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Protein Mixtures (protein + mixture)
Kinds of Protein Mixtures Selected AbstractsCo-electroosmotic capillary electrophoresis of basic proteins with 1-alkyl-3-methylimidazolium tetrafluoroborate ionic liquids as non-covalent coating agents of the fused-silica capillary and additives of the electrolyte solutionELECTROPHORESIS, Issue 11 2009Danilo Corradini Abstract The paper reports the results of a study carried out to evaluate the use of three 1-alkyl-3-methylimidazolium-based ionic liquids as non-covalent coating agents for bare fused-silica capillaries and additives of the electrolyte solutions (BGE) for CE of basic proteins in the co-EOF separation mode. The three ionic liquids are differentiated from each other by the length of the alkyl group on the imidazolium cation, consisting of either an ethyl, butyl or octyl substituent, whereas tetrafluoroborate is the common anionic component of the ionic liquids. Coating the capillary with the ionic liquid resulted in improved peak shape and protein separation, while the EOF was maintained cathodic. This indicates that each ionic liquid is effective at masking the protein interaction sites on the inner surface of the capillary, also when its adsorption onto the capillary wall has not completely neutralized all the negative charges arising from the ionization of the silanol groups and the ionic liquid is not incorporated into the BGE employed for separation. Using the coated capillaries with BGE containing the ionic liquid employed for the coating, at concentration low enough to maintaining the EOF cathodic, both peak shape and protein separation varied to different extents, based on the particular ionic liquid used and its concentration. Fast and efficient separation of the model basic protein mixture in co-electroosmotic CE is obtained with the 1-butyl-3-methylimidazolium tetrafluoroborate coated capillary and 100,mM acetate buffer (pH 4.0) containing 4.4,mM 1-butyl-3-methylimidazolium tetrafluoroborate as the BGE. [source] Microaffinity purification of proteins based on photolytic elution: Toward an efficient microbead affinity chromatography on a chipELECTROPHORESIS, Issue 3 2005Woo-Jae Chung Abstract A bead affinity chromatography system, which was based on the photolytic elution method, was integrated into a glass-silicon microchip to purify specific target proteins. CutiCore® beads, which were coupled with a photo-cleavable ligand, such as biotin and an RNA aptamer, were introduced into a filter chamber in the microchip. The protein mixture containing target protein labeled with fluorescein isothiocyanate (FITC) was then passed through the packed affinity beads in the microchamber by pressure-driven flow. During the process, the adsorbed protein on the bead was monitored by fluorescence. The concentrated target protein on the affinity bead was released by simple irradiation with UV light at a wavelength of 360 nm, and subsequently eluted with the phosphate buffer flow. The eluted target protein was quantitatively detected via the fluorescence intensity measurements at the downstream of the capillary connected to the outlet of the microchip. The microaffinity purification allowed for a successful method for the identification of specific target proteins from a protein mixture. In addition, the feasibility of this system for use as a diagnosis chip was demonstrated. [source] Exploration of ionic modification in dual-layer hollow fiber membranes for long-term high-performance protein separationAICHE JOURNAL, Issue 2 2009Yi Li Abstract Two types of ionic modification approaches (i.e., sulfonation and triethylamination) were applied with the aid of dual-layer hollow fiber technology in this work to fine tune the pore size and pore size distribution, introduce the electrostatic interaction, and reduce membrane fouling for long-term high-performance protein separation. A binary protein mixture comprising bovine serum albumin (BSA) and hemoglobin (Hb) was separated in this work. The sulfonated fiber exhibits an improved BSA/Hb separation factor at pH = 6.8 compared with as-spun fibers but at the expense of BSA sieving coefficient. On the other hand, the triethylaminated fiber reveals the best and most durable separation performance at pH = 4.8. Its BSA/Hb separation factor is maintained above 80 for 4 days and maximum BSA sieving coefficient reaches 33%. Therefore, this study documents that an intelligent combination of both size-exclusion and electrostatic interaction can synergistically enhance protein separation performance in both purity and concentration. © 2008 American Institute of Chemical Engineers AIChE J, 2009 [source] Surface-enhanced Raman sensors: early history and the development of sensors for quantitative biowarfare agent and glucose detectionJOURNAL OF RAMAN SPECTROSCOPY, Issue 6-7 2005Christy L. Haynes Abstract Surface-enhanced Raman spectroscopy (SERS) is a powerful technique for the sensitive and selective detection of low-concentration analytes. This paper includes a discussion of the early history of SERS, the concepts that must be appreciated to optimize the intensity of SERS and the development of SERS-based sensors. In order to achieve the lowest limits of detection, both the relationship between surface nanostructure and laser excitation wavelength, as well as the analyte/surface binding chemistry, must be carefully optimized. This work exploits the highly tunable nature of nanoparticle optical properties to establish the first set of optimization conditions. The SERS enhancement factor, EFSERS, is optimized when the energy of the localized surface plasmon resonance (LSPR) lies between the