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Nov 15, 2010 (Vol. 30, No. 20)

Tandem MS Zooms In on Glycosylation

Conventional Methods Are Inadequate When It Comes to Quantitative and Structural Analysis

  • Quantifying Comparability

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    The Complex Carbohydrate Research Center not only performs basic research on the synthesis, structural characterization, and medical aspects of glycosylated compounds and polysaccharides, it also operates an analytical-services facility.

    Presenting at Informa’s “Comparability for Biologics” meeting in Berlin, Parastoo Azadi, Ph.D., technical director at the Complex Carbohydrate Research Center at the University of Georgia, described the tools and techniques used for comparability studies of glycoproteins to assess batch-to-batch variability in glycosylation.

    Analytical methods can differentiate between N-linked and O-linked glycosylation, provide data on the percent and sites of glycosylation, and identify what carbohydrates are present at which sites. They can yield both qualitative and quantitative information, with the demand for quantitation increasing and techniques and technology evolving to meet that demand.

    “Quantification is still a black box for carbohydrates,” she said. The field is advancing beyond conventional MALDI-MS technology for generating molecular weight data to the more advanced MS-MS techniques that provide sequence information on glycan fractions.

    Whereas HPLC of labeled oligosaccharides remains an analytical standard, new derivatization methods to modify and stabilize the carbohydrates for analysis, coupled with MS-MS techniques, “may enable us to do accurate quantitation in the next five years,” she predicted.

    Even as they become more quantitative, analytical techniques for characterizing carbohydrates also need to evolve beyond one-sample-at-a-time analysis and enable high-throughput profiling as well as structural analysis and identification of glycans in complex mixtures.

    The nonprofit Complex Carbohydrate Research Center not only performs basic research on the synthesis, structural characterization, and medical aspects of glycosylated compounds and polysaccharides, it  also operates an analytical services facility and publishes the results of its analyses.

    In a study presented at the Society for Glycobiology’s annual conference, scientists described the glycobiological characterization of human biglycan, a proteoglycan component of the extracellular matrix present in a variety of tissue types. Altered biglycan expression has been associated with medical conditions such as osteoporosis, osteoarthritis, corneal diseases, and atherosclerosis.

    Dr. Azadi and colleagues characterized the glycan portion of human recombinant biglycan using SDS-PAGE, HPLC, and MS techniques. LC/MS-MS analysis following 18O labeling revealed that two potential N-linked glycosylation sites in the non-proteoglycan form of biglycan are fully glycosylated. The researchers used ESI-MS-MS analysis to identify all major glycosylation of the biglycan proteoglycan.

  • Glycoprotein Enrichment

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    Scientists at the Max Planck Institute of Biochemistry and Harvard Medical School have developed what they say is an easy and efficient filter-based approach for N-glycosylation analysis—N-Glyco-FASP.

    In a paper published in Cell recently,  Matthias Mann, Ph.D., and colleagues from the Max Planck Institute of Biochemistry and Harvard Medical School, described the use of a filter-aided sample preparation (FASP) method they developed to enrich for the N-glycoproteome component of tissue and plasma samples.

    The authors propose that the “830 mouse and 1998 human N-glycosylation sites” that comprise the Swiss-Prot database (“the most comprehensive resource of annotated N-glycosylation sites”) “is likely a drastic underestimate of the true extent of the mammalian N-glycoproteome.”

    In the study described, they used the FASP-based N-linked glycopeptide capture method and orbitrap mass spectrometry to analyze the peptide fragments derived from mouse tissue and plasma samples. The resulting data not only included 74% of known mouse N-glycosites, but also allowed them to identify an additional 5,753 glycosylation sites. In a single LC/MS-MS run using 200 micrograms of starting material, the researchers were able to map more than 2,000 sites.

    FASP incorporates an SDS solubilization step that enhances the enrichment efficiency for glycosylated membrane proteins. It enriches for these glycoproteins by “at least 10-fold,” said Dr. Mann.

    After on-filter protein digestion, a lectin-based peptide affinity reagent is added to the top of the filter. Glycosylated peptides bind to the lectin and are retained on the filter. They are subsequently washed through the filter and deglycosylated using PNGase F. 18O water incorporation followed by MS analysis pinpoints the glycosylation sites on the peptides.

    Dr. Mann’s group is using this method to look for plasma biomarkers in tissue samples from patients with cancer. Changes in the glycosylation status of secreted or membrane-bound glycoproteins in circulation may reveal patterns of increased or decreased glycoprotein expression that can serve as biomarkers to aid in cancer diagnosis and prognosis, and for evaluating disease progression and the risk of metastasis.

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