Mining of serum glycoproteins by an indirect approach using cell line secretome |
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Authors: | Younghee Ahn Un-Beom Kang Joon Kim Cheolju Lee |
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Institution: | 1.Life Sciences Division,Korea Institute of Science and Technology,Seoul,Korea;2.School of Life Sciences and Biotechnology,Korea University,Seoul,Korea |
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Abstract: | Glycosylation is the most important and abundant post-translational modification in serum proteome. Several specific types
of glycan epitopes have been shown to be associated with various types of disease. Direct analysis of serum glycoproteins
is challenging due to its wide dynamic range. Alternatively, glycoproteins can be discovered in the secretome of model cell
lines and then confirmed in blood. However, there has been little experi-mental evidence showing cell line secretome as a
tractable target for the study of serum glycoproteins. We used a hydrazine-based glycocapture method to selectively enrich
glycoproteins from the secretome of the breast cancer cell line Hs578T. A total of 132 glycoproteins were identified by nanoLC-MS/MS
analysis. Among the identified proteins, we selected 13 proteins that had one or more N-glycosylation motifs in the matched peptides, which were included in the Secreted Protein Database but not yet in the Plasma
Proteome Database (PPD), and whose antibodies were commercially available. Nine out of the 13 selected proteins were detected
from human blood plasma by western analysis. Furthermore, eight proteins were also detected from the plasma by targeted LC-MS/MS,
which had never been previously identified by data-dependent LC-MS/MS. Our results provide novel proteins that should be enrolled
in PPD and suggest that analysis of cell line secretome with subfractionation is an efficient strategy for discovering disease-relevant
serum proteins. |
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