Fast characterization of intact proteins using a high-throughput eight-channel parallel liquid chromatography mass spectrometry system

The preparation of protein substrates requires that a large number of chrom atographic fractions be analyzed for the presence of reactants, products an d by-products. Analyses using linear matrix-assisted laser desorption/ioniz ation mass spectrometry (MALDI-MS) or single column liquid chromatography/m ass spectrometry (LC/MS) have been inadequate because of mass resolution or throughput. Therefore, a high-throughput method employing an eight-channel parallel reverse-phase LC/MS system was developed. This system is capable of screening fractions from preparative ion-exchange chromatography with th e required mass accuracy and throughput so that the protein purification pr ocess can be monitored in a relatively short period of time. As an example, the purification and analysis of an acylated protein with a molecular weig ht of 8.9 kDa is described and the detection of a contaminating by-product that differs in size by less than 20 Da is demonstrated. Using the current instrumentation and approach, it is practical to analyze 50 protein-contain ing fractions from column chromatography in less than 1 hour using parallel LC/MS. Copyright (C) 2001 John Wiley & Sons, Ltd.