energy of the excitation wavelength and the energy of the vibrational band of interest. With the narrow LSPRs used in this work, it is straightforward to achieve EFSERS , 108. These optimization conditions were exploited to develop SERS-based sensors for two important target molecules: a Bacillus anthracis biomarker and glucose in a serum protein mixture. Using these optimized film-over-nanosphere surfaces, an inexpensive, portable Raman spectrometer was used successfully to detect the infectious dose of Bacillus subtilis spores with only a 5-s data collection. The biomarker used to detect the Bacillus subtilis spores binds irreversibly to SERS substrates, whereas other important biomolecules, such as glucose, do not have any measurable binding affinity to a bare silver surface. To overcome this difficulty, a biocompatible partition layer was self-assembled on the SERS substrate before exposure to the analyte solution. Using the partition layer approach to concentrate glucose near the SERS-active substrate, physiological glucose concentrations can be detected even in the presence of interfering serum proteins. Copyright © 2005 John Wiley & Sons, Ltd. [source] Matrigel: A complex protein mixture required for optimal growth of cell culturePROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 9 2010Chris S. Hughes Abstract Numerous cell types require a surface for attachment to grow and proliferate. Certain cells, particularly primary and stem cells, necessitate the use of specialized growth matrices along with specific culture media conditions to maintain the cells in an undifferentiated state. A gelatinous protein mixture derived from mouse tumor cells and commercialized as Matrigel is commonly used as a basement membrane matrix for stem cells because it retains the stem cells in an undifferentiated state. However, Matrigel is not a well-defined matrix, and therefore can produce a source of variability in experimental results. In this study, we present an in-depth proteomic analysis of Matrigel using a dynamic iterative exclusion method coupled with fractionation protocols that involve ammonium sulfate precipitation, size exclusion chromatography, and one-dimensional SDS-PAGE. The ability to identify the low mass and abundance components of Matrigel illustrates the utility of this method for the analysis of the extracellular matrix, as well as the complexity of the matrix itself. [source] The detection, correlation, and comparison of peptide precursor and product ions from data independent LC-MS with data dependant LC-MS/MSPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 6 2009Scott J. Geromanos Abstract The detection, correlation, and comparison of peptide and product ions from a data independent LC-MS acquisition strategy with data dependent LC-MS/MS is described. The data independent mode of acquisition differs from an LC-MS/MS data acquisition since no ion transmission window is applied with the first mass analyzer prior to collision induced disassociation. Alternating the energy applied to the collision cell, between low and elevated energy, on a scan-to-scan basis, provides accurate mass precursor and associated product ion spectra from every ion above the LOD of the mass spectrometer. The method therefore provides a near 100% duty cycle, with an inherent increase in signal intensity due to the fact that both precursor and product ion data are collected on all isotopes of every charge-state across the entire chromatographic peak width. The correlation of product to precursor ions, after deconvolution, is achieved by using reconstructed retention time apices and chromatographic peak shapes. Presented are the results from the comparison of a simple four protein mixture, in the presence and absence of an enzymatically digested protein extract from Escherichia coli. The samples were run in triplicate by both data dependant analysis (DDA) LC-MS/MS and data-independent, alternate scanning LC-MS. The detection and identification of precursor and product ions from the combined DDA search results of the four protein mixture were used for comparison to all other data. Each individual set of data-independent LC-MS data provides a more comprehensive set of detected ions than the combined peptide identifications from the DDA LC-MS/MS experiments. In the presence of the complex E. coli background, over 90% of the monoisotopic masses from the combined LC-MS/MS identifications were detected at the appropriate retention time. Moreover, the fragmentation pattern and number of associated elevated energy product ions in each replicate experiment was found to be very similar to the DDA identifications. In the case of the corresponding individual DDA LC-MS/MS experiment, 43% of the possible detectable peptides of interest were identified. The presented data illustrates that the time-aligned data from data-independent alternate scanning LC-MS experiments is highly comparable to the data obtained via DDA. The obtained information can therefore be effectively and correctly deconvolved to correlate product ions with parent precursor ions. The ability to generate precursor-product ion tables from this information and subsequently identify the correct parent precursor peptide will be illustrated in a companion manuscript. [source] Peak quantification in surface-enhanced laser desorption/ionization by using mixture modelsPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 19 2006Martijn Dijkstra Abstract Surface-enhanced laser desorption/ionization (SELDI) time of flight (TOF) is a mass spectrometry technology for measuring the composition of a sampled protein mixture. A mass spectrum contains peaks corresponding to proteins in