Identification of secreted proteins regulated by cAMP in glioblastoma cells using glycopeptide capture and label-free quantification.
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.
Hill
Jennifer J
JJ
Institute for Biological Sciences, National Research Council Canada, Ottawa, ON, Canada. jennifer.hill@nrc-cnrc.gc.ca
Moreno
Maria J
MJ
Lam
Jean C Y
JC
Haqqani
Arsalan S
AS
Kelly
John F
JF
D015153:Blotting, Western; D045744:Cell Line, Tumor; D002853:Chromatography, Liquid; D000242:Cyclic AMP / Q000494:pharmacology*; D004591:Electrophoresis, Polyacrylamide Gel; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D005909:Glioblastoma / Q000378:metabolism*; D006020:Glycopeptides / Q000737:chemistry*; D006801:Humans; D013058:Mass Spectrometry; D011485:Protein Binding; D020543:Proteome / Q000032:analysis* / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*
D006020:Glycopeptides; D020543:Proteome; D000242:Cyclic AMP