The Experts below are selected from a list of 2181 Experts worldwide ranked by ideXlab platform
Cheng S. Lee - One of the best experts on this subject based on the ideXlab platform.
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Capillary Isoelectric Focusing/reversed phase liquid chromatography/mass spectrometry.
Methods of Molecular Biology, 2009Co-Authors: Cheng S. Lee, Brian M. BalgleyAbstract:The vast number of proteins present in the proteome of a typical organism requires that separations be performed on the mixture prior to introduction into a mass spectrometer for protein identification and quantification. An integrated protein separation platform, combining Capillary Isoelectric Focusing (CIEF) with reversed phase liquid chromatography (RPLC), is described to provide high resolving power for the analysis of complex protein mixtures. Thus, the proteins are systematically resolved according to their differences in Isoelectric point and hydrophobicity using combined CIEF/RPLC separations. A key feature of the CIEF-based multidimensional separation platform is the elimination of protein loss and dilution in an integrated platform while achieving comprehensive and ultrasensitive analysis of protein profiles within small cell populations or limited tissue samples.
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Proteome analysis of microdissected tumor tissue using a Capillary Isoelectric Focusing-based multidimensional separation platform coupled with ESI-tandem MS.
Analytical Chemistry, 2005Co-Authors: Yueju Wang, Cheng S. Lee, Paul A. Rudnick, Erin L. Evans, Don L. Devoe, Zhengping Zhuang, Brian M. BalgleyAbstract:This study demonstrates the ability to perform sensitive proteome analysis on the limited protein quantities available through tissue microdissection. Capillary Isoelectric Focusing combined with nano-reversed-phase liquid chromatography in an automated and integrated platform not only provides systematic resolution of complex peptide mixtures based on their differences in Isoelectric point and hydrophobicity but also eliminates peptide loss and analyte dilution. In comparison with strong cation exchange chromatography, the significant advantages of electrokinetic Focusing-based separations include high resolving power, high concentration and narrow analyte bands, and effective usage of electrospray ionization-tandem MS toward peptide identifications. Through the use of Capillary Isoelectric Focusing-based multidimensional peptide separations, a total of 6866 fully tryptic peptides were detected, leading to the identification of 1820 distinct proteins. Each distinct protein was identified by at least one ...
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Tissue proteomics using Capillary Isoelectric Focusing-based multidimensional separations
Expert Review of Proteomics, 2005Co-Authors: Yueju Wang, Brian M. Balgley, Cheng S. LeeAbstract:The capabilities of Capillary Isoelectric Focusing-based multidimensional separations for performing proteome analysis from minute samples create new opportunities in the pursuit of biomarker discovery using enriched and selected cell populations procured from tissue specimens. In this article, recent advances in online integration of Capillary Isoelectric Focusing with nano-reversed phase liquid chromatography for achieving high-resolution peptide and protein separations prior to mass spectrometry analysis are reviewed, along with its potential application to tissue proteomics. These proteome technological advances combined with recently developed tissue microdissection techniques, provide powerful tools for those seeking to gain a greater understanding at the global level of the cellular machinery associated with human diseases such as cancer.
