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Anal. Chem. 1992, 64, 2303-2305
CORRESPONDENCE
Application of Plasma Desorption Mass Spectrometry to Molecular Weight Determination of Human Interleukin-4 Secreted by a Chinese Hamster Ovary Cell Line Anthony Tsarbopoulos,' Guor-Rong Her3 Birendra N. Pramanik, Paul P. Trotta, and Tattanahalli L. Nagabhushan Schering-Plough Research Institute, 60 Orange Street, Bloomfield, New Jersey 07003
INTRODUCTION Human interleukin-4 (IL-41, previously known as B-cell stimulatory factor-1,1p2is a T-cell derived lymphokine which mediates the proliferation and differentiation of B cells: and also stimulates mature T-cell pr~liferation.~The gene encoding human IL-4 has been cloned6 and expressed in Chinese Hamster Ovary (CHO) cells.6 The mature polypeptide is 129 amino acid residues long and contains two asparagine residues in the consensus sequence Asn-X-Serl Thr (where X can be any amino acid except possibly proline and aspartic acid) which is required for attachment of Asnlinked carbohydrate.' Not only do the carbohydrate chains give specific physiological properties on the glycoprotein molecule, but they may also play a significant role in its biological function, which is important in subsequent clinical applications of a recombinant glycoprotein. They may stabilize the protein conformation, protect the glycoprotein from proteolytic degradation, resulting in an increased lifetime in circulating blood, and participate in receptor binding and/or in subsequent activation steps. The maturation of the carbohydrate chains attached to a glycoprotein depends on the cellular expression system used, and the resulting composition and structure of the carbohydrate chains may affect the biological activity of the recombinant product. There is a need, therefore, to determine the size, as well as defiie any possible molecular heterogeneity, of the carbohydrate component rapidly and with high sensitivity. Plasma desorption (PD) time-of-flight mass spectrometry has been used previously to determine the size and the carbohydrateheterogeneity of glycosylated ovomucoids8and the mannose heterogeneity of a tryptic glycopeptide from
* Author to whom correspondence should be addressed.
Current addreas: Department of Chemistry, National Taiwan University, Taipei, Taiwan. (1) Sanderson, C. J.; O'Garra, A,; Warren, D. J.; Klaus, G . G. B. Proc. Natl. Acad. Sci. U.S.A. 1986,83,437-440. (2) Howard, M.; Farrar, J.; Hilfiker, M.; Johnson, B.; Takatsu, K.; Hamaoka, T.; Paul, W. E. J. Ezp. Med. 1982,155,914-923. (3) Coffman, R. L.; Ohara, J.; Bond, M. W.; Carty, J.; Zlotnik, A.; Paul, W.E. J. Zmmunol. 1986,4538-4541. (4) Zlotnik, A.; Ransom, J.; Frank, G.;Fischer, M.; Howard, M. Proc. Natl. Acad. Sei. U.S.A. 1987,84, 3856-3860. (5) Yokota, T.; Otsuka, T.; Mosmann, T.; Banchereau, J.; DeFrance, T.; Blanchard, D.; de Vries, J. E.; Arai, K. Proc. Natl. Acad. Sci. U.S.A. 1986,83,5894-5898. (6) Le, H. V.; Ramanathan, L.; Labdon, J. E.; Mays-Ichinco, C.; Syto, R.; Arai, N.; Hoy, P.; Takebe, Y.; Nagabhushan, T. L.; Trotta, P. P. J. Biol. Chem. 1988,263,10817-10823. " (7) Kornfeld, R.; Kornfeld, S. Annu. Reo. Biochem. 1985,54,631-664. (8)Wang, R.; Cotter, R. J.; Lin, T.-Y.; Laskowski,M. Rapid Commun. Mass Spectrom. 1988,2,71-73.
the variant surface glycoprotein of trypano~omes.~In this report, we discuss the use of PD mass spectrometry to assess the size and possible heterogeneity of the IL-4 glycoprotein expressed in CHO cells and we compare the PD mass spectral results to those obtained by SDS-gel electrophoresis. Even though there have been several reports on molecular weight (MW) measurement of glycoproteins by either eledrosprayl ion-spraylO and matrix-assisted laser desorption ionization11 mass spectrometry, this is to our knowledge the first application of PD mass spectrometry for MW determination of a glycoprotein sample weighing more than 10 OOO and having a higher than 10% carbohydrate content.
EXPERIMENTAL SECTION Materials. Recombinant human interleukin-4 (IL-4) was derived and purified from CHO cells (Schering-PloughResearch Institute, Bloomfield, NJ/DNAX Research Institute, Palo Alto, CA),as previously described! Peptides-glycosidase F (PNGase F/N-Glycanase) was purchased from Genzyme (Boston, MA), while trifluoroacetic acid (TFA) was high purity obtained from Pierce Chemical Co. (Rockford, IL). Monosaccharide Analysis. Compositional analysis of the oligosaccharide moieties of CHO IL-4,'* carried out by highperformance anion-exchange chromatography coupled with pulsed amperometric detection, resulted in the following molar concentrations: Man (1.9), GlcN (3.9), Gal (2.11, N-acetylneuraminic (sialic) acid (1.9), Fuc (0.7)and GalN (
