bluuebunny/crossref_metadata_2025 huggingface dataset:
Capillary electrophoresis of biotechnology‐derived proteins
Authors: Strege Mark A. , Lagu Avinash L.
Published: May 2005

Applications of capillary electrophoretic techniques for the analysis of biotechnology‐derived proteins continue at a steady pace. This review summarizes the analyses of these proteins achieved using capillary isoelectric focusing, micellar electrokinetic capillary chromatography, capillary electrophoresis/mass spectrometry and capillary gel electrophoresis. A very brief summary of each technique is included. Application of capillary isoelectric focusing to the analysis of recombinant tissue plasminogen activator in quality control type applications is examined in some detail. Analyses of dosage forms of biotechnology derived proteins of pharmaceutical importance as well as the identification and examination of the peptides, obtained after enzymatic cleavage of proteins, by capillary electrophoresis have been included. Examples of capillary gel electrophoresis as a substitute sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) of biotechnology‐derived proteins are also reviewed.
Capillary zone electrophoresis of proteins
Authors: Dolník Vladislav
Published: May 2005

This review article with 237 references is focused on capillary zone electrophoresis (CZE) of proteins. It includes discussion of modeling electrophoretic migration of proteins, sample pretreatment before the analysis, methods reducing the sorptions of proteins on the capillary wall, and techniques for increasing selectivity by using electrolyte additives including the sieving matrices. Significant progress in detection techniques, namely in laser‐induced fluorescence and mass spectrometry, is emphasized. Modifications of CZE using specific interactions, such as affinity capillary electrophoresis or capillary immunoelectrophoresis are debated as well as combination of CZE with other separation methods such as high performance liquid chromatography (HPLC). A number of practical applications of CZE of proteins are described.
Developments in amino acid analysis using capillary electrophoresis
Authors: Smith Joel T.
Published: May 2005

The current status in the analysis of amino acids using capillary electrophoresis is addressed. This area of biological analysis has received increased attention with more than 200 articles being published in the last five years. This review discusses pre‐, post‐, and on‐column derivatization techniques used to tag amino acids providing a detectable moiety. Several separation methodologies which provided resolution for large sets of amino acids are presented. An overview of advances in the enantiomeric resolution methodologies for amino acids is given. Both direct and indirect enantiomeric separation schemes are summarized. Recent advances in detection strategies for both derivatized and underivatized amino acids are presented. Applications utilizing amino acid analysis by capillary electrophoresis are described. This review covers articles published between 1991 and 1996.
Use of free‐flow electrophoresis for the analysis of cellular uptake of picornaviruses
Authors: Kronenberger Peter , Schober Daniela , Prchla Elisabeth , Blaas Dieter , Fuchs Renate
Published: May 2005

Free‐flow electrophoresis is a powerful tool to separate subcellular vesicles such as early and late endosomes from plasma membranes. Using this technique, the intracellular distribution of poliovirus type 2 Sabin (PV2) and its derived subviral particles was analyzed upon infection of HeLa cells. Comparison of various infection conditions showed that maximally 30% of total cell associated PV2 was found in endosomal compartments with the remainder being associated with plasma membrane fractions; 2% of viral label was recovered from the cytoplasm in form of free virions. Sucrose gradient centrifugation analysis of the viral material recovered from the respective fractions revealed that intracellular virus was exclusively in its native conformation. This is in sharp contrast to human rhinovirus serotype 2 (HRV2), which is rapidly modified to RNA‐free subviral particles upon accumulation in endosomes. The data suggest that productive poliovirus uncoating can occur at the plasma membrane whereas internalized virus is most probably aborted.
Mycobacterial phagosome maturation, rab proteins, and intracellular trafficking
Authors: Deretic V. , Via Laura E. , Fratti Rutilio A. , Deretic Dusanka
Published: May 2005

One of the most prominent features of pathogenic mycobacteria, which include the potent human pathogensMycobacterium tuberculosisandMycobacterium lepraeand their opportunistic relativesMycobacterium aviumandMycobacterium marinum, is their ability to survive and multiply in phagosomes of mononuclear phagocytic cells. The phagocytosed mycobacteria reside in a vacuolar compartment which is exempted from maturation into the phagolysosome. Recently, the arrest of the maturation of phagosomes containingM. tuberculosiscomplex organisms (Mycobacterium bovisBCG) has been linked to the accumulation on the phagosomal membrane of the small GTP binding protein rab5, specific for the control of fusion within the early endosomal compartment. Furthermore,M. bovisBCG phagosome is devoid of rab7, a rab protein associated with the late endosome. The selective accumulation of rab5 and exclusion of rab7 defines the check point that has been compromised in mycobacterial phagosome maturation. Here we summarize these observations and relates them to other phenomena in the area of membrane and protein trafficking with the emphasis on phagosomes containing intracellular pathogens.
The interaction between Mycobacterium and the macrophage analyzed by two‐dimensional polyacrylamide gel electrophoresis
Authors: Sturgill‐Koszycki Sheila , Haddix Pryce L. , Russell David G.
Published: May 2005

