Clinical applications of electrophoresis of human salivary proteins

Human salivary proteins have been studied by electrophoresis in denaturing and non-denaturing polyacrylamide gel electrophoresis (PAGE) as well as by isoelectric focusing (IEF) and two-dimensional procedures, and the clinical applications of this have been reviewed. Whilst non-denaturing PAGE is useful in studying polymorphisms, sodium dodecylsulphate PAGE appears to be otherwise preferable. Immobilized pH gradients containing carrier ampholytes (CAs) give better resolution than CA-based IEF and overcome the problems of cathode drift and loss of basic material. Proline-rich proteins stain poorly with conventional procedures and special techniques are necessary. In clinical studies, findings must be viewed over and above the large number of polymorphisms which occur normally. Studies relating salivary protein and peptide profiles to dental caries susceptibility are encouraging. Specific protein abnormalities have been associated with connective tissue disorders and could form the basis of new non-invasive diagnostic procedures. Protein differences associated with cystic fibrosis and diabetes mellitus, however, merit reinvestigation with the new procedures now available. Detection of HIV antigens in saliva is a new area of research. In the light of new techniques available and new information which has arisen from DNA studies, future prospects for the clinical applications of electrophoresis of saliva look good.     

Electrophoresis of pharmaceutical proteins: status quo

The biotechnology industry has undergone rapid growth in recent years largely due to the development and success of protein-based therapeutics for a wide range of disorders. Similar to traditional pharmaceuticals, characterization of a therapeutic protein for its physicochemical properties, process monitoring and lot release is crucial. Electrophoresis in the slab-gel format has and continues to be a mainstay of the protein laboratory; and more recently, CE has begun to make significant inroads for protein analysis in industrial settings. This review focuses on the electrophoresis of proteins with an emphasis on protein-based therapeutics in the capillary, slab-gel and to a lesser extent, the microchip format. Reported applications of electrophoresis at several stages of the biopharmaceutical industry covering the period of 2000-2005 will be discussed.     

Electrophoresis of cereal storage proteins

Cereal proteins have been studied by a number of analytical techniques over the years. One of the major methodologies utilized by cereal chemists has been electrophoresis. Starting with moving boundary electrophoresis and progressing to slab gels and high-performance capillary electrophoresis, innovative methods have been developed to provide high resolution separations of difficult to separate proteins. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE), acid-PAGE, isoelectric focusing, free zone CE, and even high-resolution two-dimensional HPLC-HPCE methods have been developed to separate cereal proteins. This review focuses on electrophoretic methods for separating and characterizing cereal storage proteins.     

Sodium dodecyl sulphate-polyacrylamide gel electrophoresis of human parotid salivary proteins.

The proteins in human parotid saliva have been separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis into 20 or more well resolved species. The Coomassie Brilliant Blue (CBB) R-250 and silver staining procedures have been modified to overcome the problems encountered with staining of proline-rich proteins. By means of the CBB R-250 procedure which stains proline-rich proteins pink-violet, immunoblotting, concanavalin A binding, periodate-Schiff staining and zinc binding, all of the major proteins have been characterised. Substantial individual-to-individual differences were observed in the protein patterns formed. Comparison of parotid, submandibular, and whole saliva from a single individual indicated that fewer proline-rich proteins are expressed in submandibular saliva than in parotid, but whole saliva contains much lower levels than either duct secretion. The results will form a useful base for future research into the functions of salivary proteins.     

Electrophoresis of megaDalton proteins inside colloidal silica

With the growth of the biopharmaceutical industry, there is a need for rapid size‐analysis of proteins on the megaDalton scale. The large pore sizes needed for such separations cannot be easily reached by pushing the current limits of size‐exclusion chromatography or gel electrophoresis. The concept detailed here is the formation of arbitrarily wide pores by packing nonporous colloidal silica in capillaries. This method can be called packed‐capillary electrophoresis, or “pCE”. Electrophoresis of protein standards (11–155 kDa) by pCE, using 345 nm diameter particles in 100 μm diameter capillaries, gives 2x higher resolution than a typical PAGE gel in 1/6 of the time. The electropherograms show that pCE is highly efficient, with half‐micrometer plate heights for all seven standards, giving 10⁵ plates for a 50 mm length. The large pore radius of 65 nm enables baseline resolution of proteins of 0.72, 1.048 and 1.236 MDa in less than 15 min. The short separation time of pCE is attributed to the absence of small pores that restrict protein migration in gels. The pCE separation is applied to the analysis of a stressed pharmaceutical‐grade IgG4 sample, giving unprecedented baseline resolution of monomer, dimer, trimer and tetramer in less than 10 min.     

Electrophoresis of mutant proteins in inherited diseases

The electrophoretic approaches for detection of mutant proteins in inherited diseases are briefly reviewed and discussed. Mutation of a protein, known to be associated with a specific inherited disease, is detected by immunoblotting, immunoprecipitation or enzyme staining, combined with various electrophoretic techniques. Some instrumental and technological devices for two-dimensional electrophoresis have been reported for the screening of mutant proteins in diseases of currently unknown etiology.     

