The Experts below are selected from a list of 1359 Experts worldwide ranked by ideXlab platform
Joseph Zaia - One of the best experts on this subject based on the ideXlab platform.
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Capillary Electrophoresis–Mass Spectrometry of Carbohydrates
Capillary Electrophoresis of Biomolecules, 2013Co-Authors: Joseph ZaiaAbstract:The development of methods for capillary electrophoresis (CE) with on-line mass spectrometric detection (CE/MS) is driven by the need for accurate, robust, and sensitive Glycomics Analysis for basic biomedicine, biomarker discovery, and Analysis of recombinant protein therapeutics. One important capability is to profile glycan mixtures with respect to the patterns of substituents including sialic acids, acetate, sulfate, phosphate, and other groups. There is additional need for an MS-compatible separation system capable of resolving carbohydrate isomers. This chapter summarizes applications of CS/MS to Analysis of carbohydrates, glycoproteins, and glycopeptides that have appeared since 2008. Readers are referred to recent comprehensive reviews covering earlier publications.
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Glycomics Analysis of mammalian heparan sulfates modified by the human extracellular sulfatase HSulf2.
PLoS ONE, 2011Co-Authors: Gregory O. Staples, Xiaofeng Shi, Joseph ZaiaAbstract:Background The Sulfs are a family of endosulfatases that selectively modify the 6O-sulfation state of cell-surface heparan sulfate (HS) molecules. Sulfs serve as modulators of cell-signaling events because the changes they induce alter the cell surface co-receptor functions of HS chains. A variety of studies have been aimed at understanding how Sulfs modify HS structure, and many of these studies utilize Sulf knockout cell lines as the source for the HS used in the experiments. However, genetic manipulation of Sulfs has been shown to alter the expression levels of HS biosynthetic enzymes, and in these cases an assessment of the fine structural changes induced solely by Sulf enzymatic activity is not possible. Therefore, the present work aims to extend the understanding of substrate specificities of HSulf2 using in vitro experiments to compare HSulf2 activities on HS from different organ tissues. Methodology/Principal Findings To further the understanding of Sulf enzymatic activity, we conducted in vitro experiments where a variety of mammalian HS substrates were modified by recombinant human Sulf2 (HSulf2). Subsequent to treatment with HSulf2, the HS samples were exhaustively depolymerized and analyzed using size-exclusion liquid chromatography-mass spectrometry (SEC-LC/MS). We found that HSulf2 activity was highly dependent on the structural features of the HS substrate. Additionally, we characterized, for the first time, the activity of HSulf2 on the non-reducing end (NRE) of HS chains. The results indicate that the action pattern of HSulf2 at the NRE is different compared to internally within the HS chain. Conclusions/Significance The results of the present study indicate that the activity of Sulfs is dependent on the unique structural features of the HS populations that they edit. The activity of HSulf2 at HS NREs implicates the Sulfs as key regulators of this region of the chains, and concomitantly, the protein-binding events that occur there.
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targeted Analysis of Glycomics liquid chromatography mass spectrometry data
Analytical and Bioanalytical Chemistry, 2011Co-Authors: Jonathan M Dreyfuss, Joseph Zaia, Gregory O. Staples, Christopher Jacobs, Yevgeniy Gindin, Gary BensonAbstract:Hydrophilic interaction chromatography (HILIC) liquid chromatography/mass spectrometry (LC/MS) is appropriate for all native and reductively aminated glycan classes. HILIC carries the advantage that retention times vary predictably according to oligosaccharide composition. Chromatographic conditions are compatible with sensitive and reproducible Glycomics Analysis of large numbers of samples. The data are extremely useful for quantitative profiling of glycans expressed in biological tissues. With these analytical developments, the rate-limiting factor for widespread use of HILIC LC/MS in Glycomics is the Analysis of the data. In order to eliminate this problem, a Java-based open source software tool, Manatee, was developed for targeted Analysis of HILIC LC/MS glycan datasets. This tool uses user-defined lists of compositions that specify the glycan chemical space in a given biological context. The program accepts high-resolution LC/MS data using the public mzXML format and is capable of processing a large data file in a few minutes on a standard desktop computer. The program allows mining of HILIC LC/MS data with an output compatible with multivariate statistical Analysis. It is envisaged that the Manatee tool will complement more computationally intensive LC/MS processing tools based on deconvolution and deisotoping of LC/MS data. The capabilities of the tool were demonstrated using a set of HILIC LC/MS data on organ-specific heparan sulfates.
