Hollow fiber flow field-flow fractionation (HF FlFFF) has been demonstrated as a tool for pre-fractionating proteomes by differences in molecular mass (Mr), where the resulting protein fractions are subsequently digested and analyzed by shotgun proteomics using two-dimensional liquid chromatography-electrospray ionization-tandem mass spectrometry (2D-LC-ESI-MS/MS). HF FlFFF is a separation device capable of fractionating proteins or cells by hydrodynamic radius, and protein fraction can be readily collected as intact conditions in aqueous buffer solutions. In this study, HF FlFFF was applied to fractionate the proteome of Corynebacterium glutamicum, a well known soil bacterium that has been widely used in bioindustry due to its remarkable ability to secrete high amounts of glutamic acid. The collected HF FlFFF fractions of different MW intervals were enzymatically digested for protein identification by 2D-LC-ESI-MS/MS. Experiments showed improvements in protein identification when HF FlFFF pre-fractionation was applied, due to decreases in the ionization suppression effect and the MS exclusion effect by spectral congestion. Pre-fractionation of C. glutamicum proteome allowed us to find 90 additional proteins by 2D-LC-ESI-MS/MS that were not found by a direct shotgun analysis without pre-fractionation. A total of 415 proteins were found overall with 203 proteins commonly found from experiments with and without pre-fractionation.
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