Fractionation and separation of human salivary proteins by pH-gradient ion exchange and reversed phase chromatography coupled to mass spectrometry

In the present work, a 2-D capillary liquid chromatography method for fractionation and separation of human salivary proteins is demonstrated. Fractionation of proteins according to their pI values was performed in the 1-D employing a strong anion exchange (SAX) column subjected to a wide-range descending pH gradient. Polystyrene-divinylbenzene (PS-DVB) RP columns were used for focusing and subsequent separation of the proteins in the 2-D. The SAX column was presaturated with a high pH buffer (A) consisting of 10 mM amine buffering species, pH 9.0, and elution was performed with a low pH elution buffer (B) having the same buffer composition and concentration as buffer A, but pH 3.5. Isoelectric point fractions eluting from the 1-D column were trapped on PS-DVB trap columns prior to back-flushed elution onto the PS-DVB analytical column for separation of the proteins. The 1-D fraction eluting at pH 9.0-8.7 was chosen for further analysis. After separation on the RP analytical column, nine RP protein fractions were collected and tryptic digested for subsequent analyses by MALDI TOF MS and column switching capillary LC coupled to ESI TOF MS and ESI QTOF MS. Eight proteins and two peptides were identified in the pH 9.0-8.7 fraction using peptide mass fingerprinting and uninterpreted MS/MS data.     

Isoelectric focusing of human parotid salivary proteins in hybrid carrier ampholyte-immobilized pH gradient polyacrylamide gels.

Isoelectric focusing of human salivary proteins with carrier ampholyte-isoelectric focusing systems requires prior desalting and concentration of samples, a procedure which is time-consuming and requires relatively large volumes of samples. By contrast, immobilized pH gradient gels are more tolerant to salt loads. Thus pretreatment of samples consists only of centrifugation prior to isoelectric focusing. If larger loads (greater than 50 micrograms) are required, the samples may be concentrated by lyophilization and reconstitution in a smaller volume of water or by dialysis against 30% w/v polyethylene glycol. Immobilized pH gradient polyacrylamide gels (incorporating a hybrid carrier ampholyte system) of two pH ranges (pH 4-9 and pH 3.5-5.0) have been used to separate the proteins in human parotid saliva. The effects of urea on focused patterns were studied; in pH 4-9 gels it gave improved resolution of protein bands, whereas in pH 3.5-5.0 gels it prevented protein precipitation. The salivary proteins were then visualized by staining with Coomassie Brilliant Blue G-250 or a silver procedure. Using the latter, 25-30 well-resolved bands were formed on a pH 4-9 gel loaded with 20 micrograms of proteins. The method offers considerable advantages compared with carrier ampholyte-isoelectric focusing.     

Electrophoresis of serum isoenzymes and proteins following acute myocardial infarction

The clinical significance of the serum enzymes creatine kinase (CK, EC 2.7.3.2), lactate dehydrogenase (LD, EC 1.1.1.27) and aspartate aminotransferase (EC 2.6.1.1), and the isoenzymes CK 1-3 and LD 1-5, in acute myocardial infarction (AMI) is reviewed. Particular attention is given to electrophoretic analysis of the isoenzymes (and the CK isoforms/subforms) following AMI and thrombolytic therapy. Other protein markers for the monitoring of AMI, including myoglobin and muscle contractile proteins, are also discussed and the potential for the detection of new marker proteins using high-resolution two-dimensional electrophoretic methods is demonstrated. Whilst emphasis is placed upon electrophoretic methods the value of complementary immunoassays is acknowledged in order to maintain a balanced perspective.     

The dynamical response of a hydrogel fiber to electrochemical stimulation

The preparation of a smart hydrogel fiber based on chitosan/poly(ethylene glycol) is presented. The dynamics of this hydrogel fiber in response to electric stimulation is reported. The effects of a number of factors have been systematically studied, including the fiber diameter, concentration of the crosslinking agent, electric potential imposed across the fiber, pH, and ionic strength of the bath solution. Fiber deformation is expressed in terms of the curvature at the midlength of the fiber for various times. The number of bending to a given extent within a given time period is used to describe the rate of cyclic deformation. Our experimental results show a stable reversibility of bending behavior under the applied electric field. The bending curvature is proportional to the intensity of the applied electric potential. Although adequate mechanical properties are maintained, the rate of deformation can be improved via the adjustment of a number of the aforementioned extrinsic factors. These observations are interpreted in terms of fiber stiffness, fixed charge density, and swelling pressure, which depend on the hydrogel equilibrium states in different pH and ionic environments along with the electrochemical reactions under the electric field. © 2000 John Wiley & Sons, Inc. J Polym Sci B: Polym Phys 39: 236–246, 2001     

Two-dimensional electrophoresis analysis of human serum proteins during the acute-phase response.

The serum of patients with meningitis, due to infection by Haemophilus influenzae type b, was analyzed. Several known acute-phase proteins were separated by two-dimensional electrophoresis and estimated quantitatively. In addition, hitherto undescribed reactants were recognized. Gels were calibrated and relevant spots related to master spot numbers in the human serum protein database.     

