Purification and MALDI-MS Characterization of Stressin, a Stress-Associated Glycoprotein
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G. Lauc
Abstract
Glycoconjugates have a whole spectrum of biological roles, from those that appear trivial to those that are crucial. Results accumulated in the past years indicate they might also play an important role in the response to stress, a complex physiological response of the human organism to various threats. We have recently identified stressin, a human serum glycoprotein, which was found to be increased under stress conditions. Here we report the purification of stressin from sera of professional soldiers and partial characterization of its protein and carbohydrate parts using lectin blotting and matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS).
Stressin was purified using a combination of ammonium sulfate precipitation, ion exchange chromatography, preparative gel electrophoresis and reverse-phase HPLC. It was found to be a highly glycosylated protein. Only 21.9 kDa (out of 36.7 kDa) was the protein part, whereas the remaining 40% of the mass originated from N-linked oligosaccharides. The carbohydrate part contained 12 sialic acids moieties, nearly 90% of which were lost due to post-source decay in the field-free tube.
Tryptic fragments were produced from glycosylated and deglycosylated stressin, separated by reverse-phase HPLC and their exact molecular masses were determined using MALDI-MS. Comparison with tryptic maps of other proteins in computer databases indicated that stressin does not correspond to any already described protein.
Copyright © 1999 by Walter de Gruyter GmbH & Co. KG
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Articles in the same Issue
- Protein Kinase C-α Produces Reciprocal Effects on the Phorbol Ester Stimulated Tyrosine Phosphorylation of a 50 kDa Kinase in Jurkat Cells
- Growth- and Development-Dependent Expression of Gangliosides in Rat Hepatocytes and Liver Tissues
- Regulated Phosphorylation of the Gal4p Inhibitor Gal80p of <I>Kluyveromyces lactis</I> Revealed by Mutational Analysis
- The WW Domain of Dystrophin Requires EF-Hands Region to Interact with β-Dystroglycan
- Purification and MALDI-MS Characterization of Stressin, a Stress-Associated Glycoprotein
- Direct Selection of EGF Mutants Displayed on Filamentous Phage Using Cells Overexpressing EGF Receptor
- Refolding Studies on the Tetrameric Loop Deletion Mutant RM6 of ROP Protein
- Secretion of Protease Nexin-II/Amyloid β Protein Precursor by Human Colorectal Carcinoma Cells and Its Modulation by Cytokines/Growth Factors and Proteinase Inhibitors
- Isolation and Primary Structure of the CCI Papain-Like Cysteine Proteinases from the Latex of Carica candamarcensis Hook
- Identification of SLPI (Secretory Leukocyte Protease Inhibitor) in Human Mast Cells Using Immunohistochemistry and In Situ Hybridisation
- Stereochemistry of the Interaction between Methionine Sulfur and the Protein Core
- Trypsin from Pacifastacus leninsculus Hepatopancreas: Purification and cDNA Cloning of the Synthesized Zymogen
