use of spacers to distance an immobilized antibody from the surface of a support matrix introduces flexibility, which can reduce steric interferences between antibodies leading to a higher antigen capture efficiency. In this paper we investigated the use of a spacer molecule, poly-ethylene glycol (PEG), between the matrix surface and antibodies for the capture of Bacillus globigii, E. coli O157:H7, and ovalbumin. The antigen capture efficiency was determined using a surface ELISA method. Antibodies against the antigens were covalently immobilized either directly or via PEG to glass surfaces using a one-step EDC reaction. The amount of antibody immobilized was determined before blocking the nonspecific binding sites with bovine serum albumin. Antibodies immobilized via a PEG spacer showed a higher capture efficiency compared to direct immobilization, which was more pronounced with large antigens. Antibodies immobilized on glass supports were stable at 65 degrees C for at least 80 min, and the capture efficiency increased with heating at 65 degrees C for 20 min.

Composition of N-linked carbohydrates from ovalbumin and co-purified glycoproteins.

Analysis of commercial samples of chicken ovalbumin by reversed-phase high performance liquid chromatography and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) showed the presence of several other co-purifying glycoproteins. These were isolated, subjected to tryptic digestion, and two of them were identified as ovomucoid and chicken riboflavin binding-protein following database matching of the peptide masses obtained by MALDI. The N-linked glycans were released from the glycoproteins and their structures were examined by MALDI-MS in combination with exoglycosidase digestion. Ovalbumin was found to be glycosylated mainly with high-mannose and hybrid structures, consistent with profiles obtained on the intact glycoprotein by electrospray. The other glycoproteins contained mainly larger, complex glycans with up to five antennae, many of which had earlier been associated with ovalbumin.

Postsource decay fragmentation of N-linked carbohydrates from ovalbumin and related glycoproteins.

N-linked glycans were released from chicken ovalbumin by hydrazinolysis and examined by matrix-assisted laser desorption/ionization mass spectrometry. Postsource decay analysis showed that most fragment ions arose as the result of internal glycosidic cleavages involving loss of nonreducing terminal residues from ions that had lost one or both GlcNAc residues from the chitobiose core [GlcNAcbeta(1 --> 4)GlcNAc]. Cross-ring fragments were abundant from the reducing-terminal GlcNAc but other cross-ring fragments were weak. The ion found to be most useful for determining the composition of the antennae attached to the 3- or 6-linked core mannose residues was an internal cleavage ion formed by loss of both the chitobiose core and the antenna linked to the 3-position of the core branching mannose. This ion was observed to lose water in the absence of a "bisecting" GlcNAc residue (beta1 --> 4 linked to the core mannose) and to lose a GlcNAc molecule (221 mass units) when a bisecting GlcNAc residue was present.

Development of recombinant ovalbumin-luteinizing hormone releasing hormone as a potential sterilization vaccine.

The objective was to develop an immunogenic chimeric ovalbumin-LHRH (ova-LHRH) molecule using genetic engineering. Hybrid ova-LHRH genes with either four or seven LHRH inserts were constructed by cassette mutagenesis and oligonucleotide mismatch mutagenesis. Recombinant ova-LHRH proteins were over-expressed in E. coli strain BL21 (DE3) using a pET expression system, which expresses a target protein with a C-terminal His-Tag. The C-terminal His-Tag allows purification by metal chelation chromatography. The antigenicity and biological effects of these recombinant proteins were tested in mice. In experiment 1, 17 female 7 wk old BALB/c mice were randomly divided into three groups. Six mice were injected with 50 microg of the recombinant ovalbumin (ova) protein. Five mice were injected with 50 microg of the recombinant protein with four LHRH inserts (ova-LHRH-7). Six mice were injected with 50 microg of the recombinant protein with seven LHRH inserts (ova-LHRH-7). One primary immunization using Freund's complete adjuvant was followed by one booster using incomplete adjuvant. Mice were killed 2 wk after the booster, blood collected, and the reproductive tract removed and weighed. Only ova-LHRH-7 decreased (P < 0.01) uterine-ovarian weight (89+/-11 mg) vs control (138+/-6 mg) and ova-LHRH-4 (126+/-16 mg). The genetically engineered molecule with seven LHRH inserts induced LHRH antibody titers which were significantly correlated (r = -0.79) with biological response. In experiment 2, the recombinant ova-LHRH-7 was evaluated at two doses with the adjuvants Zmax and Immumax. Seventy female 6-8 wk old BALB/c mice were randomly divided into seven groups of 10 mice each. Anti-LHRH titers were detected in all of the ova-LHRH-7 immunized mice. Significant decreases were shown in uterine-ovarian weight of the mice by the immunization with 30 microg of ova-LHRH-7 and Zmax (P < 0.005) or 10 microg of ova-LHRH-7 with Immumax (P < 0.025). These data show that the recombinant ova-LHRH-7 protein could have potential as an effective sterilization vaccine.