the sample. The peak areas are proportional to the measured concentrations of the corresponding proteins. Quantifying peak areas is difficult for existing methods because peak shapes are not constant across a spectrum and because peaks often overlap. We present a new method for quantifying peak areas. Our method decomposes a spectrum into peaks and a baseline using so-called statistical finite mixture models. We illustrate our method in detail on 8 samples from culture media of adipose tissue and globally on 64 samples from serum to compare our method to the standard Ciphergen method. Both methods give similar estimates for singleton peaks, but not for overlapping peaks. The Ciphergen method overestimates the heights of such peaks while our method still gives appropriate estimates. Peak quantification is an important step in pre-processing SELDI-TOF data and improvements therein will pay off in the later biomarker discovery phase. [source] Selective analysis of phosphopeptides within a protein mixture by chemical modification, reversible biotinylation and mass spectrometryRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 16 2001Maceij Adamczyk A new method combining chemical modification and affinity purification is described for the characterization of serine and threonine phosphopeptides in proteins. The method is based on the conversion of phosphoserine and phosphothreonine residues to S -(2-mercaptoethyl)cysteinyl or ,-methyl- S -(2-mercaptoethyl)cysteinyl residues by ,-elimination/1,2-ethanedithiol addition, followed by reversible biotinylation of the modified proteins. After trypsin digestion, the biotinylated peptides were affinity-isolated and enriched, and subsequently subjected to structural characterization by liquid chromatography/tandem mass spectrometry (LC/MS/MS). Database searching allowed for automated identification of modified residues that were originally phosphorylated. The applicability of the method is demonstrated by the identification of all known phosphorylation sites in a mixture of ,-casein, ,-casein, and ovalbumin. The technique has potential for adaptations to proteome-wide analysis of protein phosphorylation. Copyright © 2001 John Wiley & Sons, Ltd. [source] Replacement of fish meal with a mixture of different plant protein sources in juvenile Nile tilapia, Oreochromis niloticus (L.) dietsAQUACULTURE RESEARCH, Issue 13 2003Deyab M S D El-Saidy Abstract A plant protein mixture (PPM) was tested to replace fish meal (FM) in diets for juvenile Nile tilapia, Oreochromis niloticus. Fish averaging (±SD) 3.7±0.14 g were divided into 15 groups. Three groups were fed each of five isonitrogenous (33.6%) and isocaloric (4.7 kcal g,1) diets replacing 0%, 25%, 50%, 75% and 100% of the FM protein with similar percentages of PPM (PPM0, PPM25, PPM50, PPM75 or PPM100 respectively). The PPM consisted of 25% soybean meal, 25% cottonseed meal, 25% sunflower meal and 25% linseed meal, and 0.5% of both methionine and lysine were added to each diet except for the control. After 16 weeks of feeding, the fish fed diets PPM75 and PPM100 exhibited growth performance not differing significantly from the fish fed control diet. PPM substitution of up to 75% of the FM protein did not result in differences in the apparent protein digestibility compared with the control, whereas in the PPM100 group digestibility was significantly lower than in the other groups, except for fish fed the PPM75 diet. The incorporation of PPM in diets did not significantly affect whole-body dry matter, protein, fat or energy compared with the control. The cost,benefit analyses of the test diets indicated that the PPM diets were economically superior to FM. The protein from PPM can completely replace the FM protein in the diets for Nile tilapia, based on the results of this study. [source] Simple Purification of Immunoglobulins from Whey Proteins ConcentrateBIOTECHNOLOGY PROGRESS, Issue 2 2006Benevides C. C. Pessela We have developed a new protocol with only two steps for purification of immunoglobulins (Ig) from a protein concentrate of whey. Following this protocol, we have an 80% recovery of immunoglobulins, fairly pure. The purification was achieved by eliminating the BSA, via a strong adsorption on DEAE-agarose. Full desoprtion of the other serum proteins could be achieved without contamination with BSA. Thus, a protein solution containing only Ig and very small proteins (e.g., ,-lactoglobulins and ,-lactalbumin) was obtained. Offering this protein mixture to a lowly activated aminated support, only Ig adsorbed on the support. It has been shown that BSA is able to interact with other proteins (including Ig and lactalbumins). This ability to form complexes with other proteins prevented the success of the direct adsorption of Ig on this mildly activated support, even although Ig should be the largest protein presented in dairy whey. [source] Optimal Synthesis of Protein Purification ProcessesBIOTECHNOLOGY PROGRESS, Issue 4 2001Elsa Vásquez-Alvarez There has been an increasing interest in the development of systematic methods for the synthesis of purification steps for biotechnological products, which are often the most difficult and costly stages in a biochemical process. Chromatographic processes are extensively used in the purification of multicomponent biotechnological systems. One of the main challenges in the synthesis of purification processes is the appropriate selection and sequencing of chromatographic steps that are capable of producing the desired product at an acceptable cost and quality. This paper describes mathematical models and solution strategies based on mixed integer linear programming (MILP) for the synthesis of