The intramacrophage pathogen Mycobacterium avium resides in a vacuole which displays unusual fusion characteristics, expressed as both a failure to mature into phagolysosomes and a continued access to the early recycling pathway. In contrast, compartments containing inert IgG‐opsonized latex beads mature to phagolysosomes. Techniques were developed for the isolation of these particle‐containing phagosomes from macrophages to facilitate analysis of phagosomal constituents by electrophoresis and autoradiography. Metabolic labeling of macrophages followed by phagosome isolation and two‐dimensional polyacrylamide gel electrophoresis revealed only minor differences in the protein profiles between the M. avium and IgG‐bead phagosomes despite the marked differences in the fusigenicity of the respective vacuoles. Pulse‐chase labeling experiments revealed greater differences in the accessibility of Mycobacterium avium and IgG‐bead phagosomes to newly synthesized proteins. These phagosome isolation techniques were extended to analyze the protein synthesis profile of intracellular M. avium for comparison with bacteria that were metabolically labeled in broth culture. Not surprisingly, the majority of polypeptides in the bacilli were common to both growth conditions. However, despite these similarities, intracellular M. avium express several unique proteins, most notably one abundant protein with a molecular weight of 51 kDa. In addition, the bacteria manifest a restricted set of proteins expressed while in stasis shortly after infection.
Subcellular fractionation of polarized epithelial cells and identification of organelle‐specific proteins by two‐dimensional gel electrophoresis
Authors: Fialka Irene , Pasquali Christian , Lottspeich Friedrich , Ahorn Horst , Huber Lukas A.
Published: May 2005

Protein targeting and sorting is accomplished by complex vesicular transport processes that are tightly regulated within a cell. This is especially important for epithelial cells because correct delivery of newly synthesized proteins as well as recycling and sorting of internalized membrane proteins is essential for the establishment and preservation of cellular polarity. Many transport events, linking various subcellular compartments, have been analyzed, but many transport mechanisms still remain unresolved. In this study we attempted to identify proteins specifically associated with distinct organelles in murine mammary epithelial cells (EpH4). We isolated subcellular compartments by continuous sucrose gradient centrifugation in order to further analyze their protein composition by high‐resolution two‐dimensional gel electrophoresis (2‐DE). The successful separation of late endosomes (LE), early endosomes (EE) and most of the rough endoplasmic reticulum (RER) was confirmed by subsequent analysis of gradient fractions for compartment‐specific enzymes and marker proteins. Both Golgi and plasma membrane (PM) were found to partially co‐purify with EE in such gradients. Characteristic polypeptide patterns were revealed on 2‐DE gels for fractions enriched in membranes of different origin. Based on improved sample preparation and loading techniques (this issue, C. Pasqualiet al., Electrophoresis, 1997,18, 2573–2581), we were able to identify several proteins by immunoblotting or microsequencing of Coomassiestained spots. This will be the basis for a further characterization of organellespecific molecules in epithelial cells as well as for the establishment of a 2‐DE reference map of membrane proteins from murine mammary epithelium.
Identification of components of trans‐Golgi network‐derived transport vesicles and detergent‐insoluble complexes by nanoelectrospray tandem mass spectrometry
Authors: Shevchenko Andrej , Keller Patrick , Scheiffele Peter , Mann Matthias , Simons Kai
Published: May 2005

Epithelial cells have to deliver newly synthesized proteins to the apical and the basolateral plasma membrane domains of the polarized cell surface. Sorting takes place in the trans‐Golgi network and at least two vesicular carriers exist for apical and basolateral delivery. After immuno‐isolation, the composition of these vesicle preparations was analyzed by two‐dimensional (2‐D) gel electrophoresis and detergent extraction. In this paper we compare the constituents of detergent‐insoluble complexes in different cell lines of polarized or nonpolarized origin and present the identification of five previously uncharacterized proteins. We show that our protein identification strategy can be successfully applied to the problem of small hydrophobic proteins from organisms that have not been substantially sequenced. The high sensitivity of nanoelectrospray tandem mass spectrometry allowed us to identify two proteins that belong to the p23/p24 family of putative cargo receptors for vesicular trafficking. Furthermore we have mapped CD9 and CD81, two members of a large family of proteins consisting of highly hydrophobic four transmembrane proteins. In addition we have identified caveolin‐2 as a constituent of basolateral transport vesicles. We have also extended our analysis of immunoisolated vesicles to a more basic pI range and show that this region on 2‐D gels is devoid of proteins. With these approaches and with the previously published data we have now identified most of the major low molecular weight proteins recovered in detergent‐insoluble glycolipid‐enriched complexes.
Mapping the protein composition of trans‐Golgi network (TGN)‐derived carrier vesicles from polarized MDCK cells
Authors: Fiedler Klaus , Kellner Roland , Simons Kai
Published: May 2005

In polarized MDCK cells, proteins and lipids are sorted in the trans‐Golgi network /TGN) and packaged into different vesicular carriers that are delivered to the apical or basolateral cell surface. To gain insight into the sorting and trafficking machinery, we have previously isolated TGN‐derived carrier vesicles from perforated MDCK cells. The composition of immuno‐isolated apical and basolateral carriers was mapped by two‐dimensional (2‐D) gel electrophoresis. Here we describe the identification of several components of the vesicle fraction by using three different methods. 2‐D gel comigration was performed with carrier vesicles isolated from metabolically labeled MDCK cells and human epidermal keratinocyte lysates. This allowed us to assign eleven known components by a comparison with the comprehensive keratinocyte 2‐D gel database. These comprised two members of the 14‐3‐3 family of proteins that have been implicated in vesicular trafficking. Five proteins were purified from preparative 2‐D gels and identified by peptide microsequencing, including the β1 and β2 subunit of trimeric G proteins and an annexin II variant. A member of the SNARE family of proteins was identified by immunoblotting. The combination of 2‐D gel electrophoresis and 2‐D gel databases allows the rapid assessment of the purity of subcellular fractions and to characterize components involved in vesicular transport.
Two‐dimensional electrophoresis reveals a nuclear matrix‐associated nucleolin complex of basic isoelectric point
Authors: Gotzmann Josef , Eger Andreas , Meissner Michael , Grimm Rudolf , Gerner Christopher , Sauermann Georg , Foisner Roland
Published: May 2005

