Chip-type asymmetrical flow field-flow fractionation channel coupled with mass spectrometry for top-down protein identification

A chip-type design asymmetrical flow field-flow fractionation (AF4) channel has been developed for high-speed separation of proteins and top-down proteomic analysis using online coupled electrospray ionization mass spectrometry (ESI-MS). The new miniaturized AF4 channel was assembled by stacking multilayer thin stainless steel (SS, 1.5 mm each) plates embedded with an SS frit in such a way that the total thickness of the channel assembly was about 6 mm. The efficiency of the miniaturized AF4 channel at different channel lengths was examined with the separation of protein standards by adjusting flow rates in which an identical effective channel flow rate or an identical void time can be maintained at different channels. Detection limit, overloading effect, reproducibility, and influence of channel membrane materials on separation efficiency were investigated. Desalting and purification of proteins achieved during the AF4 operation by the action of an exiting crossflow and the use of aqueous mass-spectrometry-compatible (MS-compatible) buffer were advantageous for online coupling of the chip-type AF4 with ESI-MS. The direct coupling of AF4 and ESI-MS capabilities was demonstrated for the high-speed separation and identification of carbonic anhydrase (29 kDa) and transferrin (78 kDa) by full scan MS and for the first top-down identification of proteins with AF4-ESI-MS-MS using collision-induced fragmentation (CID). The presence of intact dimers (156 kDa) of transferrin was confirmed by AF4-ESI-MS via size separation of the dimers from monomers, followed by multiply charged ion spectral analysis of the dimers and molecular mass determinations. It was also found from these experiments that AF4-ESI-MS analysis of transferrin exhibited an increased signal-to-noise ratio compared to that of direct ESI-MS analysis due to online purification of the protein sample and size separation of dimers with AF4.