multistep purification processes. First, an optimization model is proposed that uses physicochemical data on a protein mixture, which contains the desired product, to select a sequence of operations with the minimum number of steps from a set of candidate chromatographic techniques that must achieve a specified purity level. Since several sequences that have the minimum number of steps may satisfy the purity level, it is possible to obtain the one that maximizes final purity. Then, a second model that may use the total number of steps obtained in the first model generates a solution with the maximum purity of the product. Whenever the sequence does not affect the final purity or more generally does not impact the objective function, alternative models that are of smaller size are developed for the optimal selection of steps. The models are tested in several examples, containing up to 13 contaminants and a set of 22 candidate high-resolution steps, generating sequences of six operations, and are compared to the current synthesis approaches. [source] Synthesis and Evaluation of New Thiodigalactoside-Based Chemical Probes to Label Galectin-3CHEMBIOCHEM, Issue 10 2009Monique van Scherpenzeel Abstract Light up galectin: Photoprobes based on thiodigalactoside were prepared for galectin-3, a lectin linked to cancer. The probes contained either benzophenone or acetophenone moieties as the photolabel for covalent attachment to the protein. One particular probe labeled galectin-3 selectively, even in the presence of cell lysate. New chemical probes were synthesized to label galectin-3. They are based on the high affinity thiodigalactoside ligand. The probes were synthesized with benzophenone or acetophenone moieties as the photolabel for covalent attachment to the protein. Besides labeling the protein, these aromatic photolabels also greatly enhance the affinity of the probes towards galectin-3, due to the interaction of the photolabel with two arginine guanidinium groups of the protein. The linkage between the sugar and the photolabel was varied as an ester, an amide, and a triazole. For the amide and triazole derivatives, a versatile synthetic route towards a symmetrical 3-azido-3-deoxy-thiodigalactoside was developed. The new probes were evaluated for their binding affinity of human galectin-3. They were subsequently tested for their labeling efficiency, as well as specificity in the presence of a protein mixture and a human cancer cell lysate. [source] Sedimentation as a tool for crystallization from protein mixturesCRYSTAL RESEARCH AND TECHNOLOGY, Issue 11 2006I. Dimitrov Abstract Crystals from apoferritin which is an iron-free form of protein ferritin were obtained from protein mixtures lysozyme/apoferritin using sedimentation under high gravity. Solution containing apoferritin at concentration as high as 5mg/ml in the presence of 25mg/ml lysozyme and overlaid on 5%(w/v) CdSO4 in 0,2M/L NaAC, pH=5 still favors apoferritin crystal formation under normal gravity conditions, but at apoferritin concentrations <0,5mg/ml (,1,14µM/L) in 25mg/ml (,1,71mM/L) lysozyme only the sedimentation in a centrifuge appears to be useful for separating the apoferritin molecules from the mixture followed by apoferritin crystallization in the same system. The very high molecule number ratio (,1:103) of two proteins is used to stress on the observed effect. (© 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim) [source] CE-TOF MS analysis of complex protein hydrolyzates from genetically modified soybeans , A tool for foodomicsELECTROPHORESIS, Issue 7 2010Carolina Simó Abstract A CE-TOF MS proteomic approach was applied for the analysis of hydrolyzates from complex soybean protein mixtures. After CE-TOF MS method development, the new approach provided the simultaneous analysis of more than 150 peptides from the soybean protein fraction soluble in ACN-water (80/20,v/v). The method is fast (about 30,min of analysis per sample) and is characterized by a relatively low running cost. The approach was used to study the substantial equivalence between a genetically modified variety of soybean compared with its traditional counterpart. No significant differences were found between the two studied soybeans based on the protein fraction studied. The capacity of the CE-TOF MS method to analyze complex mixtures of peptides in short times opens interesting possibilities in the growing Foodomics area. [source] Parallel isoelectric focusing IIELECTROPHORESIS, Issue 21-22 2004Gleb V. Zilberstein Abstract A miniature electrophoretic device is developed on the basis of a new isoelectric focusing (IEF) method, namely parallel isoelectric focusing. We report here the theory and the results of operation of a new parallel isoelectric device (PID). The main advantages and limitations of the method are discussed for miniaturization purposes. It is shown that the method guarantees the fast and complete separation of any complex protein mixtures under acceptable conditions, such as voltage source, temperature, size of the device, and separation process duration. It is shown that the main problem of PID miniaturization is the buffer design, and the relation between Immobiline buffer capacity and solution buffer capacity. The main experimental limitation of PID resolution is protein sensitivity to pH changes. [source] Parental relationships among three grape varieties studied by MALDI of grape seed protein profiles,JOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 9 2010Antonella Bertazzo Abstract Two Raboso cultivars, i.e. Raboso Veronese and Raboso Piave, are two black Vitis vinifera varieties. A genetic study suggested that Raboso Veronese is the progeny of