TR&D 4

Chromatographic & Electrophoretic Separations Optimized for Native MS — Leads: Susan Olesik, Lisa Holland, Florian Busch

Direct infusion of native protein samples into the mass spectrometer is problematic when the sample complexity is too high and causes ion suppression or signal overlap. We are improving methods for the separation of native/folded proteins and protein complexes using chromatographic and electrophoretic techniques, both offline and directly coupled online with MS analysis. In addition, improved ionization approaches for coupling the separation method to the mass spectrometer will optimize chance of remaining native.

 

Aim 1

Developing chromatographic separation strategies for native protein complexes
  • Develop offline and online size exclusion chromatography (SEC) and ion exchange chromatography (IEC) coupled to native MS for smaller sample sizes
  • Conversion of electrospray ionization (ESI) to nESI at column exit for fractionation and control of charge states via contained electrospray
  • Develop offline ion exchange (IEX) separations via improved planar electrospun nanofiber plates
  • Ionization and detection of native proteins separated by nanofiber plates via desorption electrospray (DESI)-MS and integrated nESI

Aim 2

Improving capillary electrophoresis for separation of native proteins/complexes
  • Adapt commercial capillaries for separation under native electrolyte conditions
  • Develop novel capillary coatings and fillings for improved separation of native proteins and protein complexes