A monoclonal antibody was raised against a salt‐extractable fraction of nuclear matrix / intermediate filament scaffolds of polarized MDCK cells. The antibody recognized an ∼ 100 kDa protein in total cell lysates and nuclear matrices of various human cells and tissues and stained nucleolar structures in immunofluorescence microscopy. By partial sequencing of five peptides derived from immunoprecipitated protein, the targeted antigen was found to be homologous to human nucleolin. After two‐dimensional electrophoresis of total HeLa cell lysates, immunoreactive bands were detected at isoelectric point (pI) 5.5–6.1, characteristic for nucleolin, and at pI 8.5–9. Whereas the protein focusing at acidic pI was found in Triton X‐100‐soluble cellular fractions, the antigen focusing at basic pI was exclusively contained in the residual nuclear fraction and was solubilized upon treatment of nuclear matrices with RNAse. The component solubilized by RNAse treatment was still detected at basic pI in two‐dimensional electrophoresis. However, upon immunoprecipitation of the antigen from the RNAse‐released fraction in the presence of sodium dodecyl sulfate (SDS), the nuclear matrix‐derived antigen was positioned at pI 5–6. The present data indicate that the nuclear matrix‐bound nucleolin is associated with ribonucleoproteins and a basic component resisting dissociation under conditions of isoelectric focusing.
Separation of early steps in endocytic membrane transport
Authors: van der Goot F. Gisou
Published: May 2005

We describe a simple subcellular fractionation scheme aimed at separating early endosomes from the plasma membrane in view of studying the possible arrival of plasma membrane‐bound toxins, proteins or other extracellular ligands in endosomes. Plasma membrane proteins were labeled with the impermeable reagent sulfosuccinimidyl‐6‐(biotinamido)hexanoate (NHS‐LC) biotin at 4°C. In a separate set of cells, early endosomes were labeled by internalization of horseradish peroxidase from the medium for 5 min. The first step of the purification, which consists of a step sucrose gradient, led to three fractions, respectively: enriched in biosynthetic membranes (interface 3), in plasma membrane and early endosomes (interface 2), and in late endosomes (interface 1). The second step, in which interface 2 was loaded at the bottom of a 17% Percoll gradient, led to the separation of the plasma membrane, including caveolae and cholesterol‐glycolipid rafts, from early endosomes. Western blot analysis of the fractions from the Percoll gradient showed that the transferrin receptor, the small GTPases rab5 and Arf6, as well as annexin II were present both at the plasma membrane and in early endosomes, whereas the caveolar marker caveolin, 1co, migrated only with the biotinylated plasma membrane proteins. We used this fractionation procedure to show that the pore‐forming toxin aerolysin does not reach the endocytic compartments of baby hamster kidney (BHK) cells. The procedure should be generally useful in rapidly determining whether extracellular proteins or ligands reach endosomes.
Unique identification of proteins from small genome organisms: Theoretical feasibility of high throughput proteome analysis
Authors: Cavalcoli James D. , VanBogelen Ruth A. , Andrews Philip C. , Moldover Brian
Published: May 2005

We evaluate current levels of accuracy for estimation of molecular weight (Mr) and isoelectric point (pI) to proteins on two‐dimensional (2‐D) gels as well as the distribution and clustering of proteins in the predicted proteome of E. coli. We also examine the ability to find single candidates within the predicted proteome for matching to a protein seen on 2‐D gels, based on the current level of accuracy. We discuss the levels of accuracy needed to match predicted proteins to observed proteins based solely on Mr and pI criteria obtained from genomic information, and propose methodology to achieve this level of accuracy. In addition, we will address the future goals of this work since the small genomes of bacteria provide a foundation and stepping stone to similar studies in higher organisms.
Melanie II – a third‐generation software package for analysis of two‐dimensional electrophoresis images: I. Features and user interface
Authors: Appel Ron D. , Palagi Patricia M. , Walther Daniel , Vargas J. Reynaldo , Sanchez Jean‐Charles , Ravier Florence , Pasquali Christian , Hochstrasser Denis F.
Published: May 2005

