Gastric cancer has one of the highest cancer mortality rates worldwide, largely because of difficulties in early-stage detection. Aberrant glycosylation in serum proteins is associated with many human diseases including inflammation and various types of cancer. Serum-based global glycan profiling using mass spectrometry has been explored and has already led to several potential glycan markers for several disease states. However, localization of the aberrant glycosylation is desirable in order to improve the specificity and sensitivity for clinical use. Here, we combined protein-specific immunoaffinity purification, glycan release, and MS analysis to examine haptoglobin glycosylation of gastric cancer patients for glyco-markers. Age- and sex-matched 60 serum samples (30 cancer patients and 30 healthy controls) were used to profile and quantify haptoglobin N-glycans. A T-test based statistical analysis was performed to identify potential glyco-markers for gastric cancer. Interestingly, abundances of several tri- and tetra-antennary fucosylated N-glycans were increased in gastric cancer patients. Additionally, structural analysis via LC/MS/MS indicated that the fucosylated complex type N-glycans were primarily decorated with antenna fucose, which can be categorized as sialyl-Le or sialyl-Le type structures. This platform demonstrates quantitative, structure-specific profiling of haptoglobin glycosylation for the purposes of biomarker discovery for gastric cancer.
Based on our previous studies, differential analysis of N-glycan expression bound on serum haptoglobin reveals the quantitative variation on gastric cancer patients. In this prospective case-control study, we explore the clinically relevant glycan markers for gastric cancer diagnosis. Serum samples were collected from patients with gastric cancer (n = 44) and healthy control (n = 44). N-glycans alteration was monitored by intact analysis of Hp using liquid chromatography–mass spectrometry followed by immunoaffinity purification with the serum samples. Intensity and frequency markers were defined depending on the mass spectrometry data analysis. Multiple markers were found with high diagnostic efficacy. As intensity markers (I-marker), six markers were discovered with the AUC > 0.8. The high efficiency markers exhibited AUC of 0.93 with a specificity of 86% when the sensitivity was set to 95%. We additionally established frequency marker (f-marker) panels based on the tendency of high N-glycan expression. The AUC to conclude patients and control group were 0.82 and 0.79, respectively. This study suggested that N-glycan variation of serum haptoglobin were associated with patients with gastric cancer and might be a promising marker for the cancer screening.
Glycosylation plays an important role in ensuring the proper structure and function of most biotherapeutic proteins. Even small changes in glycan composition, structure, or location can have a drastic impact on drug safety and efficacy. Recently, glycosylation has become the subject of increased focus as biopharmaceutical companies rush to create not only biosimilars, but also biobetters based on existing biotherapeutic proteins. Against this backdrop of ongoing biopharmaceutical innovation, updated methods for accurate and detailed analysis of protein glycosylation are critical for biopharmaceutical companies and government regulatory agencies alike. This review summarizes current methods of characterizing biopharmaceutical glycosylation, including compositional mass profiling, isomer-specific profiling and structural elucidation by MS and hyphenated techniques.
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