Selective Enrichment of Glycopeptides Using Aluminum Oxide

Protein glycosylation is significantly associated with cells cycle, immune regulation, cells recognition and cells adhesion. Aberrant glycosylation expression is involved in many diseases, such as rheumatoid arthritis, cancer, and chronic obstructive pulmonary disease. Thus, characterization of the protein glycosylation is very important to understand the life process. However, detection of glycopeptides is difficult by mass spectrometry because of the low concentration in complex sample and the suppressed signal by non-glycopeptides. Therefore, it is essential to explore an effective technique for glycopeptide enrichment. In this study, an alumina based-materials was used to selectively enrich glycopeptides. Firstly, we investigated the retention mechanism by changing the concentration of acetonitrile (ACN). With the decreased concentration of ACN, it was found that peptides were eluted according to their hydrophilicity. Moreover, most of the non-glycopeptides were eluted earlier than the glycopeptides in the high concentration of ACN fraction and the glycopeptides were found in the low concentration of ACN fraction. This result proved that hydrophilic interaction is one of the retention mechanisms for peptides retained by alumina. Secondly, we investigated the retention mechanism by changing the concentration of ammonium hydroxide. ACN were fixed at a high concentration and peptides were subsequently eluted with different concentration of ammonium hydroxide solution. At a low concentration of ammonium hydroxide solution, the peptides were no found. But at a high concentration of ammonium hydroxide solution, the glycopeptides and non-glycopeptides were eluted simultaneously. This proved that the ligand exchange is also one of the retention mechanisms, where the retention of the glycopeptides and non-glycopeptides showed no different under this mechanism. Based on the above mentioned results, the enrichment condition was optimized under the model of solid-phase extraction. High concentration of ACN mixed with a certain concentration of ammonium hydroxide solution as loading buffer and low concentration of ACN mixed with a certain concentration of ammonium hydroxide solution as elution buffer. The number of glycopeptides enriched by alumina were compared with glycopeptides before enrichment and enriched by Sepharose. In the tryptic HRP digest, 7 glycopeptides were found before enrichment, 16 glycopeptides were found by alumina and 14 glycopeptides were found by Sepharose. In the tryptic IgG digest, 2 glycopeptides were found before enrichment, 12 glycopeptides were found by alumina and 4 glycopeptides were found by Sepharose. In conclusion, alumina based-method used to enrich glycopeptides has high selection and wide coverage.