Although two‐dimensional electrophoresis (2‐DE) computer analysis software packages have existed ever since 2‐DE technology was developed, it is only now that the hardware and software technology allows large‐scale studies to be performed on low‐cost personal computers or workstations, and that setting up a 2‐DE computer analysis system in a small laboratory is no longer considered a luxury. After a first attempt in the seventies and early eighties to develop 2‐DE analysis software systems on hardware that had poor or even no graphical capabilities, followed in the late eighties by a wave of innovative software developments that were possible thanks to new graphical interface standards such as XWindows, a third generation of 2‐DE analysis software packages has now come to maturity. It can be run on a variety of low‐cost, general‐purpose personal computers, thus making the purchase of a 2‐DE analysis system easily attainable for even the smallest laboratory that is involved in proteome research. Melanie II 2‐D PAGE, developed at the University Hospital of Geneva, is such a third‐generation software system for 2‐DE analysis. Based on unique image processing algorithms, this user‐friendly object‐oriented software package runs on multiple platforms, including Unix, MS‐Windows 95 and NT, and Power Macintosh. It provides efficient spot detection and quantitation, state‐of‐the‐art image comparison, statistical data analysis facilities, and is Internet‐ready. Linked to proteome databases such as those available on the World Wide Web, it represents a valuable tool for the “Virtual Lab” of the postgenome area.
Latex allergen database
Authors: Posch Anton , Chen Zhiping , Dunn Michael J. , Wheeler Colin H. , Petersen Arnd , Leubner‐Metzger Gerhard , Baur Xaver
Published: May 2005

Two‐dimensional (2‐D) electrophoresis followed by immunoblotting and N‐terminal protein microsequencing were used to characterize and identify the IgE‐reactive proteins of Hevea latex that are the main cause of the latex type I allergy affecting especially health care workers and spina bifida children. This approach generated a comprehensive latex allergen database, which facilitated the integration of most of the latex allergen data presented in the literature. The major latex allergens Hev b 1, Hev b 3, Hev b 6 and Hev b 7 have been localized on our 2‐D maps. Moreover, we were able to identify six previously undescribed IgE‐binding latex proteins, namely enolase, superoxide dismutase, proteasome subunit C5, malate dehydrogenase, triosephosphate isomerase and endochitinase. The generated latex 2‐D maps will provide valuable information to develop strategies for the isolation of the novel IgE binding proteins in order to study the frequency of sensitization among both risk groups. Detailed knowledge of all proteins involved in latex allergy will allow better diagnosis of latex allergy and to monitor the success of prevention strategies that are needed to reduce the high prevalence of latex allergy among both risk groups.
A two‐dimensional protein map of human amniotic fluid at 17 weeks' gestation
Authors: Liberatori Sabrina , Bini Luca , de Felice Claudio , Magi Barbara , Marzocchi Barbara , Raggiaschi Roberto , Frutiger SÉVerine , Sanchez Jean‐Charles , Wilkins Marc R. , Hughes Graham , Hochstrasser Denis F. , Bracci Rodolfo , Pallini Vitaliano
Published: May 2005

Using updated technical procedures (immobilized pH gradients for isoelectric focusing followed by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis: IPG/SDS‐PAGE) we provide a two‐dimensional (2‐D) map of amniotic fluid (AF) proteins. This map comprises over 800 silver‐stained spots. Over 150 spots have been identified by matching on the net with human plasma and cerebrospinal fluid maps available from SWISS 2DPAGE database; several additional spots were assigned by immunoblotting and/or microanalytical techniques. This report details our investigation on AF proteins focusing on the 17th week of gestation, when AF is most commonly used for clinical evaluation of fetal disorders. As a whole, the map displays a number of potential markers for fetal development and for gestation abnormalities. The 2‐D electrophoretic technique allows the monitoring of all these proteins at the same time along with additional spots that may prove of diagnostic significance.
A novel standardization method for two‐dimensional DNA fingerprints
Authors: Hampe Jochen , Mrowka Ralf , Marczinek Karola , Nürnberg Peter
Published: May 2005

Two‐dimensional (2‐D) DNA fingerprinting is a technique that allows for parallel genome analysis through the simultaneous detection of up to 500 minior microsatellite loci on a 2‐D gel. Separation is performed according to size and melting temperature in the gel. In the application of this technique in genome analysis, a standardized method for the identification of individual spots is required. However, due to the polymorphic nature of up to 80% of the spots, existing standardization methods that have been primarily developed for 2‐D protein patterns are not suitable for this task. We developed a robust method that standardizes 2‐D DNA fingerprint spots on the basis of melting temperature – or denaturing gradient position – and fragment size. An external marker was used as a basis for standardization. A normalization surface was calculated over the gel dimensions by adapting an established numerical iteration technique previously used in physics termed “relaxation method”. The relaxation method works robustly with the irregularly spaced marker spots. The evaluation of the method for a spot of preknown position derived from the TP53 gene revealed a median observed error below 1% for fragment length and denaturing gradient position. The search for candidate minisatellite loci in genomic difference analysis depends on the reliable identification of alleles of this locus in different individuals. We proved experimentally that alleles of a single minisatellite locus cloned from a 2‐D gel cluster on an isothermal line can be reliably identified using the presented standardization method. In conclusion, a standardization tool for a broader application of 2‐D DNA fingerprinting in both tumor analysis and possibly parallel mutation screening is now available.
DNA sequencing with pulsed‐field capillary electrophoresis in poly(ethylene oxide) matrix
Authors: Kim Yongseong , Yeung Edward S.
Published: May 2005

DNA sequencing by using pulsed‐field capillary electrophoresis in a linear polymer matrix was performed in this work. The effects of waveform, frequency, modulation depth and heating were investigated. The separation performance for Sanger DNA fragments up to 1000 bp using a pulsed field was compared to isoelectric and isorheic conditions. A separation mechanism in the flexible poly(ethylene oxide) (PEO) gel was also suggested. We found that temperature change generated by either a pulsed field or ultrasound during DNA sequencing can have a deleterious effect on the separation efficiency.
Separation of oligonucleotides of identical size, but different base composition, by free zone capillary electrophoresis in strongly acidic, isoelectric buffers
Authors: Perego Marilena , Gelfi Cecilia , Stoyanov Alexander V. , Righetti Pier Giorgio
Published: May 2005