Ivan K. Chu - One of the best experts on this subject based on the ideXlab platform.
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online coupling of hydrophilic interaction strong cation exchange reversed phase liquid chromatography with porous graphitic carbon liquid chromatography for simultaneous proteomics and n Glycomics Analysis
Journal of Chromatography A, 2015Co-Authors: Yun Zhao, Henry C. H. Law, Zaijun Zhang, Herman C. Lam, Quan Quan, Ivan K. ChuAbstract:In this study we developed a fully automated three-dimensional (3D) liquid chromatography methodology-comprising hydrophilic interaction separation as the first dimension, strong cation exchange fractionation as the second dimension, and low-pH reversed-phase (RP) separation as the third dimension-in conjunction downstream with additional complementary porous graphitic carbon separation, to capture non-retained hydrophilic analytes, for both shotgun proteomics and N-Glycomics analyses. The performance of the 3D system alone was benchmarked through the Analysis of the total lysate of Saccharomyces cerevisiae, leading to improved hydrophilic peptide coverage, from which we identified 19% and 24% more proteins and peptides, respectively, relative to those identified from a two-dimensional hydrophilic interaction liquid chromatography and low-pH RP chromatography (HILIC-RP) system over the same mass spectrometric acquisition time; consequently, the 3D platform also provided enhanced proteome and protein coverage. When we applied the integrated technology to analyses of the total lysate of primary cerebellar granule neurons, we characterized a total of 2201 proteins and 16,937 unique peptides for this primary cell line, providing one of its most comprehensive datasets. Our new integrated technology also exhibited excellent performance in the first N-Glycomics Analysis of cynomolgus monkey plasma; we successfully identified 122 proposed N-glycans and 135 N-glycosylation sites from 122 N-glycoproteins, and confirmed the presence of 38 N-glycolylneuraminic acid-containing N-glycans, a rare occurrence in human plasma, through tandem mass spectrometry for the first time.
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Online coupling of hydrophilic interaction/strong cation exchange/reversed-phase liquid chromatography with porous graphitic carbon liquid chromatography for simultaneous proteomics and N-Glycomics Analysis.
Journal of Chromatography A, 2015Co-Authors: Yun Zhao, Henry C. H. Law, Zaijun Zhang, Herman C. Lam, Quan Quan, Ivan K. ChuAbstract:In this study we developed a fully automated three-dimensional (3D) liquid chromatography methodology-comprising hydrophilic interaction separation as the first dimension, strong cation exchange fractionation as the second dimension, and low-pH reversed-phase (RP) separation as the third dimension-in conjunction downstream with additional complementary porous graphitic carbon separation, to capture non-retained hydrophilic analytes, for both shotgun proteomics and N-Glycomics analyses. The performance of the 3D system alone was benchmarked through the Analysis of the total lysate of Saccharomyces cerevisiae, leading to improved hydrophilic peptide coverage, from which we identified 19% and 24% more proteins and peptides, respectively, relative to those identified from a two-dimensional hydrophilic interaction liquid chromatography and low-pH RP chromatography (HILIC-RP) system over the same mass spectrometric acquisition time; consequently, the 3D platform also provided enhanced proteome and protein coverage. When we applied the integrated technology to analyses of the total lysate of primary cerebellar granule neurons, we characterized a total of 2201 proteins and 16,937 unique peptides for this primary cell line, providing one of its most comprehensive datasets. Our new integrated technology also exhibited excellent performance in the first N-Glycomics Analysis of cynomolgus monkey plasma; we successfully identified 122 proposed N-glycans and 135 N-glycosylation sites from 122 N-glycoproteins, and confirmed the presence of 38 N-glycolylneuraminic acid-containing N-glycans, a rare occurrence in human plasma, through tandem mass spectrometry for the first time.
