Purification and MALDI-MS characterization of stressin, a stress-associated glycoprotein

Glycoconjugates have a whole spectrum of biological roles, from those that appear trivial to those that are crucial. Results accumulated in the past y ears indicate they might also play an important role in the response to str ess, a complex physiological response of the human organism to various thre ats. We have recently identified stressin, a human serum glycoprotein, whic h was found to be increased under stress conditions. Here we report the pur ification of stressin from sera of professional soldiers and partial charac terization of its protein and carbohydrate parts using lectin blotting and matrix assisted laser desorption/ionization time-of-flight mass spectrometr y (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 m ass originated from N-linked oligosaccharides. The carbohydrate part contai ned 12 sialic acids moieties, nearly 90% of which were lost due to post-sou rce decay in the field-free tube. Tryptic fragments were produced from glycosylated and deglycosylated stress in, separated by reverse-phase HPLC and their exact molecular masses were d etermined using MALDI-MS. Comparison with tryptic maps of other proteins in computer databases indicated that stressin does not correspond to any alre ady described protein.