A novel method for analyzing oligonucleotides of the same length, but bearing a single base substitution, is reported, based on free zone capillary electrophoresis (CZE) under rather acidic pH values. For this purpose, a set of four 18‐mers of fairly random base composition has been synthesized, bearing, in nucleotide 9, the following bases: T, C, G or A. Theoretical predictions, based on titration curves of single free nucleotides, allowed us to predict that the simultaneous separation of a mixture of all four oligonucleotides could be possible in a pH 3–4 window. In fact, electrophoresis at pH 5.7 gave a single, asymmetric peak, whereas CZE at pH 4.8 could resolve three out of four species (the T9 and G9 oligonucleotides co‐migrating into a single zone). A unique separation power could be obtained at pH 3.3 in a buffer comprising an amphoteric species (isoelectric iminodiacetic acid, IDA) and 7 M urea. Although IDA exhibited a pI of 2.23 (for a 100 mM solution), the addition of 7 M urea (necessary to denature the oligonucleotides) raised the apparent pH of the solution to 3.3.
Capillary zone electrophoresis of interconverting cis‐trans conformers of peptidyl‐proline dipeptides: Estimation of the kinetic parameters
Authors: Rathore Anurag S. , Horváth Csaba
Published: May 2005

Peptides containing proline residues, except at the N‐terminus, exist in cis and trans forms due to the rigidity of the peptidyl‐proline bond. Computer‐simulated and experimental electropherograms have been used to study the interplay of the electrophoretic migration and the kinetics of the cis‐trans interconversion. The measure of the interference by the reaction is the dimensionless Damköhler number (Da) which is the ratio of the migrant's residence time to the characteristic time of reaction in the capillary. The fraction of the trans conformer that is not separable with 90% purity is employed to quantify the extent of overlap between the concentration profiles of the two interconverting migrants and the effect of the various operational variables was examined by simulation. The deterioration of separation, as measured by the degree of overlap, is a quasi sigmoidal function of Da with the selectivity and the intrinsic efficiency of the system as the parameters. At sufficiently low temperatures the interference by reaction kinetics may vanish and the two conformers are separated. At high enough temperatures, when the rate of interconversion is very fast, the two conformers are not separated at all. A simple and rapid method is proposed for estimation of kinetic parameters for the cis‐trans isomerization on the basis of data obtained with phenylalanyl‐proline dipeptide. The procedure involves determination of the equilibrium constant by measuring equilibrium concentrations of the two conformers using nuclear magnetic resonance (NMR) or capillary zone electrophoresis (CZE) at very low temperature, correlation of the peak shapes in the electropherograms with the Da, and finally, evaluation of the forward rate constants from the assigned Da values in the domain: 0.01 < Da < 0.5. Separations using CZE were performed in the temperature range of 1–40°C by using the Beckman P/ACE unit equipped with an auxiliary cooling system. The kinetic data thus obtained showed good agreement (average error less than 5%) with those measured by NMR.
Resolution of the diastereomers of a large synthetic peptide by capillary electrophoresis using nonionic surfactants
Authors: Gilges Martin , Hadley Mark
Published: May 2005

A capillary electrophoretic method was developed for the determination of the diastereomeric composition of the bradykinin B2 antagonist, SB‐238592‐DB. The nonionic surfactant Brij 35 was demonstrated to be suitable for the baseline resolution of the three diastereoisomers of SB‐238592‐DB in the presence of a low pH phosphate buffer and small volume percentages of acetonitrile.
Identification of plasma proteins facilitated by enrichment on particulate surfaces: Analysis by two‐dimensional electrophoresis and N‐terminal microsequencing
Authors: Lück Martin , Schröder Werner , Harnisch Stephan , Thode Kai , Blunk Torsten , Paulke Bernd‐R. , Kresse Mayk , Müller Rainer H.
Published: May 2005

Plasma protein adsorption on intravenously injectable drug carriers is regarded as an important factor for the fate of the particles in the body after their administration. Therefore, the plasma protein adsorption patterns on a number of different carrier systems were analyzed in vitro employing two‐dimensional electrophoresis (2‐DE). The particulate systems presented in this study were polystyrene (PS) model particles, PS nanoparticles surface‐modified by adsorption of a surfactant, a commercial fat emulsion, and magnetic iron oxide particles used as contrast agents in magnetic resonance imaging. Most of the spots in the plasma protein adsorption patterns could be identified by matching the resulting 2‐DE gels with a reference map of human plasma proteins. Several other proteins that indicated preferentially adsorbed proteins on the surface of the particles investigated have either not been identified on the reference map, or their identity was found to be ambiguous. The relevant proteins are all present in plasma in low abundance. Since these proteins were strongly enriched on the surface of the particles, the resulting spots on the 2‐DE gels were successfully identified by N‐terminal microsequencing. With this approach, two chains of spots, designated PLS:6 and PLS:8, were determined on a plasma reference map: inter‐α‐trypsin inhibitor family heavy chain‐related protein (also named PK‐120) and a dimer of fibrinogen γ, respectively. Plasma gelsolin is presented in a 2‐DE adsorption pattern of PS model particles. One of the main proteins adsorbed by droplets of a commercial fat emulsion was identified as apoliprotein H. Moreover, the positions of apolipoproteins apoC‐II and apoC‐III were also verified on the 2‐DE protein map of human plasma. Thus, protein adsorption experiments of the kind presented in this study are increasing our insight into human plasma proteins.
Capillary ion analysis of potassium concentrations in human vitreous humor
Authors: Ferslew Kenneth E. , Hagardorn Andrea N. , Harrison M. Travis , McCormick William F.
Published: May 2005