a spontaneous cross between Raboso Piave and Marzemina Bianca cultivars. Parental relationships are usually investigated by genetic studies, which are effective to establish genetic links among different cultivars. Considering that proteome is the genome expression, in this article we evaluated the power of seed protein profiles obtained by matrix-assisted laser desorption/ionization (MALDI)/MS for parentage investigation. The three cultivars lead to very similar spectra with differences in the relative intensity of the most abundant species and the presence of very weak specific ions. In order to evaluate the analytical significance of these aspects, the variability due to instrumental factors and due to different harvesting areas and years of the same cultivars have been considered and measured by the calculation of discrepancy factor values. On one hand, the results obtained can be considered a valid confirmation of the genomic findings, whereas on the other hand, the results provide evidence for the ability of MALDI/MS to individuate minor differences in protein profiles of complex protein mixtures. Copyright © 2010 John Wiley & Sons, Ltd. [source] Efficient MILP formulations for the simultaneous optimal peptide tag design and downstream processing synthesisAICHE JOURNAL, Issue 9 2009João M. Natali Abstract Novel and efficient linear formulations are developed for the problem of simultaneously performing an optimal synthesis of chromatographic protein purification processes, and the concomitant selection of peptide purification tags, that result in a maximal process improvement. To this end, two formulations are developed for the solution of this problem: (1) a model that minimizes both the number of chromatographic steps in the final purification process flow sheet and the composition of the tag, by use of weighted objectives, while satisfying minimal purity requirements for the final product; and (2) a model that attempts to find the maximal attainable purity under constraints on the maximum number of separation techniques and tag size. Both models are linearized using a previously developed strategy for obtaining optimal piecewise linear approximations of nonlinear functions. Proposed are models to two case studies based on protein mixtures with different numbers of proteins. Results show that the models are capable of solving to optimality all the implemented cases with computational time requirements of under 1 s, on average. The results obtained are further compared with previous nonlinear and linear models attempting to solve the same problem, and, thus, show that the approach represents significant gains in robustness and efficiency. © 2009 American Institute of Chemical Engineers AIChE J, 2009 [source] Protein adsorption drastically reduces surface-enhanced Raman signal of dye moleculesJOURNAL OF RAMAN SPECTROSCOPY, Issue 9 2010Dongmao Zhang Abstract There is an increasing interest in developing surface enhancement Raman spectroscopy methods for intracellular biomolecule and for in vitro protein detection that involve dye or protein,dye conjugates. In this work, we have demonstrated that protein adsorption on silver nanoparticle (AgNP) can significantly attenuate the surface-enhanced Raman spectroscopy (SERS) signal of dye molecules in both protein/dye mixtures and protein/dye conjugates. SERS spectra of 12 protein/dye mixtures were acquired using 4 proteins [bovine serum albumin (BSA), lysozyme, trypsin, and concanavalin A] and three dyes [Rhodamine 6G, adenine, and fluorescein isothiocyanate (FITC)]. Besides the protein/dye mixtures, spectra were also obtained for the free dyes and four FITC-conjugated proteins. While no SERS signal was observed in protein/FITC mixtures or conjugates, a significantly reduced SERS intensity (up to 3 orders of magnitude) was observed for both R6G and adenine in their respective protein mixtures. Quantitative estimation of the number of dye molecules absorbed onto AgNP implied that the degree of R6G SERS signal reduction in the R6G/BSA sample is 2 to 3 orders of magnitude higher than what could be accounted for by the difference in the amount of the absorbed dyes. This finding has significant implications for both intracellular SERS analyses and in vitro protein detection using SERS tagging strategies that rely on Raman dyes as reporter molecules. Copyright © 2009 John Wiley & Sons, Ltd. [source] Nutritive value of chicken and potato mixtures for infant and preschool children feedingJOURNAL OF THE SCIENCE OF FOOD AND AGRICULTURE, Issue 12 2003Angela Sotelo Abstract Two chicken/potato protein mixtures (50:50 and 60:40) were prepared for use in formulas of high nutritive value and low cost for the diet of undernourished children and those with lactose intolerance. The proximate analysis and amino acid content of the raw materials and mixtures were determined and the chemical score (CS) was calculated. The proximate analysis and amino acid content of a commercial soybean formula were used as reference. The protein quality of the mixtures was evaluated by protein efficiency ratio (PER) and digestibility measurements. The protein content of cooked chicken and potato on a dry basis was 889 and 70 g kg,1 respectively and the carbohydrate content of potato was 762 g kg,1. Tryptophan was the limiting amino acid in chicken for infants according to the 1985 FAO pattern (CS = 76), but not for preschool children. Valine was limiting in potato for both infants and preschool children (CS = 56 and 88 respectively). Tryptophan was limiting in both 50:50 and 60:40 mixtures for infants; also the PER was higher in the 60:40 mixture and not significantly different from the control (casein), but