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Integrated Capillary Isoelectric Focusing/Nano-reversed Phase Liquid Chromatography Coupled with ESI−MS for Characterization of Intact Yeast Proteins
Journal of Proteome Research, 2005Co-Authors: Yueju Wang, Brian M. Balgley, Paul A. Rudnick, Erin L. Evans, Don L. Devoe, Cheng S. LeeAbstract:An integrated protein concentration/separation platform, combining Capillary Isoelectric Focusing (CIEF) with nano-reversed phase liquid chromatography (nano-RPLC), is developed to provide significant protein concentration and high resolving power for the analysis of complex protein mixtures. Upon completion of protein Focusing, the proteins are sequentially and hydrodynamically loaded into individual trap columns using a group of microinjection and microselection valves. Repeated pro-tein loadings and injections into trap columns are carried out automatically until the entire CIEF cap-illary content is sampled and fractionated. Each CIEF fraction “parked” in separate trap columns is further resolved using nano-RPLC, and the eluants are analyzed using electrospray ionization-mass spectrometry. Keywords: Capillary Isoelectric Focusing • ESI−MS • intact proteins • proteomics • reversed-phase liquid chromatography
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Capillary Isoelectric Focusing based multidimensional concentration separation platform for proteome analysis
Analytical Chemistry, 2003Co-Authors: Jinzhi Chen, Brian M. Balgley, Don L. Devoe, Cheng S. LeeAbstract:An integrated proteome concentration/separation approach involving on-line combination of Capillary Isoelectric Focusing (CIEF) with Capillary reversed-phase liquid chromatography (CRPLC) is developed for providing significant analyte concentration and extremely high resolving power toward protein and peptide mixtures. Upon completion of analyte Focusing, the self-sharpening effect greatly restricts analyte diffusion and contributes to analyte stacking in narrowly focused bands with a concentration factor of ∼240. In addition to analyte Focusing, CIEF as the first separation dimension resolves proteins/peptides on the basis of their differences in pI and offers greater resolving power than that achieved in strong cation exchange chromatography. The grouping of two highly resolving and completely orthogonal separation techniques of CIEF and CRPLC, together with analyte Focusing and concentration, significantly enhances the dynamic range and sensitivity of conventional mass spectrometry toward the identific...
Brian M. Balgley - One of the best experts on this subject based on the ideXlab platform.
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Capillary Isoelectric Focusing/reversed phase liquid chromatography/mass spectrometry.
Methods of Molecular Biology, 2009Co-Authors: Cheng S. Lee, Brian M. BalgleyAbstract:The vast number of proteins present in the proteome of a typical organism requires that separations be performed on the mixture prior to introduction into a mass spectrometer for protein identification and quantification. An integrated protein separation platform, combining Capillary Isoelectric Focusing (CIEF) with reversed phase liquid chromatography (RPLC), is described to provide high resolving power for the analysis of complex protein mixtures. Thus, the proteins are systematically resolved according to their differences in Isoelectric point and hydrophobicity using combined CIEF/RPLC separations. A key feature of the CIEF-based multidimensional separation platform is the elimination of protein loss and dilution in an integrated platform while achieving comprehensive and ultrasensitive analysis of protein profiles within small cell populations or limited tissue samples.
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Proteome analysis of microdissected tumor tissue using a Capillary Isoelectric Focusing-based multidimensional separation platform coupled with ESI-tandem MS.
Analytical Chemistry, 2005Co-Authors: Yueju Wang, Cheng S. Lee, Paul A. Rudnick, Erin L. Evans, Don L. Devoe, Zhengping Zhuang, Brian M. BalgleyAbstract:This study demonstrates the ability to perform sensitive proteome analysis on the limited protein quantities available through tissue microdissection. Capillary Isoelectric Focusing combined with nano-reversed-phase liquid chromatography in an automated and integrated platform not only provides systematic resolution of complex peptide mixtures based on their differences in Isoelectric point and hydrophobicity but also eliminates peptide loss and analyte dilution. In comparison with strong cation exchange chromatography, the significant advantages of electrokinetic Focusing-based separations include high resolving power, high concentration and narrow analyte bands, and effective usage of electrospray ionization-tandem MS toward peptide identifications. Through the use of Capillary Isoelectric Focusing-based multidimensional peptide separations, a total of 6866 fully tryptic peptides were detected, leading to the identification of 1820 distinct proteins. Each distinct protein was identified by at least one ...