Capillary ion analysis (CIA) is a form of capillary electrophoresis which uses the differential electrophoretic mobility of ions to perform a separation of an ionic mixture. Application of this technique for direct detection of potassium concentrations in human vitreous humor was the purpose of this investigation. CIA was performed using a Waters Quanta 4000 Capillary Electrophoresis System with a 745 Data Module using a 75 μm × 60 cm capillary and a run electrolyte of 67.7 mg hydroxyisobutyric acid (HIBA), 52.8 mg 18‐crown‐6 ether and 64 μL UV‐CAT‐1 reagent (4‐ethylbenzylamine) in a volume of 100 mL water (18 Mohm) with a voltage of 20 kV using ultraviolet absorption detection at 214 nm. Migration times were: ammonium ion, 2.86 min; potassium, 3.24 min; calcium, 3.84 min; sodium, 3.98 min; barium (internal standard), 4.68 min; and lithium, 4.79 min. Correlation coefficients (r) between peak area ratios and concentration ranges of 2.5–144 mmole/L (100–1000 ppm) were from 0.9855 to 0.9999. Coefficients of variation (CV) ranged from 1.45 to 13.8% between days and from 1.38 to 9.43% within‐day. Application of this methodology to twenty‐five vitreous humor specimens from forensic cases was compared to analysis by ion‐specific electrode for potassium concentration. Comparison of CIA to ion‐specific electrode analysis of vitreous humor potassium concentrations revealed a correlation coefficient of 0.9642. CIA is applicable to forensic analysis of potassium concentration in forensic vitreous humor specimens. Quantitation of numerous cation concentrations is possible by direct CIA of vitreous humor.
Strategies for the capillary electrophoretic separation of indole alkaloids in Psilocybe semilanceata
Authors: Pedersen‐Bjergaard Stig , Rasmussen Knut Einar , Sannes Ellen
Published: May 2005

While the hallucinogenic mushrooms Psilocybe semilanceata have previously been analyzed for the indole alkaloids psilocybin and baeocystin by capillary zone electrophoresis (CZE) at pH 11.5, the present work focused on the development of an alternative and complementary capillary electrophoretic method for their identification. Owing to their structural similarity and zwitterionic nature, the compounds were difficult to resolve based on different interactions with cationic or anionic micelles. However, while the attempts with micellar electrokinetic chromatography (MEKC) were unsuccessful, rapid derivatization with propyl chloroformate and reanalysis by CZE at pH 11.5 was effective to support identification of the two indole alkaloids. Psilocin was difficult to analyze by CZE at pH 11.5 owing to comigration with the electroosmotic flow. For this compound, the pH of the running buffer was reduced to 7.2 to effectively enhance the electrophoretic mobility.
The analysis of fountain pen inks by capillary electrophoresis with ultraviolet/visible absorbance and laser‐induced fluorescence detection
Authors: Rohde Ellen , Vogt Carla , Heineman William R.
Published: May 2005

Information on the identity of inks adds to the circumstantial evidence in legal cases involving fraudulent documents. In combination with optical methods, multiple thin‐layer chromatography (TLC) is currently the analytical tool used by forensic chemists to separate, compare and distinguish inks based on their dye composition. In our studies, capillary electrophoresis (CE) was used for the analysis of water‐soluble fountain pen inks. Inks are complex mixtures of synthetic organic and inorganic dyes, surfactants, resins and other components. The investigations included the development of an electrophoretic separation method, the optimization of an extraction procedure for inks from paper as well as the evaluation of ultraviolet/visible (UV/VIS) absorbance and laser‐induced fluorescence (LIF) detection for the analysis of inks by CE. Good results for the separation of 17 blue and black inks of different manufacturers and countries of origin were obtained with 100 mM borate buffer, pH 8.0, containing 20% methanol. The electropherograms of the inks and their extracts from paper showed patterns that were in most cases distinctly different from each other. Ultraviolet/visible scans can be used to compare spectra of the separated main and trace components of inks. Fluorescence detection at different excitation and emission wavelengths was more sensitive, but added to the complexity of the electropherograms due to the excitation of coextracted fluorescing paper components. The resolution power of CE combined with the information content provided by the detection modes investigated prove CE to be a powerful tool for the identification of water‐soluble inks used on paper documents.
DNA fingerprinting of Listeria monocytogenes using enterobacterial repetitive intergenic consensus (ERIC) motifs‐polymerase chain reaction/capillary electrophoresis
Authors: Sciacchitano Carl J.
Published: May 2005