Yun Zhao - One of the best experts on this subject based on the ideXlab platform.
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online coupling of hydrophilic interaction strong cation exchange reversed phase liquid chromatography with porous graphitic carbon liquid chromatography for simultaneous proteomics and n Glycomics Analysis
Journal of Chromatography A, 2015Co-Authors: Yun Zhao, Henry C. H. Law, Zaijun Zhang, Herman C. Lam, Quan Quan, Ivan K. ChuAbstract:In this study we developed a fully automated three-dimensional (3D) liquid chromatography methodology-comprising hydrophilic interaction separation as the first dimension, strong cation exchange fractionation as the second dimension, and low-pH reversed-phase (RP) separation as the third dimension-in conjunction downstream with additional complementary porous graphitic carbon separation, to capture non-retained hydrophilic analytes, for both shotgun proteomics and N-Glycomics analyses. The performance of the 3D system alone was benchmarked through the Analysis of the total lysate of Saccharomyces cerevisiae, leading to improved hydrophilic peptide coverage, from which we identified 19% and 24% more proteins and peptides, respectively, relative to those identified from a two-dimensional hydrophilic interaction liquid chromatography and low-pH RP chromatography (HILIC-RP) system over the same mass spectrometric acquisition time; consequently, the 3D platform also provided enhanced proteome and protein coverage. When we applied the integrated technology to analyses of the total lysate of primary cerebellar granule neurons, we characterized a total of 2201 proteins and 16,937 unique peptides for this primary cell line, providing one of its most comprehensive datasets. Our new integrated technology also exhibited excellent performance in the first N-Glycomics Analysis of cynomolgus monkey plasma; we successfully identified 122 proposed N-glycans and 135 N-glycosylation sites from 122 N-glycoproteins, and confirmed the presence of 38 N-glycolylneuraminic acid-containing N-glycans, a rare occurrence in human plasma, through tandem mass spectrometry for the first time.
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Online coupling of hydrophilic interaction/strong cation exchange/reversed-phase liquid chromatography with porous graphitic carbon liquid chromatography for simultaneous proteomics and N-Glycomics Analysis.
Journal of Chromatography A, 2015Co-Authors: Yun Zhao, Henry C. H. Law, Zaijun Zhang, Herman C. Lam, Quan Quan, Ivan K. ChuAbstract:In this study we developed a fully automated three-dimensional (3D) liquid chromatography methodology-comprising hydrophilic interaction separation as the first dimension, strong cation exchange fractionation as the second dimension, and low-pH reversed-phase (RP) separation as the third dimension-in conjunction downstream with additional complementary porous graphitic carbon separation, to capture non-retained hydrophilic analytes, for both shotgun proteomics and N-Glycomics analyses. The performance of the 3D system alone was benchmarked through the Analysis of the total lysate of Saccharomyces cerevisiae, leading to improved hydrophilic peptide coverage, from which we identified 19% and 24% more proteins and peptides, respectively, relative to those identified from a two-dimensional hydrophilic interaction liquid chromatography and low-pH RP chromatography (HILIC-RP) system over the same mass spectrometric acquisition time; consequently, the 3D platform also provided enhanced proteome and protein coverage. When we applied the integrated technology to analyses of the total lysate of primary cerebellar granule neurons, we characterized a total of 2201 proteins and 16,937 unique peptides for this primary cell line, providing one of its most comprehensive datasets. Our new integrated technology also exhibited excellent performance in the first N-Glycomics Analysis of cynomolgus monkey plasma; we successfully identified 122 proposed N-glycans and 135 N-glycosylation sites from 122 N-glycoproteins, and confirmed the presence of 38 N-glycolylneuraminic acid-containing N-glycans, a rare occurrence in human plasma, through tandem mass spectrometry for the first time.