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Tissue proteomics using Capillary Isoelectric Focusing-based multidimensional separations
Expert Review of Proteomics, 2005Co-Authors: Yueju Wang, Brian M. Balgley, Cheng S. LeeAbstract:The capabilities of Capillary Isoelectric Focusing-based multidimensional separations for performing proteome analysis from minute samples create new opportunities in the pursuit of biomarker discovery using enriched and selected cell populations procured from tissue specimens. In this article, recent advances in online integration of Capillary Isoelectric Focusing with nano-reversed phase liquid chromatography for achieving high-resolution peptide and protein separations prior to mass spectrometry analysis are reviewed, along with its potential application to tissue proteomics. These proteome technological advances combined with recently developed tissue microdissection techniques, provide powerful tools for those seeking to gain a greater understanding at the global level of the cellular machinery associated with human diseases such as cancer.
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Integrated Capillary Isoelectric Focusing/Nano-reversed Phase Liquid Chromatography Coupled with ESI−MS for Characterization of Intact Yeast Proteins
Journal of Proteome Research, 2005Co-Authors: Yueju Wang, Brian M. Balgley, Paul A. Rudnick, Erin L. Evans, Don L. Devoe, Cheng S. LeeAbstract:An integrated protein concentration/separation platform, combining Capillary Isoelectric Focusing (CIEF) with nano-reversed phase liquid chromatography (nano-RPLC), is developed to provide significant protein concentration and high resolving power for the analysis of complex protein mixtures. Upon completion of protein Focusing, the proteins are sequentially and hydrodynamically loaded into individual trap columns using a group of microinjection and microselection valves. Repeated pro-tein loadings and injections into trap columns are carried out automatically until the entire CIEF cap-illary content is sampled and fractionated. Each CIEF fraction “parked” in separate trap columns is further resolved using nano-RPLC, and the eluants are analyzed using electrospray ionization-mass spectrometry. Keywords: Capillary Isoelectric Focusing • ESI−MS • intact proteins • proteomics • reversed-phase liquid chromatography
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Capillary Isoelectric Focusing based multidimensional concentration separation platform for proteome analysis
Analytical Chemistry, 2003Co-Authors: Jinzhi Chen, Brian M. Balgley, Don L. Devoe, Cheng S. LeeAbstract:An integrated proteome concentration/separation approach involving on-line combination of Capillary Isoelectric Focusing (CIEF) with Capillary reversed-phase liquid chromatography (CRPLC) is developed for providing significant analyte concentration and extremely high resolving power toward protein and peptide mixtures. Upon completion of analyte Focusing, the self-sharpening effect greatly restricts analyte diffusion and contributes to analyte stacking in narrowly focused bands with a concentration factor of ∼240. In addition to analyte Focusing, CIEF as the first separation dimension resolves proteins/peptides on the basis of their differences in pI and offers greater resolving power than that achieved in strong cation exchange chromatography. The grouping of two highly resolving and completely orthogonal separation techniques of CIEF and CRPLC, together with analyte Focusing and concentration, significantly enhances the dynamic range and sensitivity of conventional mass spectrometry toward the identific...
Richard D. Smith - One of the best experts on this subject based on the ideXlab platform.
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Capillary Isoelectric Focusing-Mass Spectrometry of Proteins and Protein Complexes
Methods of Molecular Biology, 2004Co-Authors: Suzana Martinović, Ljiljana Paša-tolić, Richard D. SmithAbstract:Complex proteome samples require efficient separation and detection methods in order to characterize their protein components. On-line combination of Capillary Isoelectric Focusing (CIEF) with electrospray ionization (ESI) mass spectrometry (MS) is shown as an effective method to analyze complex protein mixtures. Our experience with several microorganisms allowed us to establish successful experimental protocol. Here we use the example of E. coli whole cell lysate for the CIEF separation and MS detection on the intact protein level. The protocol was further adapted for the analysis of the mixture of non-covalent complexes on the intact complex level.
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Capillary Isoelectric Focusing of yeast cells.