both were different from the 50:50 mixture. Since both chicken and potato are available even for low-income people, a formula prepared in the 60:40 ratio is of potential benefit for infants or preschool children who have lactose intolerance, mainly in developing countries. Copyright © 2003 Society of Chemical Industry [source] Quantitative specificity-based display library screening identifies determinants of antibody-epitope binding specificity,PROTEIN SCIENCE, Issue 9 2009Sejal S. Hall Abstract Despite the critical importance of molecular specificity in bimolecular systems, in vitro display technologies have been applied extensively for affinity maturation of peptides and antibodies without explicitly measuring the specificity of the desired interaction. We devised a general strategy to measure, screen, and evolve specificity of protein ligand interactions analogous to widely used affinity maturation strategies. The specificity of binding to target and nontarget antibodies labeled with spectrally distinct fluorophores was measured simultaneously in protein mixtures via multiparameter flow cytometry, thereby enabling screening for high target antibody specificity. Isolated antibody specific ligands exhibited varying specificity, revealing critical amino acid determinants for target recognition and nontarget avoidance in complex mixtures. Molecular specificity in the mixture was further enhanced by quantitative directed evolution, yielding a family of epitopes exhibiting improved specificities equivalent, or superior to, the native peptide antigen to which the antibody was raised. Specificity screening simultaneously favored affinity, yielding ligands with three-fold improved affinity relative to the parent epitope. Quantitative specificity screening will be useful to screen, evolve, and characterize the specificity of protein and peptide interactions for molecular recognition applications. [source] Equalizer technology , Equal rights for disparate beadsPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 11 2010Eva-Maria Keidel Abstract One major limitation in proteomics is the detection and analysis of low-abundant proteins, i.e. in plasma. Several years ago, a technique to selectively enrich the relative concentration of low-abundant proteins was introduced by Boschetti and co-workers. It is based on a specific and saturable interaction of proteins to a high diversity of binding sites, realized by a hexapeptide library coupled to beads. This technology was commercialized as Equalizer beads or ProteoMiner. However, during application of ProteoMiner beads to plasma samples unexpected results questioned the proposed mode of action. Therefore, ProteoMiner beads were compared with chromatographic beads exhibiting completely different surface chemistry. Sepabeads FP-OD400 octadecyl, FP-DA400 diethylamine, FP-BU400 butyl, FP-HG400 hydroxyl and EXE056 epoxy were used. The results show that ProteoMiner or the different Sepabeads behave surprisingly similarly in the separation of complex protein mixtures. ProteoMiner beads interact with protein mixtures according to a general hydrophobic binding mechanism, where diversity in surface ligands plays only a negligible role. [source] Phosphoproteome analysis of the human Chang liver cells using SCX and a complementary mass spectrometric strategyPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 10 2008Shaohui Sui Abstract The liver is the largest organ in the body, with many complex, essential functions, such as metabolism, deintoxication, and secretion, often regulated via post-translational modifications, especially phosphorylation. Thus, the detection of phosphoproteins and phosphorylation sites is important to comprehensively explore human liver biological function. The human Chang liver cell line is among the first derived from non-malignant tissue, and its phosphoproteome profile has never been globally analyzed. To develop the complete phosphoproteome and probe the roles of protein phosphorylation in normal human liver, we adopted a shotgun strategy based on strong cation exchange chromatograph, titanium dioxide and LC-MS/MS to isolate and identify phosphorylated proteins. Two types of MS approach, Q-TOF and IT, were used and compared to identify phosphosites from complex protein mixtures of these cells. A total of 1035 phosphorylation sites and 686 phosphorylated peptides were identified from 607 phosphoproteins. A search using the public database of PhosphoSite showed that approximately 344 phosphoproteins and 760 phosphorylation sites appeared to be novel. In addition, N-terminal phosphorylated peptides were a greater fraction of all identified phosphopeptides. With GOfact analysis, we found that most of the identified phosphoproteins are involved in regulating metabolism, consistent with the liver's role as a key metabolic organ. [source] Immunoaffinity separation of plasma proteins by IgY microbeads: Meeting the needs of proteomic sample preparation and analysisPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 13 2005Lei Huang Abstract Separation of complex protein mixtures that have a wide dynamic range of concentration, such as plasma or serum, is a challenge for proteomic analysis. Sample preparation to remove high-abundant proteins is essential for proteomics analysis. Immunoglobulin yolk (IgY) antibodies have unique and advantageous features that enable specific protein removal to aid in the detection of low-abundant proteins and biomarker discovery. This report describes the efficiency and effectiveness of IgY microbeads in separating 12 abundant proteins from plasma with an immunoaffinity spin column or LC column. The protein separation and