| Abstract
| - A stepwise mobilization strategy has been developed forthe elution of complex protein mixtures, separated bycapillary isoelectric focusing (CIEF) for detection usingon-line electrospray ionization mass spectrometry (ESI-MS). Carrier polyampholytes are used to establish a pHgradient as well as to control the electroosmotic flowarising from the use of uncoated fused-silica capillaries.Elution of focused protein zones is achieved by controllingthe mobilization pressure and voltage, leaving the remaining protein zones focused inside the capillary. Proteinzones are stepwise eluted from the capillary by changingthe mobilization conditions. Stepwise mobilization improves separation resolution and simplifies coupling withmultistage MS (i.e., MSn) analysis since it allows moreeffective temporal control of protein elution from the CIEFcapillary. We also describe a modified configuration forcoupling CIEF with ESI-MS using a coaxial sheath flowinterface that facilitate the automation of on-line CIEF-ESI-MS analyses. The stepwise mobilization strategy isdemonstrated for the analysis of standard protein mixtures and soluble E. coli lysate proteins using CIEF-ESI-MS. These results indicate that inlet pressure or voltageprogramming to control the elution of the protein zonesfrom the capillary (i.e., gradient mobilization) may allowfor the optimization of the mobilization conditions andprovide higher resolution for CIEF separation of complexmixtures with on-line MS.
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