Detection of Aberrant Glycosylation of Serum Haptoglobin for Gastric Cancer Diagnosis Using a Middle-Up-Down Glycoproteome Platform

Abstract

Gastric cancer is a frequently occurring cancer and is the leading cause of cancer-related deaths. Recent studies have shown that aberrant glycosylation of serum haptoglobin is closely related to gastric cancer and has enormous potential for use in diagnosis. However, there is no platform with high reliability and high reproducibility to comprehensively analyze haptoglobin glycosylation covering microheterogeneity to macroheterogeneity for clinical applications. In this study, we developed a middle-up-down glycoproteome platform for fast and accurate monitoring of haptoglobin glycosylation. This platform utilizes an online purification of LC for sample desalting, and an in silico haptoglobin glycopeptide library constructed by combining peptides and N-glycans to readily identify glycopeptides. In addition, site-specific glycosylation with glycan heterogeneity can be obtained through only a single MS analysis. Haptoglobin glycosylation in clinical samples consisting of healthy controls (n = 47) and gastric cancer patients (n = 43) was extensively investigated using three groups of tryptic glycopeptides: GP1 (including Asn184), GP2 (including Asn207 and Asn211), and GP3 (including Asn241). A total of 23 individual glycopeptides were determined as potential biomarkers (p < 0.00001). In addition, to improve diagnostic efficacy, we derived representative group biomarkers with high AUC values (0.929 to 0.977) through logistic regression analysis for each GP group. It has been found that glycosylation of haptoglobin is highly associated with gastric cancer, especially the glycosite Asn241. Our assay not only allows to quickly and easily obtain information on glycosylation heterogeneity of a target glycoprotein but also makes it an efficient tool for biomarker discovery and clinical diagnosis.

High‐throughput immunoaffinity enrichment and N‐glycan analysis of human plasma haptoglobin

“…In conclusion, optimal conditions for Hp N-glycans release and et al, 2012). Not all of these less complex glycans reported by O'Flaherty et al were recently confirmed on a glycopeptide level (Chandler et al, 2013;Jeong et al, 2021;Pompach et al, 2013), so their presence in low abundance could be explained by contamination from other plasma proteins, as well as at least partially by the differences in used methodological approaches.…”

“…Before lectin microarray enrichment (Miura et al, 2018) and 2‐DE, another option was a depletion of plasma of most abundant proteins like albumin and immunoglobulin G (IgG) or even sequential capturing of plasma proteins and their glycosylation analysis (Klein et al, 2010; O'Flaherty et al, 2019; Wilson et al, 2002; Zhao et al, 2007). Moreover, methods for Hp purification and enrichment established so far were generally low‐throughput, limited to individual sample preparation using affinity columns, and applied to a few dozen samples (Jeong et al, 2021; Pompach et al, 2013), therefore we are still lacking a fast and efficient method for large‐scale studies tackling Hp N‐glycosylation changes in different diseases or population studies.…”

First bioelectronic immunoplatform for quantitative secretomic analysis of total and metastasis-driven glycosylated haptoglobin

“…In addition to abundant alterations in the proteome, posttranslational modifications (PTMs) of proteins deserve attention in early detection of diseases and their monitoring. Glycosylation is one of the most important PTMs and plays a critical role in various biological processes, such as protein function and cell-cell interaction, which makes it as an important biomarker for cancer and infectious diseases [1][2][3].…”

“…In this way, they function as important modulators for cellcell interaction and influence the fate and behaviors of these cells [4]. To improve the specificity and sensitivity of diagnosis, some studies have been directed toward the in-depth characterization of glycosylation of specific target glycoproteins [3,5]. It is important to note that direct analysis of intact glycoproteins, rather than glycan profiling, which is still considered the gold standard for its high throughput and sensitivity, accounts advantageous because of its simplicity, ease of sample preparation, and analysis time saving.…”

“…Among them, MS is widely used, and it is considered one of the most precise techniques for qualitative and quantitative analyses of glycosylation [6]. However, it shows limitations for its implementation in the clinic due to the cost and multiple steps required for sample preparation, the difficulties of analysis and the complex interpretation of the data [2,3]. Therefore, it would be ideal to have tools for the rapid, easy, and accurate determination of glycosylated biomarkers more compatible with their use in the clinic.…”