Two-dimensional SEC/RPLC coupled to mass spectrometry for the analysis of peptides.

A two-dimensional liquid chromatography system is described here which uses size exclusion liquid chromatography (SEC) followed by reversed phase liquid chromatography (RPLC) to separate the mixture of peptides resulting from the enzymatic digestion of a protein. A novel LC/LC interface, using two RPLC columns in parallel rather than storage loops, joins the two chromatographic dimensions. This new interface design permits the use of conventional analytical diameter HPLC columns, 7.8 mm for SEC and 4.6 mm for RPLC, making construction and maintenance of this system very easy. The reversed phase chromatography utilizes 1.5 microns diameter, nonporous C-18 modified silica particles, which produce fast and efficient analyses. Following the high-resolution two-dimensional chromatographic separation, an electrospray mass spectrometer detects the peptide fragments. The mass spectrometer scans a 2000 m/z range to identify the analytes from their molecular weights. The analyses of tryptic digests of ovalbumin and serum albumin are each described.

Hybrid selection with cDNA-silica.

A partial length ovalbumin cDNA-silica was produced using primer extension of (dT)18-silica with annealed partial ovalbumin RNA and reverse transcriptase. This cDNA-silica was used to test whether full-length ovalbumin RNA could be selectively purified in the presence of a large excess of other (mouse muscle) RNA. The cDNA-silica synthesized had minimally 60 pmol cDNA per gram silica and had a capacity for full-length ovalbumin RNA of minimally 38 micrograms/g. Even when other RNA was present in greater than 1000-fold excess, ovalbumin RNA was selectively retained by the cDNA-silica and was eluted in yields of 43% with an enrichment which varied over the range of 29-162-fold in various experiments. These results show that even rare RNAs can be selectively purified in high yield using cDNA-silica. The importance of these results to hybrid selection and subtractive library preparation is discussed.

The transmembrane and flanking sequences of beta 1,2-N-acetylglucosaminyltransferase I specify medial-Golgi localization.

UDP-GlcNAc:alpha 3-D-mannoside beta 1,2-N-acetylglucosaminyltransferase I (GnTI) is an N(in)/C(out) (type II) membrane protein, localized in the medial-Golgi, that initiates the conversion of high mannose N-glycans to complex N-glycans. Anti-rabbit GnTI antibodies were generated using a purified, enzymatically active, bacterial recombinant fusion protein as immunogen. Rabbit GnTI was effectively retained in the Golgi complex of transfected COS-1 cells and murine L cells, as assessed by indirect immunofluorescence using the species-specific anti-GnTI antibodies; no surface expression of rabbit GnTI could be detected in the transfected cells. Rabbit GnTI, stably expressed in murine L cells, was localized by immunoperoxidase electron microscopy to the medial-cisternae of the Golgi stack. The role of the transmembrane domain of GnTI in Golgi localization was examined by generation of a hybrid construct containing the amino-terminal 31 amino acids of GnTI, corresponding to the 25-residue transmembrane (signal/anchor) domain and flanking hydrophilic sequences, fused with ovalbumin; this ovalbumin/GnTI hybrid molecule was retained in the Golgi complex of transfected COS cells and stably transfected murine L cells. No surface expression of ovalbumin/GnTI was detected. In contrast, ovalbumin fused to the equivalent domains of the human transferrin receptor, a type II cell-surface protein, was efficiently expressed on the cell surface of transfected cells. The ovalbumin/GnTI hybrid molecules in the transfected L cells were N-glycosylated, indicating an N(in)/C(out) membrane orientation, and were localized by immunoperoxidase electron microscopy to one or two cisternae of the medial-Golgi (90% of stained Golgi profiles showed medial-cisternae staining). These results show that a signal contained within the transmembrane domain and flanking residues of GnTI specifies medial-Golgi localization.

The behavior of hydration water of protein with the protectant in the view of 1HNMR.

The alterations of the amount of hydration water and the extent of the interaction with hydration water and protein during freezing and after freeze-drying were investigated with or without cryoprotectant using 1HNMR and other methods. Ovalbumin was used as a representative protein that was stable against freezing and drying. Myosin was also used as a typical unstable protein. It was found that myosin had no stable hydration layer compared with ovalbumin. It was considered that this difference might be the major cause of the difference in the stability of both proteins against freezing and drying. With cryoprotectant, the amount of hydration water was decreased and the extent of the interaction of hydration water with protein was increased. The results suggested the effective protectant substituted for part of the hydration water and strengthened the interaction during freezing and drying. It is also possible that the formation of a "quasi-hydration layer" might protect protein against denaturation due to freezing and drying.

