Automated Intact Glycopeptide Enrichment Method Facilitating Highly Reproducible Analysis of Serum Site-Specific N-Glycoproteome

Abstract

Bottom-up proteomics has been increasingly applied in clinical research to study the disease pathophysiology and to discover disease biomarkers. However, glycoproteomic analysis always requires tedious experimental steps for intact glycopeptide enrichment, which has been the technique bottleneck for large-scale analysis of clinical samples. Herein, we developed an automated glycopeptide enrichment method for the analysis of serum site-specific N-glycoproteome. This automated method allowed for processing one sample within 20 min. It showed higher enrichment specificity, more intact glycopeptide identifications, and better quantitative reproducibility than the traditional manual method using microtip enrichment devices. We further applied this method to investigate the serum site-specific N-glycosylation changes between four patients with pancreatic cancer and seven healthy controls. The principal component analysis of intact N-glycopeptides showed good clustering across cancer and normal groups. Furthermore, we found that the site-specific glycoforms, monofucosylated and nonsialylated oligosaccharides, on IgG1 site 180 expressed a significant decrease in pancreatic cancer patients compared to healthy controls. Together, the automated method is a powerful tool for site-specific N-glycoproteomic analysis of complex biological samples, and it has great potential for clinical utilities.

Methods for quantification of glycopeptides by liquid separation and mass spectrometry

“…HILIC displays broad applications and outstanding performance in comprehensive glycoproteomic analyses of biological samples (L. Liu et al, 2021;Q. Shu et al, 2020;Zacharias et al, 2016;.…”

Advances in mass spectrometry‐based glycoproteomics: An update covering the period 2017–2021

“…Recently, Liu et al. [87] developed an automated enrichment method based on a HILIC column to analyze serum site‐specific N‐ glycoproteomes. A total of 2081 N‐ glycopeptides from 190 glycoproteins were identified.…”

“…Recently, various hydrophilic materials, such as hydrophilic interaction chromatography (HILIC) materials, metal-organic frameworks (MOFs), polymer, and magnetic materials have been used for glycopeptide enrichment. Recently, Liu et al [87] developed an automated enrichment method based on a HILIC column to analyze serum site-specific N-glycoproteomes. A total of 2081 Nglycopeptides from 190 glycoproteins were identified.…”

Synthesis of Surface-Functionalized Molybdenum Disulfide Nanomaterials for Efficient Adsorption and Deep Profiling of the Human Plasma Proteome by Data-Independent Acquisition

“…Blood has long been considered as an optimal sample to monitor the physiological and pathological changes of the whole body. − As a form of “liquid biopsy”, protein tests on blood have received increasing attention for the discovery of disease biomarkers and monitoring of the therapeutic response to treatment. − Compared with single-plex immunoassays, multiplexing, highly specific, antibody-independent, and hypothesis-free mass spectrometry (MS)-based proteomic technology is the dominant method for high-throughput blood proteome profiling and biomarker screening. − Although great efforts have been devoted to discovering blood biomarkers to aid in early disease diagnosis and therapeutic response monitoring, few proteins have been successfully validated and have received FDA approval in recent years; thus, there is extensive research interest in developing new sample treatment and mass spectrometry (MS)-based methods for biomarker screening. ,− The major challenge in MS-based blood biomarker development is the highly complex nature of plasma/serum samples. Plasma/serum proteins have an extremely large abundance dynamic range that spans more than 10 orders of magnitude, with disease-derived proteins often present at the lower end of this range .…”