Strategies for locating disulfide bonds in a monoclonal antibody via mass spectrometry

✍ Scribed by Rohin Mhatre; James Woodard; Chenhui Zeng

• Publisher: John Wiley and Sons
• Year: 1999
• Tongue: English
• Weight: 125 KB
• Volume: 13
• Category: Article
• ISSN: 0951-4198
• DOI: 10.1002/(sici)1097-0231(19991230)13:24<2503::aid-rcm818>3.0.co;2-w

✦ Synopsis

The location of the disulfide bonds in a recombinant monoclonal antibody was confirmed by matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) and electrospray ionization (ESI) mass spectrometry (MS). A non-reduced Endoproteinase Lys-C (Endo Lys-C) digest of the antibody was analyzed directly by MALDI-TOFMS. The sample was then reduced on-plate by depositing dithiothreitol (DTT) on the sample spot and re-analyzed by MALDI-TOFMS. The disulfide bonds were assigned based on the disappearance of certain mass ions in the non-reduced digest and the appearance of product ions in the reduced digest. A rapid LC/ESI-MS protocol was also developed to determine the location of the disulfide bonds. The peptides generated from the Endo Lys-C digest of the antibody were partially separated on a high performance liquid chromatography (HPLC) column by utilizing a steep gradient and analyzed by ESI-MS. The masses of the partially resolved peptides were determined by deconvoluting the mass spectra.