Stepwise mobilization of focused proteins in capillary isoelectric focusing mass spectrometry

A stepwise mobilization strategy has been developed for the elution of comp lex protein mixtures, separated by capillary isoelectric focusing (CIEF) fo r detection using on line electrospray ionization mass spectrometry (ESI-MS ). Carrier polyampholytes are used to establish a pH gradient as well as to control the electroosmotic flow arising from the use of uncoated fused-sil ica capillaries, Elution of focused protein zones is achieved by controllin g the mobilization pressure and voltage, leaving the remaining protein zone s focused inside the capillary. Protein zones are stepwise eluted from the capillary by changing the mobilization conditions. Stepwise mobilization im proves separation resolution and simplifies coupling with multistage MS (i. e., MSn) analysis since it allows more effective temporal control of protei n elution from the CIEF capillary. We also describe a modified configuratio n for coupling CIEF with ESI-MS using a coaxial sheath flow interface that facilitate the automation of on-line CIEF-ESI-MS analyses. The stepwise mob ilization strategy is demonstrated for the analysis of standard protein mix tures and soluble E, coli lysate proteins using CIEF-ESI-MS. These results indicate that inlet pressure or voltage programming to control the elution of the protein zones from the capillary (i,e., gradient mobilization) may a llow for the optimization of the mobilization conditions and provide higher resolution for CIEF separation of complex mixtures with on-line MS.