Abstract
The Human Proteome Organisation Human Disease Glycomics/Proteome Initiative recently coordinated a multi-institutional study that evaluated methodologies that are widely used for defining the N-glycan content in glycoproteins. The study convincingly endorsed mass spectrometry as the technique of choice for glycomic profiling in the discovery phase of diagnostic research. The present study reports the extension of the Human Disease Glycomics/Proteome Initiative's activities to an assessment of the methodologies currently used for O-glycan analysis. Three samples of IgA1 isolated from the serum of patients with multiple myeloma were distributed to 15 laboratories worldwide for O-glycomics analysis. A variety of mass spectrometric and chromatographic procedures representative of current methodologies were used. Similar to the previous N-glycan study, the results convincingly confirmed the pre-eminent performance of MS for O-glycan profiling. Two general strategies were found to give the most reliable data, namely direct MS analysis of mixtures of permethylated reduced glycans in the positive ion mode and analysis of native reduced glycans in the negative ion mode using LC-MS approaches. In addition, mass spectrometric methodologies to analyze O-glycopeptides were also successful.
Highlights
The Human Proteome Organisation Human Disease Glycomics/Proteome Initiative recently coordinated a multiinstitutional study that evaluated methodologies that are widely used for defining the N-glycan content in glycoproteins
In this study, the O-glycan content of three IgA1 samples was analyzed by 15 participating laboratories. Six of these laboratories used either MALDI or ESI to obtain mass fingerprints of the hinge glycopeptide, three laboratories eliminated the O-glycans without reduction and tagged the free reducing ends prior to chromatographic analysis, and nine laboratories used reductive elimination to release the glycans, which were analyzed by MS after permethylation or as native glycans by LC-MS or MS alone
The fact that most of the sialic acid was preserved demonstrates that MALDI is a suitable tool for profiling sialylated glycoforms provided the linear ion mode is used in the TOF analysis
Summary
The Human Proteome Organisation Human Disease Glycomics/Proteome Initiative recently coordinated a multiinstitutional study that evaluated methodologies that are widely used for defining the N-glycan content in glycoproteins. IgA1 preparations from three patients with multiple myeloma were delivered by HGPI to 15 experienced academic laboratories, and the results of O-glycomics analysis, especially the total glycoform profiles, obtained using different analytical methodologies were assessed.
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