sample preparation process was monitored via SDS-PAGE, 2-DE, LC-MS/MS, or clinical protein assays. The data demonstrate the high specificity of the protein separation, with removal of 95,99.5% of the abundant proteins. IgY microbeads against human proteins can also selectively remove orthologous proteins of other mammals such as mouse, rat, etc. Besides the specificity and reproducibility of the IgY microbeads, the report discusses the factors that may cause potential variations in protein separation such as protein,protein interactions (known as "Interactome"), binding and washing conditions of immunoaffinity reagents, etc. A novel concept of Seppromics is introduced to address methodologies and science of protein separation in a context of proteomics. [source] Quantitative profiling of LNCaP prostate cancer cells using isotope-coded affinity tags and mass spectrometryPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 4 2004Katie L. Meehan Abstract Androgens are involved in the pathogenesis of diseases including adenocarcinoma of the prostate. These hormones are important for growth, maintenance, and integrity of structure and function of the prostate. Androgen-deprivation is currently the only effective systemic therapy for prostate cancer but the effects of androgens on the proteome are still poorly described. Here we quantitatively profile changes in the proteome of LNCaP human prostate cancer cells in response to androgen using the newly developed isotope-coded affinity tag (ICAT) labeling and two-dimensional liquid chromatography-tandem mass spectroscopy (2-D LC-MS/MS). ICAT enables the concurrent identification and comparative quantitative analysis of proteins present in various biological samples including human cell and tissue extracts. Quantification and identification of 139 proteins in complex protein mixtures obtained from androgen-stimulated and unstimulated LNCaP cells were achieved. Changes in levels of 77 proteins in response to androgens were detected. Some of these proteins have been previously reported to be regulated by androgens and include spermine synthase, fatty acid synthase and calreticulin precursor. A large number of proteins that have not been previously reported to be expressed in prostate cells were also quantitatively identified. Examples of these include members of the dual specificity protein phosphatase subfamily, "similar" to hypothetical protein DKFZp434B0328.1, "similar" to 14-3-3 protein zeta and "similar" to hypothetical protein 458, components of the actin cytoskeleton and a range of unknown/uncharacterized proteins. This catalogue of proteins provides an overview of the proteome of prostate cancer cells and the global changes that occur in response to androgens. [source] Improving peptide identification using an empirical peptide retention time databaseRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 1 2009Wei Sun Peptide retention time (RT) is independent of tandem mass spectrometry (MS/MS) parameters and can be combined with MS/MS information to enhance peptide identification. In this paper, we utilized peptide empirical RT and MS/MS for peptide identification. This new approach resulted in the construction of an Empirical Peptide Retention Time Database (EPRTD) based on peptides showing a false-positive rate (FPR) ,1%, detected in several liquid chromatography (LC)/MS/MS analyses. In subsequent experiments, the RT of peptides with FPR >1% was compared with empirical data derived from the EPRTD. If the experimental RT was within a specified time range of the empirical value, the corresponding MS/MS spectra were accepted as positive. Application of the EPRTD approach to simple samples (known protein mixtures) and complex samples (human urinary proteome) revealed that this method could significantly enhance peptide identification without compromising the associated confidence levels. Further analysis indicated that the EPRTD approach could improve low-abundance peptides and with the expansion of the EPRTD the number of peptide identifications will be increased. This approach is suitable for large-scale clinical proteomics research, in which tens of LC/MS/MS analyses are run for different samples with similar components. Copyright © 2008 John Wiley & Sons, Ltd. [source] Identification and characterization of a new , -casein variant in goat milk by high-performance liquid chromatography with electrospray ionization mass spectrometry and matrix-assisted laser desorption/ionization mass spectrometryRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 17 2004Francesco Galliano A new variant of , -casein was detected in the casein fraction obtained from milk of a goat belonging to an autochthonous breed of southern Italy, ,Argentata dell'Etna'. Reversed-phase high-performance liquid chromatography/electrospray ionization mass spectrometry (RP-HPLC/ESI-MS) analysis indicated that the new , -casein variant, here named D, has a Mr 15,Da higher than that of variant C previously described. The modification in the amino acid sequence responsible for the 15,Da difference in Mr between variants C and D was determined by coupling trypsin digestion with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and RP-HPLC/ESI-MS, and it was demonstrated that it is due to the point mutation Val207,,,Asn207. The phosphorylation pattern of the new variant D was shown to be identical to that of variant C, as the protein shows two phosphorylation levels, 5 and 6P, occurring with comparable relative abundances. Ser35 was determined as one of the phosphorylation sites, whereas the others were probably analogous to those determined previously for the , -Cn variant C, at Thr12 and Ser15, 17,19. The