An international comparability study on quantification of total methyl cytosine content.

Various methods have been developed for quantitative analysis of DNA methylation. However, there is currently no reference analysis system regarding DNA methylation with which other analytical approaches can be compared and evaluated. A standard measurement system that includes reference methods and reference materials may improve comparability and credibility of data obtained from different analytical environments. In an effort to establish a standard system for measurement of DNA methylation, the Korea Research Institute of Standards and Science (KRISS) coordinated an international comparison study among different national metrology institutes. An initial stage of the study involved an intercomparison regarding quantitative measurement of total methyl cytosine contents in artificially constructed DNA samples. The measurement principle involved measurement of dNMP contents following enzymatic hydrolysis of DNA samples. Results of the study showed good comparability among four of five participants and close agreement with reference values assigned by the coordinating laboratory. Conflicting data from one participant may have resulted from incomplete hydrolysis of samples due to use of insufficient amounts of enzymes. These results indicate that comparable and accurate results can be obtained from different measurement environments if digestion conditions are controlled appropriately and valid calibration systems are employed.

Separation-based glycoprofiling approaches using fluorescent labels.

Glycoprotein analysis is essential within the biopharmaceutical industry, as the structure of the different glycans present can affect the safety and efficacy of products. However analysis of cleaved glycans presents a major analytical challenge, due to their inherent complexity, lack of chromophore and the existence of various isoforms (both position and linkage). In addition, almost all glycoproteins consist of a heterogeneous collection of differently glycosylated variants, so the released glycan pool contains a range of structures. Both normal phase chromatography and capillary gel electrophoresis offer excellent selectivity for the analysis of fluorescently labelled glycans. The normal phase (NP) chromatographic approach is sensitive, reliable and well established, with databases available for searching structures assigned relative to retention times. Capillary gel electrophoresis with laser induced fluorescence (CGE-LIF) offers faster analysis times, though currently no databases are available to search mobilities against structures, therefore data has to be cross-correlated with either normal phase chromatography or mass spectrometry approaches when developing and validating methods. The principles of both methods are described and a review is presented that includes evaluation against a set of criteria established through consultation with the biopharmaceutical industry.