Glycomics-Guided Glycoproteomics Facilitates Comprehensive Profiling of the Glycoproteome in Complex Tumor Microenvironments

被引:0
作者
Chau, The Huong [1 ]
Bansal, Naaz [1 ]
Chernykh, Anastasia [1 ]
Kawahara, Rebeca [1 ,2 ]
Thaysen-Andersen, Morten [1 ,2 ]
机构
[1] Macquarie Univ, Fac Sci & Engn, Sch Nat Sci, Macquarie Pk, Australia
[2] Nagoya Univ, Inst Glyco Core Res iGCORE, Nagoya, Japan
来源
JOVE-JOURNAL OF VISUALIZED EXPERIMENTS | 2025年 / 216期
基金
澳大利亚研究理事会;
关键词
PAIRED COLORECTAL-CANCER; N-GLYCOPROTEOME; GLYCOSYLATION; GLYCAN; IDENTIFICATION; GLYCOPEPTIDE; PROTEINS; GRANULES; TOOLS;
D O I
10.3791/67405
中图分类号
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号
07 ; 0710 ; 09 ;
摘要
Glycosylation is a common and structurally diverse protein modification that impacts a wide range of tumorigenic processes. Mass spectrometry-driven glycomics and glycoproteomics have emerged as powerful approaches to analyze liberated glycans and intact glycopeptides, respectively, offering a deeper understanding of the heterogeneous glycoproteome in the tumor microenvironment (TME). This protocol details the glycomics-guided glycoproteomics method, an integrated omics technology, which firstly employs porous graphitized carbon-LC-MS/MS-based glycomics to elucidate the glycan structures and their quantitative distribution in the glycome of tumor tissues, cell populations, or bodily fluids being investigated. This allows for a comparative glycomics analysis to identify altered glycosylation across patient groups, disease stages, or conditions, and, importantly, serves to enhance the downstream glycoproteomics analysis of the same sample(s) by creating a library of known glycan structures, thus reducing the data search time and the glycoprotein misidentification rate. Focusing on N-glycoproteome profiling, the sample preparation steps for the glycomics-guided glycoproteomics method are detailed in this protocol, and key aspects of the data collection and analysis are discussed. The glycomicsguided glycoproteomics method provides quantitative information on the glycoproteins present in the TME and their glycosylation sites, site occupancy, and site-specific glycan structures. Representative results are presented from formalin-fixed paraffin- embedded tumor tissues from colorectal cancer patients, demonstrating that the method is sensitive and compatible with tissue sections commonly found in biobanks. Glycomics-guided glycoproteomics, therefore, offers a comprehensive view into the heterogeneity and dynamics of the glycoproteome in complex TMEs, generating robust biochemical data required to better understand the glycobiology of cancers.
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页数:24
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