Analytical Chemistry, 2000Co-Authors: Yufeng Shen, Scott J. Berger, Richard D. SmithAbstract:In the present work, Capillary Isoelectric Focusing (CIEF) methods were developed for the separation and identification of yeast cells. Yeast cells (∼4-μm diameter) cultured to various phases of growth were shown to be reproducibly resolved by CIEF using 100-μm-i.d. fused-silica capillaries coated with hydroxypropyl methylcellulose. Separation efficiencies corresponding to peak capacities of >4000 were obtained. The suitable cell concentration range for obtaining repeatable elution in CIEF separations was found to be quite low (
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Capillary Isoelectric Focusing of yeast cells
Analytical Chemistry, 2000Co-Authors: Yufeng Shen, Scott J. Berger, Richard D. SmithAbstract:In the present work, Capillary Isoelectric Focusing (CIEF) methods were developed for the separation and identification of yeast cells. Yeast cells (∼4-μm diameter) cultured to various phases of growth were shown to be reproducibly resolved by CIEF using 100-μm-i.d. fused-silica capillaries coated with hydroxypropyl methylcellulose. Separation efficiencies corresponding to peak capacities of >4000 were obtained. The suitable cell concentration range for obtaining repeatable elution in CIEF separations was found to be quite low (<3 cells/μL). CIEF experiments showed that yeast cell populations at early log, mid log, and stationary growth phases differ in Isoelectric point, with values ranging from 5.2 to 6.4. The broader application of CIEF are projected for microorganism identification and separation based upon growth conditions.
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High-efficiency Capillary Isoelectric Focusing of peptides.
Analytical Chemistry, 2000Co-Authors: Yufeng Shen, Scott J. Berger, Gordon A. Anderson, Richard D. SmithAbstract:Several approaches are presently being developed for global proteome characterization that are based upon the analysis of polypeptide mixtures resulting from digestion of (often complex) mixtures of proteins. Improved methods for peptide analysis are needed that provide for sample concentration, higher resolution separations, and direct compatibility with mass spectrometry. In this work, methods for the high-efficiency Capillary Isoelectric Focusing (CIEF) separation of peptides have been developed that provide for simultaneous sample concentration and separation according to peptide Isoelectric point. Under typical nondenaturing CIEF conditions, peptides are concentrated approximately 500-fold, and peptides present at < 1 ng/ microL were detectable using conventional UV detection. CIEF separations of peptides provided much faster measurements of Isoelectric points compared with conventional Isoelectric Focusing in gels. Very small differences in peptide Isoelectric points (deltapI approximately 0.01) could be resolved, High-efficiency CIEF separations for complex peptide mixtures from tryptic digestion of yeast cytosol fractions were obtained and showed significant improvement over those obtained using Capillary zone electrophoresis and packed Capillary reversed-phase liquid chromatography.
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Stepwise Mobilization of Focused Proteins in Capillary Isoelectric Focusing Mass Spectrometry
Analytical Chemistry, 2000Co-Authors: Chao-xuan Zhang, Ljiljana Paša-tolić, Gordon A. Anderson, Fan Xiang, Timothy D. Veenstra, Richard D. SmithAbstract:A stepwise mobilization strategy has been developed for the elution of complex protein mixtures, separated by Capillary Isoelectric Focusing (CIEF) for detection using on-line electrospray ionization mass spectrometry (ESI-MS). Carrier polyampholytes are used to establish a pH gradient as well as to control the electroosmotic flow arising from the use of uncoated fused-silica capillaries. Elution of focused protein zones is achieved by controlling the mobilization pressure and voltage, leaving the remaining protein zones focused inside the Capillary. Protein zones are stepwise eluted from the Capillary by changing the mobilization conditions. Stepwise mobilization improves separation resolution and simplifies coupling with multistage MS (i.e., MSn) analysis since it allows more effective temporal control of protein elution from the CIEF Capillary. We also describe a modified configuration for coupling CIEF with ESI-MS using a coaxial sheath flow interface that facilitate the automation of on-line CIEF-ESI...