Hui Zhang - One of the best experts on this subject based on the ideXlab platform.
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QUANTITY: An Isobaric Tag for Quantitative Glycomics
Scientific Reports, 2015Co-Authors: Shuang Yang, Meiyao Wang, Lijun Chen, Bojiao Yin, Guoquiang Song, Illarion V. Turko, Karen W. Phinney, Michael J. Betenbaugh, Hui ZhangAbstract:Glycan is an important class of macromolecules that play numerous biological functions. Quantitative Glycomics - Analysis of glycans at global level - however, is far behind genomics and proteomics owing to technical challenges associated with their chemical properties and structural complexity. As a result, technologies that can facilitate global glycan Analysis are highly sought after. Here, we present QUANTITY (Quaternary Amine Containing Isobaric Tag for Glycan), a quantitative approach that can not only enhance detection of glycans by mass spectrometry, but also allow high-throughput glycomic Analysis from multiple biological samples. This robust tool enabled us to accomplish glycomic survey of bioengineered Chinese Hamster Ovary (CHO) cells with knock-in/out enzymes involved in protein glycosylation. Our results demonstrated QUANTITY is an invaluable technique for glycan Analysis and bioengineering.
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Solid‐phase glycan isolation for Glycomics Analysis
PROTEOMICS - Clinical Applications, 2012Co-Authors: Shuang Yang, Hui ZhangAbstract:Glycosylation is one of the most significant protein PTMs. The biological activities of proteins are dramatically changed by the glycans associated with them. Thus, structural Analysis of the glycans of glycoproteins in complex biological or clinical samples is critical in correlation with the functions of glycans with diseases. Profiling of glycans by HPLC-MS is a commonly used technique in analyzing glycan structures and quantifying their relative abundance in different biological systems. Methods relied on MS require isolation of glycans from negligible salts and other contaminant ions since salts and ions may interfere with the glycans, resulting in poor glycan ionization. To accomplish those objectives, glycan isolation and clean-up methods including SPE, liquid-phase extraction, chromatography, and electrophoresis have been developed. Traditionally, glycans are isolated from proteins or peptides using a combination of hydrophobic and hydrophilic columns: proteins and peptides remain on hydrophobic absorbent while glycans, salts, and other hydrophilic reagents are collected as flowthrough. The glycans in the flowthrough are then purified through graphite-activated carbon column by hydrophilic interaction LC. Yet, the drawback in these affinity-based approaches is nonspecific binding. As a result, chemical methods by hydrazide or oxime have been developed for solid-phase isolation of glycans with high specificity and yield. Combined with high-resolution MS, specific glycan isolation techniques provide tremendous potentials as useful tools for Glycomics Analysis.
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solid phase glycan isolation for Glycomics Analysis
Proteomics Clinical Applications, 2012Co-Authors: Shuang Yang, Hui ZhangAbstract:Glycosylation is one of the most significant protein PTMs. The biological activities of proteins are dramatically changed by the glycans associated with them. Thus, structural Analysis of the glycans of glycoproteins in complex biological or clinical samples is critical in correlation with the functions of glycans with diseases. Profiling of glycans by HPLC-MS is a commonly used technique in analyzing glycan structures and quantifying their relative abundance in different biological systems. Methods relied on MS require isolation of glycans from negligible salts and other contaminant ions since salts and ions may interfere with the glycans, resulting in poor glycan ionization. To accomplish those objectives, glycan isolation and clean-up methods including SPE, liquid-phase extraction, chromatography, and electrophoresis have been developed. Traditionally, glycans are isolated from proteins or peptides using a combination of hydrophobic and hydrophilic columns: proteins and peptides remain on hydrophobic absorbent while glycans, salts, and other hydrophilic reagents are collected as flowthrough. The glycans in the flowthrough are then purified through graphite-activated carbon column by hydrophilic interaction LC. Yet, the drawback in these affinity-based approaches is nonspecific binding. As a result, chemical methods by hydrazide or oxime have been developed for solid-phase isolation of glycans with high specificity and yield. Combined with high-resolution MS, specific glycan isolation techniques provide tremendous potentials as useful tools for Glycomics Analysis.
