Fast characterization of intact proteins using a high-throughput eight-channel parallel liquid chromatography/mass spectrometry system |
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Authors: | Feng B Patel A H Keller P M Slemmon J R |
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Institution: | Department of Protein Biochemistry, GlaxoSmithKline, King of Prussia, PA 19406, USA. bingbing_2_feng@sbphrd.com |
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Abstract: | The preparation of protein substrates requires that a large number of chromatographic fractions be analyzed for the presence of reactants, products and by-products. Analyses using linear matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) or single column liquid chromatography/mass 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 the required mass accuracy and throughput so that the protein purification process can be monitored in a relatively short period of time. As an example, the purification and analysis of an acylated protein with a molecular weight 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-containing fractions from column chromatography in less than 1 hour using parallel LC/MS. |
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