Glycopeptide Enrichment Methods And Establishment Of Disease-related Research Glycopeptides

Glycosylation is one of the most common post-translational modifications (PTMs). A variety of biological functions have been proposed for the carbohydrate moiety of the glycoproteins. Detailed structural analysis of glycoproteins requires methods capable of isolating glycopeptides from tryptic digests of purified glycoproteins and complex protein mixtures. It is common knowledge that approximately50%of proteins in plasma are glycosylated, and abnormal glycosylation is closely related with the development of disease. Leukemia is a malignant tumor in blood systeme. The changes in glycoproteins and oligosaccharide structures according to therapeutic stages of leukemia patients can provide new experimental clues for leukemia treatment.In present study, we developed a simple, effective and economical approach to enrich glycopeptides, which named GA-ziptip. Then GA-chromtogrophy were developed, which can analyze glycopeptides on line with mass spectrometry. Glycoproteins in plasma from normal and leukemia patients were enriched using multi-lectin affinity chromatography, followed by separation by two-dimensional gel electrophroesis. The profiles of glycoprotein derived from leukemia patients with different stages were compared with the healthy, which revealed that haptoglobin-P chain was related with the process of leukemia. Then the glycopeptides structure of haptoglobin-β chain in plasma derived from patients with leukemia were analyzed using GA-ziptip and LC-MS. Mass spectrometric analyses demonstrated that the attachments of sialylated glycans of haptoglobin-β chain abundantly increased in the plasma of leukemia patients, and decreased along with the treatment progress, revealing that the amount changes of sialylated glycans attached to the haptoglobin were related with the prognosis of leukemia.These findings suggest that haptoglobin-β chain and the binding glycans could serve as a novel marker for treatment monitoring and prognosis for leukemia. The strategy used in present study is a novel approach for analysis of glycoproteins.