An integrated ten-pump, eight-channel parallel LC/MS system for automated high-throughput analysis of proteins

An integrated 10-pump eight-channel LC/MS system has been developed for aut omated high-throughput analysis of intact proteins in recombinant protein p urification processes. The key features of the system include (1) a compact 10-pump HPLC module that uses two pumps to generate a binary gradient and 8 pumps to deliver the mixed gradient to eight independent flow channels; ( 2) a TDF mass spectrometer with an eight-channel multiplexed ESI interface, which records separate data for all eight channels over each HPLC run cycl e; and (3) highly automated data processing software that allows unattended calculation of protein molecular weight (in Da) from original mass spectra l data (in m/z). This system was used in the routine screening of fractions from preparative scale chromatography to monitor the purification process with the required mass accuracy and throughput. As an example, the producti on and purification of an acylated protein with a molecular weight of 9 kDa is described. Using this off-line approach, it is practical to fully chara cterize protein-containing fractions from column chromatography with an ove rall analytical throughput of I min/protein sample with minimum operator in volvement.