Two-dimensional electrophoresis of human parotid salivary proteins from normal and connective tissue disorder subjects using immobilised pH gradients.

Two-dimensional electrophoretic analysis of human salivary proteins using immobilised pH gradients in the first dimension, thin-layer gradient horizontal sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the second, and modified staining procedures has resulted in a substantial improvement in their resolution. Unlike carrier ampholyte-based techniques, immobilised pH gradients prevent the loss of proteins of pI greater than 8; accordingly, basic components, including basic proline-rich proteins, can now be resolved. A two-dimensional map showing the locations and identities of most of the major proteins has been constructed. Narrow-range pH gradients can be constructed to give increased resolution of proteins of particular interest. By means of a pH 3.5-5.0 gradient, the abnormal salivary proteins associated with connective tissue disorders were found to be a highly heterogeneous group of pI approximately 3.75-4.75 and Mr approximately 32,000; although low levels occurred in some normal individuals, there was less heterogeneity (pI approximately 3.75-4.25). The technique should form a base for future structural, functional, and clinical studies on human salivary proteins.     

Dipolar response of hydrated proteins

The paper presents an analytical theory and numerical simulations of the dipolar response of hydrated proteins in solution. We calculate the effective dielectric constant representing the average dipole moment induced at the protein by a uniform external field. The dielectric constant shows a remarkable variation among the proteins, changing from 0.5 for ubiquitin to 640 for cytochrome c. The former value implies a negative dipolar susceptibility, that is a dia-electric dipolar response and negative dielectrophoresis. It means that ubiquitin, carrying an average dipole of ?240 D, is expected to repel from the region of a stronger electric field. This outcome is the result of a negative cross-correlation between the protein and water dipoles, compensating for the positive variance of the intrinsic protein dipole in the overall dipolar susceptibility. In contrast to the neutral ubiquitin, charged proteins studied here show para-electric dipolar response and positive dielectrophoresis. The study suggests that the dipolar response of proteins in solution is strongly affected by the coupling of the protein surface charge to the hydration water. The protein-water dipolar cross-correlations are long-ranged, extending ~2 nm from the protein surface into the bulk. A similar correlation length of about 1 nm is seen for the electrostatic potential produced by the hydration water inside the protein. The analysis of numerical simulations suggests that the polarization of the protein-water interface is highly heterogeneous and does not follow the standard dielectric results for cavities carved in dielectrics. The polarization of the water shell gains in importance, relative to the intrinsic protein dipole, at high frequencies, above the protein Debye peak. The induced interfacial dipole can be either parallel or antiparallel to the protein dipole, depending on the distribution of the protein surface charge. As a result, the high-frequency absorption of the protein solution can be either higher or lower than the absorption of water. Both scenarios have been experimentally observed in the THz window of radiation.     

Homochiral expression of proteins:a discussion on the natural chirality related to the origin of life

Homochirality in life has always been a driving force in scientific research and natural exploration. It has not been satisfactorily explained, and systematic investigations are necessary. This paper reported a homochiral expression of proteins dependent on the stirring direction of growing media. By controlling the stirring direction clockwise (CW) and anticlockwise (ACW) of the culture medium, proteins with distinct secondary structures were obtained, and D-amino acid may be included in the protein cultur...     

Homochiral expression of proteins: a discussion on the natural chirality related to the origin of life

Homochirality in life has always been a driving force in scientific research and natural exploration. It has not been satisfactorily explained, and systematic investigations are necessary. This paper reported a homochiral expression of proteins dependent on the stirring direction of growing media. By controlling the stirring direction clockwise (CW) and anticlockwise (ACW) of the culture medium, proteins with distinct secondary structures were obtained, and D-amino acid may be included in the protein cultured with the stirring direction of ACW. Considering the effect of force fields, which might affect the process of folding and refolding of cellular protein in this report, the control of force fields might be a good way to prepare asymmetric drugs, and the rotational direction of the earth is possibly related to the chirality in primitive life molecules.     

Binding of salivary proteins and oral bacteria to hydrophobic and hydrophilic surfaces in vivo and in vitro

Chemical modifications of mineral surfaces were performed in order to gain insight into what surface properties are decisive of the accumulation of dental plaque. A non-charged, hydrophilic surface was made by two consecutive plasma polymerizations, firstly with allyl alcohol, secondly with acrylic acid, followed by adsorption of a poly(ethylene glycol)-poly(ethylene imine) adduct. A strongly hydrophobic surface was obtained by plasma polymerization of hexamethyldisiloxane. Ellipsometry was used to monitor protein interaction with the surfaces. The hydrophilic surface gave very little adsorption of both a model protein, IgG, and of saliva proteins. The hydrophobic surface, on the other hand, adsorbed high amounts of both types of proteins. In vitro adhesion of an oral bacterium,S. mutans, as well as in vivo studies, gave the opposite result, the hydrophobic surface giving less adhesion and less plaque accumulation than the hydrophilic surface. A tentative explanation of this behavior is that the saliva proteins that bind to the hydrophobic surface adsorb in an unnatural conformation which does not favor bacteria adherence.