Herman C. Lam - One of the best experts on this subject based on the ideXlab platform.
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online coupling of hydrophilic interaction strong cation exchange reversed phase liquid chromatography with porous graphitic carbon liquid chromatography for simultaneous proteomics and n Glycomics Analysis
Journal of Chromatography A, 2015Co-Authors: Yun Zhao, Henry C. H. Law, Zaijun Zhang, Herman C. Lam, Quan Quan, Ivan K. ChuAbstract:In this study we developed a fully automated three-dimensional (3D) liquid chromatography methodology-comprising hydrophilic interaction separation as the first dimension, strong cation exchange fractionation as the second dimension, and low-pH reversed-phase (RP) separation as the third dimension-in conjunction downstream with additional complementary porous graphitic carbon separation, to capture non-retained hydrophilic analytes, for both shotgun proteomics and N-Glycomics analyses. The performance of the 3D system alone was benchmarked through the Analysis of the total lysate of Saccharomyces cerevisiae, leading to improved hydrophilic peptide coverage, from which we identified 19% and 24% more proteins and peptides, respectively, relative to those identified from a two-dimensional hydrophilic interaction liquid chromatography and low-pH RP chromatography (HILIC-RP) system over the same mass spectrometric acquisition time; consequently, the 3D platform also provided enhanced proteome and protein coverage. When we applied the integrated technology to analyses of the total lysate of primary cerebellar granule neurons, we characterized a total of 2201 proteins and 16,937 unique peptides for this primary cell line, providing one of its most comprehensive datasets. Our new integrated technology also exhibited excellent performance in the first N-Glycomics Analysis of cynomolgus monkey plasma; we successfully identified 122 proposed N-glycans and 135 N-glycosylation sites from 122 N-glycoproteins, and confirmed the presence of 38 N-glycolylneuraminic acid-containing N-glycans, a rare occurrence in human plasma, through tandem mass spectrometry for the first time.
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Online coupling of hydrophilic interaction/strong cation exchange/reversed-phase liquid chromatography with porous graphitic carbon liquid chromatography for simultaneous proteomics and N-Glycomics Analysis.
Journal of Chromatography A, 2015Co-Authors: Yun Zhao, Henry C. H. Law, Zaijun Zhang, Herman C. Lam, Quan Quan, Ivan K. ChuAbstract:In this study we developed a fully automated three-dimensional (3D) liquid chromatography methodology-comprising hydrophilic interaction separation as the first dimension, strong cation exchange fractionation as the second dimension, and low-pH reversed-phase (RP) separation as the third dimension-in conjunction downstream with additional complementary porous graphitic carbon separation, to capture non-retained hydrophilic analytes, for both shotgun proteomics and N-Glycomics analyses. The performance of the 3D system alone was benchmarked through the Analysis of the total lysate of Saccharomyces cerevisiae, leading to improved hydrophilic peptide coverage, from which we identified 19% and 24% more proteins and peptides, respectively, relative to those identified from a two-dimensional hydrophilic interaction liquid chromatography and low-pH RP chromatography (HILIC-RP) system over the same mass spectrometric acquisition time; consequently, the 3D platform also provided enhanced proteome and protein coverage. When we applied the integrated technology to analyses of the total lysate of primary cerebellar granule neurons, we characterized a total of 2201 proteins and 16,937 unique peptides for this primary cell line, providing one of its most comprehensive datasets. Our new integrated technology also exhibited excellent performance in the first N-Glycomics Analysis of cynomolgus monkey plasma; we successfully identified 122 proposed N-glycans and 135 N-glycosylation sites from 122 N-glycoproteins, and confirmed the presence of 38 N-glycolylneuraminic acid-containing N-glycans, a rare occurrence in human plasma, through tandem mass spectrometry for the first time.