The molecular technique, enterobacterial repetitive intergenic consensus (ERIC)‐polymerase chain reaction (PCR) produces genomic DNA fingerprint that discriminate bacterial species and strains. This technique was applied to the characterization of Listeria monocytogenes, an important food‐borne pathogen implicated in numerous cases of listeriosis. The ERIC‐PCR resulted in distinct DNA fingerprinting patterns of all L. monocytogene serotypes and Listeria species. Analysis of the genomic DNA fingerprints was accomplished using capillary electrophoresis (CE), an alternative technique to the conventional agarose gel method. The optimization of CE conditions (electrokinetic injection, applied voltage) resulted in the resolution of amplified DNA fragments up to 1000 bp. Comparisons of electropherograms provided genomic fingerprint templates which could be further used for supplementary information. The ERIC‐PCR method coupled to CE provides a rapid technique in differentiating bacterial spp., and may contribute relevant information in food‐borne outbreak studies.
Analysis of the D1S80 locus by capillary electrophoresis
Authors: Mitchell Joseph W. , Walsh David
Published: May 2005

We have demonstrated in a systematic manner that the capillary electrophoresis (CE) method of genotyping the human D1S80 locus is an effective replacement for the commonly used gel electrophoresis method. The CE method is fast, with a total run time of less than 22 min per sample. Separation has been optimized so that resolution for all pairs of alleles from 14 through 41 is greater than 1, providing unambiguous identification. The method utilizes run buffer containing 0.30% hydroxyethyl cellulose as the sieving polymer in a 50 μm internal diameter (ID) column coated with a 0.1 μm DB‐17 film. Laserinduced fluorescence (LIF) with YO‐PRO‐1 intercalating dye is used for detec‐tion. Internal standards of 300 and 1000 bp bracket the DlS80 region in the electropherogram. Two typing procedures were evaluated: matching of the normalized migration times of sample alleles to ladder alleles; and software alignment of sample and ladder electropherograms using the internal standard peaks as references. Using the first procedure, 91.5% of the D1S80 genotypes were unambiguous, while 100% were unambiguous using the second procedure. Seventy‐nine routine samples were analyzed in a side‐by‐side comparison of the CE and slab gel methods, with complete agreement of the results. Twenty‐two samples were selected from a large database previously analyzed by the slab gel method to demonstrate that all alleles from 14 through 41 were typed correctly, including samples containing adjacent alleles. Additionally, 43 samples containing alleles greater than the 41 repeat number were typed correctly.
Nonradioactive sequence‐tagged microsatellite site analyses: A method transferable to the tropics
Authors: Lagoda Pierre J. L. , Dambier Dominique , Grapin Agnès , Baurens Franc‐Christophe , Lanaud Claire , Noyer Jean‐Louis
Published: May 2005

Utilization of existing isozyme analysis facilities to detect sequence‐tagged microsatellite site (STMS) polymorphism or any simple sequence repeat (SSR) variation is described. Different parameters concerning the difficulties in transferring molecular techniques to less sophisticated laboratory infrastructures (i. e. tropical outstations) are discussed (e.g. reproducibility, efficacy, precision). Nonradioactive STMS analysis is bound to foster collaborative research between “biodiversity” and “biotechnology” centers.
Expression and subcellular distribution of filamin isotypes in endothelial cells and pericytes
Authors: Shojaee Negin , Patton Wayne F. , Chung‐Welch Nancy , Su Qin , Hechtman Herbert B. , Shepro David
Published: May 2005

Two principal forms of the actin binding protein, filamin, are expressed in mammalian cells: nonmuscle and muscle isotypes (FLN‐1 and FLN‐2). A protein that copurifies with an α‐naphthyl acetate hydrolyzing esterase from human omentum microvessel endothelial cells (EC) is isolated by nondenaturing electrophoresis, sodium dodecyl sulfate (SDS)‐polyacrylamide gel electrophoresis and electroblotting. The purified protein is subjected to in situ trypsin cleavage, reversed‐phase high performance liquid chromatography (HPLC) and automated Edman degradation. Six peptide fragments from the protein are identified to have 60—66% identity with nonmuscle filamin (ABP‐280). Two of these peptides are 100% identical to a previously sequenced human muscle filamin fragment. Polyclonal antibody is produced using a 16‐residue synthetic peptide corresponding to a structural β‐sheet region of muscle filamin. Compared with a variety of vascular cells evaluated, retinal pericytes express an abundance of both muscle and non‐muscle filamin isotypes. Pericytes contain at least 10 times more muscle filamin than human umbilical vein EC and at least three times the amount expressed in human omentum micro‐vessel and bovine pulmonary artery EC. Differential detergent fractionation indicates that both filamin isotypes are primarily localized in the cytosol and membrane/organelle fractions of pericytes. Another actin crosslinking protein, α‐actinin, is primarily found in the cytosol and cytoskeletal fractions. The dynamic regulation of actin microfilament organization in pericytes may be controlled in part by the two filamin isotypes, which in turn may contribute to pericyte contractility.
Analysis of differential protein expression in normal and neoplastic human breast epithelial cell lines
Authors: Williams Katherine , Chubb Cynthia , Huberman Eliezer , Giometti Carol S.
Published: May 2005