Janusz Pawliszyn - One of the best experts on this subject based on the ideXlab platform.
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Dynamic kinetic Capillary Isoelectric Focusing : A powerful tool for studying protein-DNA interactions
Analytical Chemistry, 2007Co-Authors: Zhen Liu, Andrei P. Drabovich, Sergey N. Krylov, Janusz PawliszynAbstract:A new method called dynamic kinetic Capillary Isoelectric Focusing (DK-CIEF) is presented for the study of protein−DNA interactions. The method is based on CIEF with laser-induced fluorescence-whole column imaging detection in which protein−DNA complexes are separated with spatial resolution while dissociations of the complexes are dynamically monitored using a CCD camera with temporal resolution. This method allows for the discrimination of different complexes and the measurement of the individual dissociation rate constants.
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Capillary Isoelectric Focusing coupled with dynamic imaging detection: A one-dimensional separation for two-dimensional protein characterization.
Journal of Proteome Research, 2006Co-Authors: Zhen Liu, Tibebe Lemma, Janusz PawliszynAbstract:Here, we devised a novel approach for two-dimensional (2D) protein characterization using a single one-dimensional separation followed by a second characterization in the same instrument. The approach combines Capillary Isoelectric Focusing (CIEF), which separates proteins according to pI, with dynamic imaging detection, which permits monitoring of protein diffusion in real time and thereby allows estimation of molecular weight from diffusion coefficient. Compared with classical 2D gel electrophoresis, the approach provided several significant advantages including speed and ease in operation and automation, while yielding comparable accuracy. The approach was applicable for protein samples of low to moderate complexity. Keywords: two-dimensional characterization • protein • diffusion • Capillary Isoelectric Focusing • whole-column imaging detection
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Fluorescence imaging detection for Capillary Isoelectric Focusing.
Electrophoresis, 2005Co-Authors: Janusz PawliszynAbstract:A simple laser-induced fluorescence (LIF) imaging detector and an ultrasensitive LIF imaging detector are described for Capillary Isoelectric Focusing (CIEF). An argon ion laser beam of 496.5 nm is used as excitation source. In the simple LIF imaging detector, the excitation beam is directed into a Capillary column by an optic fiber array. In the ultrasensitive LIF imaging detector, the laser beam is first expanded, then is focused into the 4.5 cm long Capillary column by a cylindrical lens. Fluorescence emission is detected by a charge-coupled device (CCD) camera. The feasibility and performance of the LIF imaging detector system for CIEF are first verified with a naturally fluorescent protein, b-phycoerythrin. Then, the ultrasensitive LIF imaging system is used as a detector for CIEF of proteins labeled with fluorescein isothiocyanate (FITC). Three FITC-labeled proteins (i) α-D-galatosylated FITC-albumin, (ii) insulin-FITC, and (iii) casein-FITC, are used as model samples. Fluorescence images of the model samples are measured during the CIEF process. The Focusing of the protein samples is complete in about 1.5 min. The ultrasensitive detector's detection limits for the FITC-labeled proteins are at the level of 10−10M, and the mass detection limits are about 4.5 × 10−17 mole, even though only 10% of the fluorescence emission is collected. Therefore, the method is capable of separating and detecting 10−11M or amole (10−18 mole) level protein samples with a band-pass filter more specific to the fluorescence light. Potential applications of the LIF imaging system in addition to quantitation of separated fluorescent species in various Capillary electrophoresis methods can also include investigation of interaction between analytes focused in a Capillary column.
