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Site-Specific Intact N-Linked Glycopeptide Characterization of Prostate-Specific Membrane Antigen from Metastatic
Stephen Mackay1,2,3, Naomi L Hitefield1,2,4, Ian O Oduor1
1Leroy T. Canoles Jr. Cancer Research Center, Eastern Virginia Medical School, Norfolk, Virginia 23507, United States.
ACS Omega
|September 5, 2022
Summary
This study characterizes N-linked glycans on prostate-specific membrane antigen (PSMA) in prostate cancer cells. Differences in PSMA glycosylation were identified, offering potential targets for disease progression.
Area of Science:
- Biochemistry
- Proteomics
- Glycomics
Background:
- The N-linked glycan composition of prostate-specific membrane antigen (PSMA) and its role in prostate cancer progression remain incompletely understood.
- PSMA is a key biomarker in prostate cancer, and its glycosylation may influence its function and behavior.
Purpose of the Study:
- To comprehensively analyze and compare the site-specific N-linked glycopeptides of PSMA from two distinct metastatic prostate cancer cell lines.
- To establish a methodology for detailed glycopeptide analysis that can be applied to clinical samples.
Main Methods:
- Isolation of PSMA from LNCaP and MDAPCa2b prostate cancer cell lines.
- Intact glycopeptide analysis using liquid chromatography-high-collision-energy-dissociation-electrospray-ionization-tandem-mass-spectrometry (LC-HCD-EThcD-MS/MS) on a Tribrid Orbitrap Fusion Lumos mass spectrometer.
- Differential quantitative analysis of site-specific N-glycopeptides using Byonic and Byologic software, with confirmation by endoglycosidase treatment and lectin capture.
Main Results:
- Significant differences in the abundance of multiple glycopeptides at specific asparagine residues (51, 76, 121, 195, 336, 459, 476, and 638) were observed between the two cell lines (p < 0.05).
- Biochemical validation confirmed the mass spectrometry-based glycopeptide and site occupancy data.
- The developed strategy proved effective for comprehensive PSMA glycopeptide analysis.
Conclusions:
- The study successfully characterized site-specific N-linked glycosylation of PSMA in different prostate cancer cell lines, revealing significant quantitative differences.
- This analytical approach provides a foundation for investigating PSMA glycan alterations in clinical samples to identify potential biomarkers for disease progression and metastasis.
