2University of Zagreb, Faculty of Pharmacy and Biochemistry, A. Kovačića 1, 10000 Zagreb, Croatia
* To whom correspondence should be addressed.
Received February 2, 2015; Revision received February 24, 2015
Glycosylation affects structure, folding, and function of numerous proteins. Aberrant glycosylation has been shown to be associated with different diseases. A wide range of analytical methods is available for glycan analysis of antibodies (mainly IgG), but analysis of plasma glycans is less established due to additional challenges encountered with higher complexity of the sample. Here we describe development and optimization of a high-throughput sample preparation method for hydrophilic interaction liquid chromatography and ultra-performance liquid chromatography analysis of plasma N-glycans. Clean-up of labeled glycans was found to be the largest source of variation, and we tested cellulose, silica gel, Bio-Gel, and hydrophilic GHP filter as stationary phases for solid-phase extraction. All stationary phases were shown to be suitable for purification of labeled glycans, but GHP filter plate in combination with cold 96% acetonitrile had the highest reproducibility and was easiest to work with. The method was further optimized with Plackett–Burman screening design and validated in terms of analysis of major step variation and between-day and between-person variation. The developed method is fast, cost-effective, and easy to perform, and it has very good reproducibility during long period of time, enabling the detection of biological variability of the plasma N-glycome.
KEY WORDS: plasma, glycans, high-throughput, glycome analysis, HILIC, UPLC