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Updated: Jun 26, 2026
Glycopeptide Capture for Cell Surface Proteomics
Published on: May 9, 2014
Identification of secreted proteins regulated by cAMP in glioblastoma cells using glycopeptide capture and label-free
Jennifer J Hill1, Maria J Moreno, Jean C Y Lam
1Institute for Biological Sciences, National Research Council Canada, Ottawa, ON, Canada. jennifer.hill@nrc-cnrc.gc.ca
Abstract:
Exposure of glioblastoma U87MG cells to a cAMP analog leads to a decrease in proliferation, invasion, and angiogenic potential. Here, we apply a label-free MS-based approach to identify formerly N-linked glycopeptides that change in abundance upon cAMP treatment. Over 150 unique glycopeptides in three biological repetitions were quantified, leading to the identification of 14 upregulated proteins and 21 downregulated proteins due to cAMP treatment. Of these, eight have been validated, either through comparison with microarray data or by Western blot. We estimate our ability to identify differentially expressed peptides at greater than 85% in a single biological repetition, while the analysis of multiple biological repetitions lowers the false positive rate to approximately 2%. Many of the proteins identified in this study are involved in cell signaling and some, such as Tenascin C, Cathepsin L, Neuroblastoma suppressor of tumorigenicity, and AXL/UFO tyrosine-protein kinase receptor, have been previously shown to be involved in glioblastoma progression. We also identify several semitryptic peptides that increase in abundance upon cAMP treatment, suggesting that cAMP regulates protease activity in these cells. Overall, these results demonstrate the benefits of using a highly specific enrichment method for quantitative proteomic experiments.
Insights
Cyclic adenosine monophosphate (cAMP) treatment reduces glioblastoma cell growth and invasion. This study used mass spectrometry to identify key proteins affected by cAMP, revealing potential new therapeutic targets for glioblastoma.
Area of Science:
- Proteomics
- Cancer Biology
- Molecular Signaling
Background:
- Glioblastoma is an aggressive brain tumor with limited treatment options.
- Cyclic adenosine monophosphate (cAMP) is a crucial second messenger involved in various cellular processes.
- Understanding how cAMP affects glioblastoma at a molecular level is vital for developing new therapies.
Purpose of the Study:
- To investigate the proteomic changes in glioblastoma U87MG cells upon treatment with a cAMP analog.
- To identify specific N-linked glycopeptides and proteins whose abundance is altered by cAMP.
- To validate the identified differentially expressed proteins.
Main Methods:
- Label-free, mass spectrometry (MS)-based quantitative proteomic approach.
- Analysis of N-linked glycopeptides.
- Validation using microarray data and Western blot.
- Utilized three biological repetitions for robust data analysis.
Main Results:
- Quantified over 150 unique glycopeptides.
- Identified 14 upregulated and 21 downregulated proteins following cAMP treatment.
- Validated eight differentially expressed proteins.
- Demonstrated high accuracy (>85%) in identifying differentially expressed peptides, with a low false positive rate (~2%) using multiple repetitions.
- Identified proteins involved in cell signaling, including those known to impact glioblastoma progression (e.g., Tenascin C, Cathepsin L).
- Observed an increase in semitryptic peptides, suggesting cAMP regulates protease activity.
Conclusions:
- cAMP analog treatment significantly impacts glioblastoma cell proliferation, invasion, and angiogenic potential.
- The study highlights the utility of a label-free MS-based approach with specific enrichment for quantitative proteomics.
- Identified proteins and pathways regulated by cAMP offer potential targets for glioblastoma therapy.
