Related Experiment Video
Updated: Jul 5, 2025
Glycomics-Guided Glycoproteomics Facilitates Comprehensive Profiling of the Glycoproteome in Complex Tumor Microenvironments
Published on: February 7, 2025
Comparative Analysis of Site-Specific N-glycosylation of LAMP1 from Breast Cancer Tissues
Shoko Ohashi1, Daisuke Takakura1, Noritoshi Kobayashi2
1Graduate School of Medical Life Science, Yokohama City University, 1-7-29 Suehiro-cho, Tsurumi-ku, Yokohama 230-0045, Japan.
Abstract:
Glycosylation changes in cancer proteins have been associated with malignant transformation. However, techniques for analyzing site-specific glycosylation changes in target proteins obtained from clinical tissue samples are insufficient. To overcome these problems, we developed a targeted N-glycoproteomic approach consisting of immunoprecipitation, glycopeptide enrichment, LC/MS/MS and structural assignment using commercially available analytical software followed by manual confirmation. This approach was applied to the comparative site-specific glycosylation analysis of lysosome-associated membrane glycoprotein 1 (LAMP1) between breast cancer (BC) tumors and normal tissues adjacent to tumors. Extensive determination of glycan heterogeneity from four N-glycosylation sites (Asn84/103/249/261) in LAMP1 identified 262 glycoforms and revealed remarkable diversity in tumor glycan structures. A significant increase in N-glycoforms with multiple fucoses and sialic acids at Asn84/249 and high-mannose-type glycans at Asn103/261 were observed in the tumor. Principal component analysis revealed that tumors of different subtypes have independent distributions. This approach enables site-specific glycopeptide analysis of target glycoprotein in breast cancer tissue and become a powerful tool for characterizing tumors with different pathological features by their glycan profiles.
Insights
New N-glycoproteomic methods reveal distinct glycosylation patterns in breast cancer (BC) proteins. These findings offer a powerful tool for characterizing tumor subtypes based on their unique glycan profiles.
Area of Science:
- Biochemistry
- Proteomics
- Cancer Research
Background:
- Aberrant protein glycosylation is a hallmark of cancer.
- Existing methods for analyzing site-specific glycosylation in clinical samples are limited.
Purpose of the Study:
- To develop and validate a targeted N-glycoproteomic approach for analyzing site-specific glycosylation in clinical breast cancer tissues.
- To compare the N-glycosylation profiles of lysosome-associated membrane glycoprotein 1 (LAMP1) between breast cancer tumors and adjacent normal tissues.
Main Methods:
- Developed a targeted N-glycoproteomic workflow involving immunoprecipitation, glycopeptide enrichment, LC/MS/MS, and structural assignment.
- Applied the method to analyze site-specific glycosylation of LAMP1 at four N-glycosylation sites (Asn84/103/249/261) in breast cancer and normal tissues.
Main Results:
- Identified 262 distinct glycoforms of LAMP1, revealing significant differences between tumor and normal tissues.
- Observed increased fucosylated and sialylated N-glycoforms at Asn84/249 and high-mannose glycans at Asn103/261 in tumors.
- Principal component analysis showed distinct glycan profile distributions for different breast cancer subtypes.
Conclusions:
- The developed targeted N-glycoproteomic approach is effective for site-specific glycopeptide analysis in breast cancer tissue.
- Glycan profiling of LAMP1 can serve as a powerful tool for characterizing tumors and distinguishing between different pathological features and subtypes.