Use of polymeric reversed-phase columns for the characterization of polypeptides extracted from human pancreata. II. Effect of the stationary phase.

The potential value of eight commercial available polymer-based reversed-phase (RP) columns for peptide and protein separations was evaluated using crude acetic acid extracts of normal and diabetic human pancreata and mixtures of pure polypeptides as samples. All columns were characterized with acetic acid gradients in water as mobile phase, and different chromatographic profiles were obtained depending on the type of polymer column (bare or derivatized) and the type of ligand. Some of the columns were virtually free from effects related to the polymer skeleton whereas in others the separation was influenced by both the ligand and the polymeric backbone. Two selected polymeric RP columns were, together with a silica-based C4 column, further characterized with acetonitrile gradients in trifluoroacetic acid (TFA), and the separation temperature was found to have a drastic effect on the separation efficiency for proteins with mol. wt. greater than 6000 dalton. No such effect was seen for polypeptides with mol. wt. less than 6000 dalton. Mixtures of pure peptides and proteins were separated using acetic acid gradients in water, acetonitrile or isopropanol, and normally the highest efficiency was found with the use of acetonitrile as mobile phase modifier. Isopropanol was less suitable as an organic modifier. The separation of the beta-lactoglobulin A- and B-chains may be used to give a rapid estimate of the chromatographic usability of polymer-based RP-columns for peptide and protein separations in acetic acid gradients in water and in acetonitrile gradients. Recoveries for insulin, proinsulin, growth hormone, ovalbumin and human serum albumin were measured for several polymer-based RP columns eluted with acetic acid gradients in water and with acetonitrile-based mobile phases. The highest recoveries of serum albumin and ovalbumin were found after elution with acetic acid gradients in water.

Microbore flow-rates and protein chromatography.

Reversed-phase chromatography of proteins on microbore columns can achieve sensitivities that exceed those for standard-bore columns by a factor of 10-20, when operated at the same linear velocities. These gains in sensitivity are accompanied by proportional reductions in peak volume. Sensitivities on standard- (4.6 mm I.D.) and narrow-bore (2.1 mm I.D.) columns have been further improved by reducing the flow-rates to those typical for microbore (1 mm I.D.) columns. We have investigated the role of flow-rate in determining peak volumes for a constant time gradient and found that flow-rate affects peak volume to a much greater extent than column diameter. Column length was not found to have a significant effect on either peak volume or sensitivity. We have found that a four-fold reduction in flow-rate results in at least a two-fold reduction in peak volume over the flow-rate range from 25 to 400 microliters/min. Recovery of proteins in smaller volumes should prove beneficial to subsequent protein characterization methodologies.

Effect of column degradation on the reversed-phase high-performance liquid chromatographic separation of peptides and proteins.

Many reversed-phase separations of proteins and peptides are currently performed in acidic mobile phases, e.g., 0.1% trifluoroacteic acid in water (pH 2) with organic modifiers. Such conditions are known to promote the cleavage of the silane from the silica in bonded-phase columns, especially for monomeric stationary phases. The stability of some columns commonly used for proteins and peptides has been examined, and it has been shown by both chromatographic and elemental analysis that degradation occurs very rapidly with fresh, "totally covered" column materials. Despite the loss of over half of the bonded phase in some cases, certain columns still exhibit adequate chromatographic performance, although reproducibility can be affected. The implications of these results with respect to both bonded-phase synthesis and mechanistic interpretation of chromatographic data is discussed.

A sodium dodecyl sulfate--polyacrylamide gel electrophoresis system that separates peptides and proteins in the molecular weight range of 2500 to 90,000.

A discontinuous sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis system is described which provides superior resolution of polypeptides with molecular weights from approximately 2500 to 90,000. The system utilizes a relatively low-mobility acetate ion in the stacking gel and high-mobility strong anions, sulfate and chloride, as leading and trailing ions in the separating gel. The entire system is run at pH 7.8. The separating gel contains 8 M urea, and can be used at acrylamide concentrations from 5 to 18%, all with 5% crosslinker concentrations. Using a number of protein standards, the calibration curves obtained with this system are linear over the molecular weight range from 2500 to 90,000, regardless of acrylamide concentration. These studies indicate that by providing good resolution of small peptides, this system greatly extends the utility of one-dimensional peptide mapping techniques.