A proteomic approach to characterize protein shedding
| Autor: | Mamoun Ahram, David L. Springer, Deanna L. Auberry, David S. Wunschel, Joshua N. Adkins |
|---|---|
| Rok vydání: | 2005 |
| Předmět: |
Protein Denaturation
Proteome Proteolysis Peptide Proteomics Biochemistry Mass Spectrometry Phorbol Esters medicine Humans Biotinylation Endoplasmic Reticulum Chaperone BiP Molecular Biology Cells Cultured Heat-Shock Proteins chemistry.chemical_classification Membrane Glycoproteins medicine.diagnostic_test Membrane Proteins Epithelial Cells Trypsin Transmembrane protein Protein Structure Tertiary Transmembrane domain Hyaluronan Receptors Membrane protein chemistry Intercellular Signaling Peptides and Proteins Tetradecanoylphorbol Acetate Proteoglycans Syndecan-4 Glycolysis Chromatography Liquid Molecular Chaperones medicine.drug |
| Zdroj: | PROTEOMICS. 5:123-131 |
| ISSN: | 1615-9861 1615-9853 |
| DOI: | 10.1002/pmic.200400912 |
| Popis: | Shedding (i.e., proteolysis of ectodomains of membrane proteins) plays an important pathophysiological role. In order to study the feasibility of identifying shed proteins, we analyzed serum-free media of human mammary epithelial cells by mass spectrometry following induction of shedding by the phorbol ester, 4β-phorbol 12-myristate 13-acetate (PMA). Different means of sample preparation, including biotinylation of cell surface proteins, isolation of glycosylated proteins, and preparation of crude protein fraction, were carried out to develop the optimal method of sample processing. The collected proteins were digested with trypsin and analyzed by reversed-phase capillary liquid chromatography interfaced to an ion-trap mass spectrometer. The resulting peptide spectra were interpreted using the program SEQUEST. Analyzing the sample containing the crude protein mixture without chemical modification or separation resulted in the greatest number of identifications, including putatively shed proteins. Overall, 93 membrane-associated proteins were identified including 57 that contain at least one transmembrane domain and 36 that indirectly associate with the extracellular surface of the plasma membrane. Of the 57 transmembrane proteins, 43 were identified by extracellular peptides providing strong evidence for them originating from regulated proteolysis or shedding processes. We combined results from the different experiments and used a peptide count method to estimate changesmore » in protein abundance. Using this approach, we identified 2 proteins, syndecan-4 and hepatoma-derived growth factor, whose abundances increased in media of cells treated with PMA. We also detected proteins whose abundances decreased after PMA treatment such as 78-kDa glucose-regulated protein and calreticulin. Further analysis using immunoblotting validated the abundance changes for syndecan-4 and 78-kDa glucose-regulated protein as a result of PMA treatment. These results demonstrate that mass spectrometry can be used to identify low-abundance shed proteins and to estimate changes in protein abundances.« less |
| Databáze: | OpenAIRE |
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