| Despite remarkable progress made by modern science in cancer research in the past decades, cancer is still a leading cause of death in many countries. Early diagnosis is very important for cancer treatment, significantly improving the therapy efficacy and survival rate. Cancer biomarkers are usually specific endogenous biomolecules which can be monitored to provide disease related information for the diagnosis and treatment of cancers. They are usually used for cancer screening, early diagnosis, efficacy assessment and cancer patient’s prognosis monitoring. Most of the biomarkers approved by FDA for cancer diagnosis and administration response monitoring are glycosylated proteins. Glycoproteins involved in various biological processes are formed when glycans covalently attach to polypeptide side chains. Proteins with abnormal glycosylation have been revealed to play a significant role in cancer occurrence, and some of them have been developed into cancer biomarkers.Human chorionic gonadotropin (hCG) is a very important glycoprotein hormone mainly produced by the placental syncytiotrophoblast cells. It is a heterodimer consisted of an a subunit and a β subunit, combining together by non-covalent hydrophobic and ionic interactions. Hyper-glycosylated hCG (hCG-H), subjected to the so-called "hyperglycosylation", is a major glycosylation variant of hCG with more complex oligosaccharide chains. hCG-H is produced by cytotrophoblast cells during implantation or choriocarcinoma cells. hCG-H is also an important cancer biomarker and functions in invasion and growth of various germ cell tumors.The accuracy and sensitivity of cancer diagnosis will be greatly improved, if we can establish an accurate and rapid approach for both hCG concentration measurement and hCG-H monitoring. A lot of commercial diagnostic products have been developed for hCG detection, but they do not meet the current needs of related disease diagnosis with the following reasons.1) Current immunodiagnostic products are based on the hypothesis that the binding ability of antibodies is not affected by the glycosylation status of hCG, and the individual variation of hCG’s glycosylation pattern is ignored.2) The existing diagnostic products are limited to analyze the concentrations of hCG in the samples, lacking in further consideration whether the hCG is abnormally glycosylated.Screening antibody which could distinguish hCG-H from normal hCG is the key point to solve the above problems. One goal of this paper is to screen out satisfactory and specific monoclonal antibodies (mAbs), followed by epitope analysis with hydrogen/deuterium exchange-mass spectrometry approach. We illustrated the influence on binding ability of mAbs by glycosylated status from the perspective of spatial location between the oligosaccharides and epitopes. This mechanism provided valuable theoretical basis for antibody screening of other tumor-related glycoprotein biomarkers, and lay the foundation for the development of new generation of immunodiagnostic kits.Low molecular weight heparins (LMWHs) are oligosaccharide drugs prepared by chemical or enzymatic depolymerization of heparin, they are mainly used for prevention and treatment of deep vein thrombosis and pulmonary thrombosis clinically. Recent studies show that LMWHs are potential anticancer drugs due to their activities aiming at cancer cell for proliferation inhibition, apoptosis induction, and tumor-antiangiogenesis interference. The oligosaccharide components of LMWHs are very complex, featured by strong heterogeneity. Structural characterization is essential for preparative process optimization, safety evaluation and generic drugs approval. There is important application value to establish a sensitive and efficient mass spectrometry analysis method. Another purpose of this dissertation is to develop a liquid chromatography-mass spectrometry (LC-MS) method for LMWHs.The main achievements of this paper are as follows:1. Method establishment to purify hCG from biological samples.Filtration, ultraflltration and immuno-affinity chromatography were combined to develop an approach for hCG purification. The results of SDS-PAGE demonstrated hCG purified from invasive mole patients smeared to a much broader band than that from healthy pregnant women, indicating its possible hyperglycosylated pattern. With help of in-gel digestion and mass spectrometry identification, we confirmed the purified protein was hCG.2. Comprehensive analysis of glycosylated sites and N-linked oligosaccharides of hCG.O-glycopeptides obtained after tryptic digestion were subjected to a series of specific exoglycosidases containing β-N-acetylglucosaminidase, sialidase A and β (1-3,4) galactosidase, leaving a single GalNAc residue attached to the Ser/Thr. Followed by nanoLC-MS/MS analysis, Thr-54 of a subunit in hCG was originally found O-glycosylated.N-glycans of hCG and hCG-H released by PNGase F and recovered by PGC-SPE (Porous graphitized carbon, PGC) were analyzed through PGC-ESI-MS method. The results showed the percentage of N-glycans with more branched antennas, primarily tetra-antennary, was significantly larger, suggesting hCG-H purified from the cancer patients was hyperglycosylated.3. Screening out two types of mAb based on whether the KD value is affected by hCG glycosylated status or not.The BLI technique (Bio-layer interferometry, BLI) was adopted to determine affinity dissociation constants (Kd) of different mAbs with hCG and hCG-H. We found that the KD values of MCA329 with hCG and hCG-H were similar, illustrating that the binding between MCA329 and hCG is not influenced by glycosylation status. The KD value of MCA1024 with hCG is much lower than that of MCA329 with hCG-H, showing that the binding force between MCA 1024 and normal hCG is stronger and affected by its glycosylation status.4. Epitope mapping analysis of different mAbs using hydrogen-deuterium exchange (HDX) and LC-MS.We analyzed the epitopes of MCA329 and MCA1024 using HDX and LC-MS. The deuterium exchange efficiency of three peptides,β65-77,β78-83 and β116-133, decreased obviously after the combination of hCG β and MCA329. The peptide β109-145 deficiency at C-terminal of hCG βdid not affect the antibody binding associated with epitope β2, which indicates peptide β116-133 is not included in the epitope of MCA329. The decrease in deuterium exchange efficiency of β116-133 occurred probably because the binding of MCA329 to one terminal of hairpin structure within hCG β65-83 happens near the C-terminal, which limits the rotation of β116-133. While the deuterium exchange efficiency of peptides β65-77 and β78-83 decreased apparently after the combination of hCG β and MCA 1024, suggesting the epitope of MCA1024 is also located within β65-77. The affinity of MCA1024 is affected by hCG glycosylation status while three N-glycosylation sites (Asn-52 of a subunit, Asn-13 and Asn-30 of β subunit) are distributed to the other terminal of hairpin structure within hCG 065-83. The follow-up analysis of these three specific N-glycosylation sites shows that the Asn-13 and Asn-30 on hCG-H β subunit are obviously hyperglycosylated, while the Asn-52 on a subunit is not glycosylated. We inferred the hyperglycosylation of Asn-13 and Asn-30 led to the decreased affinity of MCA 1024, which is evidenced by affinity increase after partial deglycosylation of hCG-H.5. Intact LMWH chain analysis by a robust RPIP-ESI-MS method.A RPIP-ESI-MS (Reversed phase ion pair electrospray ionization mass spectrometry) method is established for intact LMWHs (Low molecular weight heparins) chain analysis. This approach can be adopted for both the high-quality fingerprint mapping and compositional analysis of LMWHs. With the help of a semi-automatic spectrum interpreting software, more than 140 enoxaparin oligosaccharides and 80 nadroparin oligosaccharides ranging from dp4 to dp26 are identified with mass error within 20 ppm. Furthermore, this method can also be used for the identification of trace amount of impurities. |
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