WCID iCIEF coupled to MS and fractionation
WCID iCIEF has provided unique, reliable and high resolution protein charged based separation for biologics. Characterization of individual peaks is essential for comprehending the nature of product isomers and protein degradation pathway. It is therefore very desirable if WCID iCIEF can be coupled to MS or peak fractionation to provide structure characterization of the separated protein isomer. Unfortunately, there weren’t any solutions until the development of AES’ proprietary cartridge technology.
Coupling WCID iCIEF to MS or peak fractionation was a big technical challenging. WCID iCIEF is normally conducted with a short capillary about 5 cm in length. A long enough outlet transfer capillary connected to the separation capillary in WCID iCIEF is necessary to introduce separated proteins into the MS interface or fractionation collection vials. This is impossible to realize with conventional WCID iCIEF cartridges. The resolution achieved in the short separation capillary will be lost during the transfer process when the focused proteins are forced into the transfer capillary.
Coupling WCID iCIEF to MS or peak fractionation is realized with AES’ proprietary cartridges. AES developed the cartridge utilizing a column diameter transformation technique, in which a large (inner diameter) ID capillary is used for separation and small ID capillary for sample transfer. One typical cartridge has a 200 µm ID separation capillary and 50 µm ID transfer capillary. The focused protein zones inside the separation capillary will be continuously pushed out to the outlet transfer capillary after desired iCIEF separation is achieved. With this cartridge, the ID of the separation capillary is 4 times that of the outlet capillary. When 1 mm of protein zone from the separation capillary is forced directly into the outlet transfer capillary, it will occupy about 16 mm in length (illustrated in Figure 2).
The process of transferring a small section of focused protein into a much longer section effectively minimizes the potential remixing of separated proteins inside the transfer capillary. During syringe mobilization, the electric field can be adjusted to preserve the separation resolution. The difference in capillary ID greatly minimizes the remixing of separated protein isomers. Therefore, the resolution achieved during iCIEF will essentially be preserved. The longer and smaller transfer capillary, combined with electrical isolation between the separation capillary and transfer capillary allows great flexibility when applying reliable high resolution WCID iCIEF to other fields, such as protein fractionation, and spotting the separated protein elution to a MALDI target plate. Figure 3 shows high resolution iCIEF fractionation, fraction collection and fraction iCIEF of hemoglobin control AFSC, demonstrating the speed and efficiency of scanning iCIEF protein fractionation.