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 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.
Feng
B
B
Department of Protein Biochemistry, GlaxoSmithKline, King of Prussia, PA 19406, USA. bingbing_2_feng@sbphrd.com
Patel
A H
AH
Keller
P M
PM
Slemmon
J R
JR
D000213:Acyl Carrier Protein / Q000032:analysis / Q000235:genetics / Q000302:isolation & purification; D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation / Q000379:methods*; D002852:Chromatography, Ion Exchange; D004867:Equipment Design; D008970:Molecular Weight; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000737:chemistry* / Q000302:isolation & purification; D021241:Spectrometry, Mass, Electrospray Ionization / Q000295:instrumentation / Q000379:methods*; D013056:Spectrophotometry, Ultraviolet; D013997:Time Factors
D000213:Acyl Carrier Protein; D011994:Recombinant Proteins