Browsing by Author "Yang, Shuang"
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Item Multidimensional Microfluidic Bioseparation Systems With Spatial Multiplexing(2008-12-19) Yang, Shuang; DeVoe, Donald L; Mechanical Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Despite the refinement of liquid chromatography and peptide mass fingerprinting techniques for protein analysis, two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) separations of intact proteins remain a core technology for proteomic studies due to their high peak capacities and resolving power. In 2-D PAGE, denatured intact proteins are separated on the basis of their charge state by isoelectric focusing (IEF), followed by a size-based separation using sodium dodecyl sulfate (SDS)-PAGE. While 2-D PAGE is most commonly practiced with backend analysis of proteins by mass spectrometry, 2-D PAGE expression maps alone can provide valuable insight for differential studies, including the analysis of post-translational modifications, by yielding information about the approximate isoelectric point (pI) and molecular weight (MW) of differentially expressed species within complex samples. However, conventional slab-gel 2-D PAGE remains a labor intensive and low throughput process, which significantly constrains its utility. In this dissertation, a novel microfluidic 2-D PAGE platform is developed which employs a combination of multifunctional photopolymerized polyacrylamide (PAAm) gels and a discontinuous sodium dodecyl sulfate (SDS)-PAGE buffer system. The PAAm gel is used as a highly-resolving separation medium for gel electrophoresis, while discrete PAAm gel plugs integrated into specific regions of the chip enable acid, base, and ampholyte solutions to be fully isolated prior to chip operation. The gel plugs allow different separation buffers to be stored within the chip, enabling the use of a discontinuous buffer system chosen to provide sample stacking during the second-dimension separation. The gel plugs are also employed as on-chip SDS containers, allowing defined volumes of SDS to be repeatably injected and complexed with the IEF-focused proteins, without the need for external intervention. The IEF channel itself possesses an angled geometry to minimize sample tailing, and the chip design employs backbiasing channels which eliminate sample leakage and enable uniform sample transfer between the separation dimensions. Validation of the full 2-D system is presented using fluorescently-labeled E. coli cell lysate as a model system.Item QUANTITY: An Isobaric Tag for Quantitative Glycomics(2015) Yang, Shuang; Wang, Meiyao; Chen, Lijun; Yin, Bojiao; Song, Guoqiang; Turko, Illarion V.; Phinney, Karen W.; Betenbaugh, Michael J.; Zhang, Hui; Li, ShuweiGlycan 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.Item QUANTITY: An Isobaric Tag for Quantitative Glycomics(Nature Publishing Group, 2015-11-30) Yang, Shuang; Wang, Meiyao; Chen, Lijun; Yin, Bojiao; Song, Guoquiang; Turko, Illarion V.; Phinney, Karen W.; Betenbaugh, Michael J.; Zhang, Hui; Li, ShuweiGlycan 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.Item Simultaneous analyses of N-linked and O-linked glycans of ovarian cancer cells using solid-phase chemoenzymatic method(Springer Nature, 2017-01-13) Yang, Shuang; Höti, Naseruddin; Yang, Weiming; Liu, Yang; Chen, Lijun; Li, Shuwei; Zhang, HuiGlycans play critical roles in a number of biological activities. Two common types of glycans, N-linked and O-linked, have been extensively analyzed in the last decades. N-glycans are typically released from glycoproteins by enzymes, while O-glycans are released from glycoproteins by chemical methods. It is important to identify and quantify both N- and O-linked glycans of glycoproteins to determine the changes of glycans. The effort has been dedicated to study glycans from ovarian cancer cells treated with O-linked glycosylation inhibitor qualitatively and quantitatively. We used a solid-phase chemoenzymatic approach to systematically identify and quantify N-glycans and O-glycans in the ovarian cancer cells. It consists of three steps: (1) immobilization of proteins from cells and derivatization of glycans to protect sialic acids; (2) release of N-glycans by PNGase F and quantification of N-glycans by isobaric tags; (3) release and quantification of O-glycans by β-elimination in the presence of 1-phenyl-3-methyl-5-pyrazolone (PMP). We used ovarian cancer cell lines to study effect of O-linked glycosylation inhibitor on protein glycosylation. Results suggested that the inhibition of O-linked glycosylation reduced the levels of O-glycans. Interestingly, it appeared to increase N-glycan level in a lower dose of the O-linked glycosylation inhibitor. The sequential release and analyses of N-linked and O-linked glycans using chemoenzymatic approach are a platform for studying N-glycans and O-glycans in complex biological samples. The solid-phase chemoenzymatic method was used to analyze both N-linked and O-linked glycans sequentially released from the ovarian cancer cells. The biological studies on O-linked glycosylation inhibition indicate the effects of O-glycosylation inhibition to glycan changes in both O-linked and N-linked glycan expression.