Purification of Derivatized Oligosaccharides by Solid Phase Extraction for Glycomic Analysis

Citation: Zhang Q, Li H, Feng X, Liu B-F, Liu X ( Purification of Derivatized Oligosaccharides by Solid Phase Extraction for Glycomic Analysis Qiwei Zhang 0 Henghui Li 0 Xiaojun Feng 0 Bi-Feng Liu 0 Xin Liu 0 Nikos K. Karamanos, University of Patras, Greece 0 Britton Chance Center for Biomedical Photonics at Wuhan National Laboratory for Optoelectronics-Hubei Bioinformatics and Molecular Imaging Key Laboratory, Systems Biology Theme, Department of Biomedical Engineering, College of Life Science and Technology, Huazhong University of Science and Technology , Wuhan , China Profiling of glycans released from proteins is very complex and important. To enhance the detection sensitivity, chemical derivatization is required for the analysis of carbohydrates. Due to the interference of excess reagents, a simple and reliable purification method is usually necessary for the derivatized oligosaccharides. Various SPE based methods have been applied for the clean-up process. To demonstrate the differences among these methods, seven types of self-packed SPE cartridges were systematically compared in this study. The optimized conditions were determined for each type of cartridge and it was found that microcrystalline cellulose was the most appropriate SPE material for the purification of derivatized oligosaccharide. Normal phase HPLC analysis of the derivatized maltoheptaose was realized with a detection limit of 0.12 pmol (S N21 = 3) and a recovery over 70%. With the optimized SPE method, relative quantification analysis of N-glycans from model glycoproteins were carried out accurately and over 40 N-glycans from human serum samples were determined regardless of the isomers. Due to the high stability and sensitivity, microcrystalline cellulose cartridge showed potential applications in glycomics analysis. - Glycosylation represents one of the most complex and widespread post-translational modifications of human proteins. N-linked and O-linked glycosylations are two major patterns that are related to the structure and function of the glycoproteins [13]. Aberrant glycosylation profile may lead to dramatic changes of the proteins in the activity, distribution as well as stability [47]. Thus, the analysis of glycosylation is extensively conducted in biological and clinical research as well as in pharmaceutical industry [810]. Different methods have been reported, including high performance liquid chromatography (HPLC) [11,12], capillary electrophoresis [13,14] and high pH anion-exchange chromatography [15,16] with electrochemical, optical or mass spectrometric (MS) detection [17,18]. To enhance the detection sensitivity, oligosaccharides are usually derivatized prior to analysis. Most of such derivatization reactions can be accomplished by coupling the reagents with the reducing end of saccharides [17,19]. During this step, reagents, including salts, derivatization reagents and solvent are normally presented in large excess. Thus, a clean-up procedure has to be implemented to remove the excess reagents that often interfere with the following detection [20]. Various methods have been applied for the clean-up processes, such as paper chromatography, gel filtration, precipitation and solid-phase extraction (SPE) [2124]. Among these strategies, SPE based approaches have been widely employed due to its diverse stationary phases, rapid and simple operation, and high throughput [25]. There are two major types of stationary phases for SPE methods: carbon and hydrophilic materials. As a typical carbon material, porous graphitized carbon (PGC) shows good performance in the purification of non-derivatized glycans [8,26]. However, it presents some drawbacks for labeled samples. The elution for PGC required the use of solutions containing 0.1% trifluoroacetic acid (TFA), which could cause the loss of sialic acid residues if it was not removed properly [27]. Moreover, most of the labels consist of aromatic nucleus, which have strong adsorption to PGC, resulting in difficulty in the removal of excess labels from the derivatized oligosaccharides. In contrast, hydrophilic interaction chromatography (HILIC) SPE method can overcome this problem. In this method, the glycans are retained on stationary phases by hydrophilic interaction, whereas excess labels can be removed due to their lower hydrophilicity than the glycans [20]. Therefore, HILIC SPE method is becoming the most suitable strategy for the purification of derivatized glycans. Several stationary phases have been reported for this method, including DPA-6S, microcrystalline cellulose (MCC) and cotton [2729]. Discovery DPA-6S column showed good performance in the recovery of labeled oligosaccharides [28]. The MCC-based SPE method showed high reproducibility for the fast sample purification [27]. But MCC was always used in the loose state without being compressed, which might reduce sample recovery. Cotton SPE microtips could remove salts, most nonglycosylated peptides, and detergents such as sodium dodecyl sulfate from microscale samples [29]. Although these HILIC SPE methods were widely employed for the purification of derivatized oligosaccharides, a complete comparison of these methods on terms of recovery and reproducibility has not yet been reported before. In this study, we 95% ACN (v/v), 5.0 mL 80% ACN and 3% FA (v/v), 5.0 mL 95% ACN (v/v), 10.0 mL Acetone/Ethanol = 1/1 (v/v), 15.0 mL 80% ACN (v/v), 15.0 mL Hexane/Acetic acid = 3/2 (v/v), 8.0 mL 80% ACN and 5% FA (v/v), 10.0 mL systematically compared the performances of different materials as the stationary phases of SPE cartridges, including DPA-6S, MCC, DSC-CN, DSC-Si, DSC-NH2, DSC-Diol and ZIC-HILIC. Since it is difficult to fill cotton into the cartridge evenly, we exclude the test of cotton. The aim of this study is to find out the most appropriate HILIC SPE method and to establish a standard strategy for the purification of labeled oligosaccharides. By comparing the recovery and reproducibility, it was found that MCC was the most appropriate material and chosen for the next experiments. The washing conditions were optimized and the differences of two reaction conditions were demonstrated by a relative quantitative research. The MCC cartridge was further applied to the purification of biological samples followed by HPLC analysis. Results showed that MCC held high potential for the analysis of complex and trace-level samples such as the human serum. Materials and Methods Chemicals and Reagents DSC-Si, DSC-CN, DSC-NH2, DSC-Diol, ZIC-HILIC, DPA6S, maltoheptaose, 2-aminobenzoic acid (2-AA), Fetuin, ribonuclease B (RNase B) and human serum were purchased from Sigma-Aldrich (MO, U.S.A.). N-glycosidase F (PNGase F) and endoglycosidase buffer pack (EBP) were obtained from New England Biolabs (MA, U.S.A.). Deionized water was purified using a Milli-Q device (Millipore, MA, U.S.A.). Acetic acid, formic acid (FA), TFA, dimethyl sulfoxide (DMSO), sodium cyanoborohydride, ammonium forma (...truncated)