High‐resolution two‐dimensional gel electrophoresis (2‐DE) and database analysis was used to establish protein expression patterns for cultured normal human mammary epithelial cells and thirteen breast cancer cell lines. The Human Breast Epithelial Cell database contains the 2‐DE protein patterns, including relative protein abundances, for each cell line, plus a composite pattern that contains all the common and specifically expressed proteins from all the cell lines. Significant differences in protein expression, both qualitative and quantitative, were observed not only between normal cells and tumor cells, but also among the tumor cell lines. Eight percent (56/727) of the consistently detected proteins were found in significantly (P < 0.001) variable levels among the cell lines. Eight proteins present in normal cultured breast epithelial cells were not detected in any of the tumor cell lines. We identified a subset of the differentially expressed proteins using a combination of immunostaining, protein sequencing, comigration, and subcellular fractionation. These identified proteins include the intermediate filament components vimentin and cytokeratins. The cell lines can be classified into four distinct groups based on their intermediate filament protein profile. We also identified heat shock proteins; hsp27 and hsp60 varied in abundance and in some cases in the relative phosphorylation levels among the cell lines. Many of the differentially expressed proteins we identified have roles in cellular proliferation and differentiation, including annexin V, elongation initiation factor 5A, Rho GDP dissociation inhibitor, and prohibitin. We identified inosine‐5‐monophosphate dehydrogenase in each of the cell lines, and found the levels of this enzyme in the tumor cell lines elevated 2‐ to 20‐fold relative to the levels in normal cells. These results expand the human breast epithelial cell protein database (http://www.anl.gov/CMB/PMG) which is being built to assist researchers with the identification of abnormal patterns of expression and pathways associated with malignancy.
TMIG‐2DPAGE: A new concept of two‐dimensional gel protein database for research on aging
Authors: Toda Tosifusa , Kaji Kazuhiko , Kimura Narimichi
Published: May 2005

Cellular proteins of a normal human diploid fibroblast line (TIG‐3) at various stages of replicative aging were resolved by horizontal isoelectric focusing on an immobilized pH gradient, followed by vertical sodium dodecyl sulfate (SDS)‐polyacrylamide gel electrophoresis (PAGE). Spot proteins were visualized by silver staining and quantitated by image processing. All corresponding spots were matched among two‐dimensional gel images, and variation profiles in relative abundance of individual proteins during in vitro aging were classified into five categories, i.e., (i) increase, (ii) decrease, (iii) increase followed by decrease, (iv) decrease followed by increase, and (v) irregular or nonsignificant variation. The new concept of the Tokyo Metropolitan Institute of Gerontology two‐dimensional gel protein database (TMIG‐2DPAGE) was prepared from the above data to support research on cellular aging. The database was put on our World Wide Web home page at the URL of http://www.tmig.or.jp/2D/ to allow free access through the Internet. The individual protein data entries were linked to the standard spot protein map of the two‐dimensional gel image in order to be accessible by clicking the mouse on it.
The effects of a mixture of charged and neutral additives on analyte migration behavior in capillary electrophoresis
Authors: Kranack Andrea R. , Bowser Michael T. , Britz‐Mckibbin Philip , Chen David D. Y.
Published: May 2005

Multicomponent additives, such as derivatized cyclodextrins with various degrees of substitution, can be considered single‐component additives as long as the fraction of each component remains constant. In this paper, equations are derived describing the effect of such additives on the migration behavior of analytes. These equations are used in the study of capillary electrophoresis (CE) systems with differentially charged cyclodextrins as additives. For weakly acidic analytes, the binding with highly negatively charged sulfobutyl ether β‐cyclodextrin (SBE‐β‐CD) increases their negative electrophoretic mobility, while the binding with neutral hydroxypropyl‐β‐cyclodextrin (HP‐β‐CD) decreases their negative mobility. By obtaining the equilibrium constants and mobilities for each additive with each analyte (in this case, phenol, 2‐naphthol and 1‐naphthol), the migration behavior of these analytes in CE systems is quantitatively predicted at various concentrations of mixtures of the two additives. The properties of the contour lines in the binding isotherm surfaces of such CE systems are discussed.
Base‐specific separation of oligodeoxynucleotides by capillary affinity gel electrophoresis
Authors: Baba Yoshinobu , Sawa Tomohiro , Kishida Akio , Akashi Mitsuru
Published: May 2005

Capillary affinity gel electrophoresis was applied to sequence‐specific and base composition‐specific recognition of oligodeoxynucleotides, utilizing the formation of heteroduplexes between a nucleic acid analogue immobilized into the capillary gel and soluble oligodeoxynucleotides with different sequences. Capillary affinity gel electrophoresis using capillaries filled with a conjugated gel of polyacrylamide and a synthetic nucleic acid analogue [poly(9‐vinyladenine)] was effective for the selective separation of hexathymidylic acid from a mixture of four homopolymers of A6, C6, G6, and T6 and for the complete resolution of five heteropolymers of hexadeoxynucleotides (TAAAAA, TTAAAA, TTTAAA, TTTTAA, TTTTTA). We also demonstrated that capillary affinity gel electrophoresis was useful for the selective and the sensitive sequence‐specific recognition of sequence isomers of DNA (TTTTAA, TTTTAAT, TTTATA, and TTTTAA).
High‐throughput robotic system for sequencing of microbial genomes
Authors: Hilbert Helmut , Schäfer Andreas , Collasius Michael , Düsterhöft Andreas
Published: May 2005

A high‐throughput robotic workstation system was used for double‐stranded plasmid DNA template preparation and sequencing reaction setup to streamline the sequencing process in genome projects. All 96‐well miniprep kits that were tested provided high quality plasmid DNA suitable for fluorescent DNA sequencing. After quantitation in a 96‐well UV spectrophotometer, the plasmid DNA was used as template to automatically set up sequencing reactions. The setup was controlled by spread sheets that were imported into the robotic system. We utilized this integrated system to prepare all necessary shotgun templates for our contributions to a number of large‐scale genome projects as well as a full‐length cDNA sequencing project.