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Jun 1, 2010 (Vol. 30, No. 11)

Maximizing HPLC Power and Throughput

Optimizing Separation Speed and Efficiency Gives Tremendous Boost to Critical Parameters

  • The plasma glycome represents a potential source of disease biomarkers. Changes in glycosylation of glycoproteins are often associated with inflammatory and other acute-phase disease responses and may provide a tool for early disease detection. In breast cancer, detectable glycan changes associated with inflammatory activity in tumors typically precede metastatic activity.

    It is also possible to characterize changes in the glycosylation of proteins from cells shed by tumors, and such glycan-based biomarkers might prove useful for detecting circulating tumor cells. In prostate cancer, although the relative level of prostate specific antigen (PSA) is a commonly used biomarker for prostate cancer screening, it is neither sufficiently sensitive nor specific.

    Pauline Rudd, Ph.D., NIBRT professor of glycobiology at University College, Dublin, and colleagues at the National Institute for Bioprocessing Research and Training (NIBRT), have identified a subset of PSA that has an altered glycosylation pattern in the prostate cancer setting.

    “Altered glycosylation of acute-phase proteins and IgG suggests that cancer regulates certain pathways favoring cancer cell survival,” Dr. Rudd observes. In ovarian cancer, for example, N-glycosylation changes in serum glycoproteins have been associated with a decrease in galactosylation of IgG and an increase in sialyl Lewis X.

    Dr. Rudd’s group has developed a robotic HPLC-based platform designed to perform quantitative glycan analysis and glycoprofiling to support both research on glycoproteins and bioprocessing applications from early-stage design through process analytical technology and biopharmaceutical production. Their work has led to the development of a glycome database called GlycoBase that contains structures of plasma glycans compiled from 1,008 individuals. The database includes more than 400 glycan structures, including 117 from the plasma glycome.

    Dr. Rudd’s group used HPLC analysis of fluorophore-labeled glycans combined with sialidase digestion to separate and quantify individual glycans into 33 chromatographic peaks. Dr. Rudd reports “surprisingly large biological variability at the population level.”

    GlycoBase provides the elution positions for labeled N-glycan structures and the predicted products of exoglycosidase digestion. The database is being used to study the variability, heritability, and lifestyle factors that affect the composition of the human plasma N-glycan.

    Dr. Rudd cites several advantages of using HPLC for glycome analysis, including the sensitivity and reproducibility of the technology and its ability to yield quantitative results. Furthermore, HPLC resolves molecules on the basis of their hydrophobic/hydrophilic properties and is able to analyze both charged and neutral glycans simultaneously and to distinguish between isomers.

    Dr. Rudd’s group automated the HPLC analytical workflow in a 96-well plate format from sample processing through data interpretation. N-linked sugars can be analyzed at concentrations as low as the femtomole range and isolated from microgram quantities of glycoprotein mixtures using traditional amide columns.

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