results reported here indicate that the combined use of RP-HPLC/ESI-MS, MALDI-TOFMS and MS/MS represents a powerful tool for the detection and characterization of minor components present in complex protein mixtures. Copyright © 2004 John Wiley & Sons, Ltd. [source] Nano-high-performance liquid chromatography in combination with nano-electrospray ionization Fourier transform ion-cyclotron resonance mass spectrometry for proteome analysisRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 12 2003Christian Ihling Fourier transform ion-cyclotron resonance (FTICR) mass spectrometry offers several advantages for the analysis of biological samples, including excellent mass resolution, ultra-high mass measurement accuracy, high sensitivity, and wide mass range. We report the application of a nano-HPLC system coupled to an FTICR mass spectrometer equipped with nanoelectrospray source (nano-HPLC/nano-ESI-FTICRMS) for proteome analysis. Protein identification in proteomics is usually conducted by accurately determining peptide masses resulting from enzymatic protein digests and comparing them with theoretically digested protein sequences from databases. A tryptic in-solution digest of bovine serum albumin was used to optimize experimental conditions and data processing. Spots from Coomassie Blue and silver-stained two-dimensional (2D) gels of human thyroid tissue were excised, in-gel digested with trypsin, and subsequently analyzed by nano-HPLC/nano-ESI-FTICRMS. Additionally, we analyzed 1D-gel bands of membrane preparations of COS-6 cells from African green monkey kidney as an example of more complex protein mixtures. Nano-HPLC was performed using 1-mm reverse-phase C-18 columns for pre-concentration of the samples and reverse-phase C-18 capillary columns for separation, applying water/acetonitrile gradient elution conditions at flow rates of 200,nL/min. Mass measurement accuracies smaller than 3,ppm were routinely obtained. Different methods for processing the raw data were compared in order to identify a maximum number of peptides with the highest possible degree of automation. Parallel identification of proteins from complex mixtures down to low-femtomole levels makes nano-HPLC/nano-ESI-FTICRMS an attractive approach for proteome analysis. Copyright © 2003 John Wiley & Sons, Ltd. [source] Analysis of protein ions in the range 3000,12000,Th under partial (no discharge) atmospheric pressure chemical ionization conditions using ion trap mass spectrometryRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 12 2002Simone Cristoni A new approach, based on the use of atmospheric pressure chemical ionization ion trap mass spectrometry (APCI-ITMS), but without a corona discharge, was investigated for application to creating and monitoring protein ions. It must be emphasized that APCI is not usually used in protein analysis. In order to verify the applicability of the proposed method to the analysis of proteins, two standard proteins (horse cytochrome c and horse myoglobin) were analyzed. A mixture of the two proteins was also analyzed showing that this novel approach, based on the use of APCI, can be used in the analysis of protein mixtures. Copyright © 2002 John Wiley & Sons, Ltd. [source] Liquid chromatography and electron-capture dissociation in Fourier transform ion cyclotron resonance mass spectrometryRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 10 2002Magnus Palmblad Liquid separation methods in combination with electrospray mass spectrometry as well as the recently introduced fragmentation method electron capture dissociation (ECD) have become powerful tools in proteomics research. This paper presents the results of the first successful attempts to combine liquid chromatography (LC) and Fourier transform ion cyclotron resonance mass spectrometry (FTICRMS) with ECD in the analysis of a mixture of standard peptides and of a bovine serum albumin tryptic digest. A novel electron injection system provided conditions for ECD sufficient to yield extensive sequence information for the most abundant peptides in the mixtures on the time-scale of the chromatographic separation. The results suggest that LC/ECD-FTICRMS can be employed in the characterization of peptides in enzymatic digests of proteins or protein mixtures and identify and localize posttranslational modifications. Copyright © 2002 John Wiley & Sons, Ltd. [source] A cylindrical capacitor ionization source: droplet generation and controlled charge reduction for mass spectrometryRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 6 2001Daniel D. Ebeling A cylindrical capacitor ionization source was used in conjunction with corona discharge charge reduction for generation of singly charged ions for mass spectrometric analysis. The source consists of a fused-silica capillary threaded with a platinum wire and placed inside a stainless steel tube. Application of an electric potential to the wire results in the production of a linear stream of charged droplets when an aqueous solution is pumped through the capillary. Subsequent solvent evaporation yields ions, providing a continuous ion source for mass spectrometry. Passage of the ions through a corona discharge charge reduction chamber permits reduction of the charge state to predominantly singly charged species, facilitating analysis of DNA and protein mixtures. The change from production of multiply charged ions to production of singly charged ions is extremely simple, requiring only modulation of the voltage applied to the corona discharge electrode. A simple technique for construction of the ionization source is reported. Copyright © 2001 John Wiley & Sons, Ltd. [source] | ||||||||||||||||||||||||||||