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Laser‐induced fluorescence detection of non‐covalently labeled protein in Capillary Isoelectric Focusing
Journal of Separation Science, 2002Co-Authors: Newman S.-k. Sze, Tiemin Huang, Janusz PawliszynAbstract:Non-Covalent labeling for fluorescence detection of proteins has been investigated to increase the sensitivity of Capillary Isoelectric Focusing using laser-induced fluorescence (LIF) detection. Non-covalently labeling fluorescent dyes, NanoOrange, Sypro red, Sypro orange, and Sypro tangerine were explored for the coupling of bovine serum albumin (BSA) and hemoglobin. Labeled proteins were studied by two complementary detection methods, viz. whole column UV and LIF detection instruments. The studies using a commercial Capillary Isoelectric Focusing (CIEF) instrument with UV detection gave accurate pI point determination of the labeled protein, and it was confirmed that non-covalently labeled BSA focused to well characterized peaks and the related calculated pI values did not change significantly. The axial LIF detection system confirmed the formation of fluorescent labeled BSA, and an improvement of detection sensitivity of at least 10 times was achieved using LIF as compared to the UV absorption instrument.
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Miniaturization of Capillary Isoelectric Focusing.
Electrophoresis, 2001Co-Authors: Newman S.-k. Sze, Janusz PawliszynAbstract:Miniaturization of whole-column imaging Capillary Isoelectric Focusing (CIEF) is discussed. A 1.2 cm Capillary was used as a separation column for CIEF. The experimental results for the analysis of two pI markers and the protein myoglobin showed that good CIEF separation results could be obtained. Secondly, a light-emitting diode (LED) was used as the light source for the whole-column absorbance imaging detection. The Focusing of both the pI markers and myoglobin were observed with the LED light source. The whole-column imaging CIEF instrument was simplified and miniaturized by the use of the LED. Further developments are also discussed.
Yingru Zhang - One of the best experts on this subject based on the ideXlab platform.
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A Middle-Up Approach with Online Capillary Isoelectric Focusing/Mass Spectrometry for In-Depth Characterization of Cetuximab Charge Heterogeneity
Analytical Chemistry, 2018Co-Authors: Jun Dai, Yingru ZhangAbstract:Previously, we reported a new online Capillary Isoelectric Focusing/mass spectrometric (CIEF/MS) method for intact monoclonal antibody (mAb) charge variant analysis that uses an electrokinetically pumped sheath-flow nanospray ion source on a time-of-flight (TOF) MS with a pressure-assisted chemical mobilization. The direct online CIEF/MS method exhibited excellent resolution of charge variants conforming to those of imaged Capillary Isoelectric Focusing with ultraviolet detection (iCIEF/UV). However, for complex mAbs, CIEF/MS spectra of the intact charge variant peaks may be too convoluted to be effectively interpreted. In the present study, we implemented a middle-up approach to enhance the capability of the CIEF/MS method for characterizing complex mAb charge variants by reducing sample complexity. To demonstrate such a strategy, we fragmented cetuximab through IdeS enzymatic cleavage and dithiothreitol (DTT) reduction. For the first time, online CIEF/MS resolved the complex charge variants of cetuximab...
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a middle up approach with online Capillary Isoelectric Focusing mass spectrometry for in depth characterization of cetuximab charge heterogeneity
Analytical Chemistry, 2018Co-Authors: Jun Dai, Yingru ZhangAbstract:Previously, we reported a new online Capillary Isoelectric Focusing/mass spectrometric (CIEF/MS) method for intact monoclonal antibody (mAb) charge variant analysis that uses an electrokinetically pumped sheath-flow nanospray ion source on a time-of-flight (TOF) MS with a pressure-assisted chemical mobilization. The direct online CIEF/MS method exhibited excellent resolution of charge variants conforming to those of imaged Capillary Isoelectric Focusing with ultraviolet detection (iCIEF/UV). However, for complex mAbs, CIEF/MS spectra of the intact charge variant peaks may be too convoluted to be effectively interpreted. In the present study, we implemented a middle-up approach to enhance the capability of the CIEF/MS method for characterizing complex mAb charge variants by reducing sample complexity. To demonstrate such a strategy, we fragmented cetuximab through IdeS enzymatic cleavage and dithiothreitol (DTT) reduction. For the first time, online CIEF/MS resolved the complex charge variants of cetuximab...
