Identification of proteins in complexes by solid-phase microextraction/multistep elution/capillary electrophoresis/tandem mass spectrometry.
A method to directly identify proteins in complex mixtures by solid-phase microextraction (micro-SPE)/multistep elution/capillary electrophoresis (CE)/tandem mass spectrometry (MS/MS) is described. A sheathless liquid-metal junction interface is used to interface CE and electrospray ionization MS/MS. A subfemtomole detection limit is achieved for protein identification through database searching using MS/MS data. The SPE serves as a semiseparation dimension using an organic-phase step-elution gradient in combination with the second separation dimension for increased resolving power of complex peptide mixtures. This approach improves the concentration detection limit for CE and allows more proteins in complex mixtures to be identified. A 75-protein complex from yeast ribosome is analyzed using this method and 80-90% of the proteins in the complex can be identified by searching the database using the MS/MS data from a complete analysis. This multidimensional CE/MS/MS methodology provides an alternative to multidimensional liquid chromatography/MS/MS for direct identification of small amounts of protein in mixtures.
Department of Molecular Biotechnology, University of Washington, Seattle 98195-7730, USA.
W
W
Tong
A
A
Link
J K
JK
Eng
J R
JR
Yates
D019075:Electrophoresis, Capillary / Q000379:methods*; D006868:Hydrolysis; D013058:Mass Spectrometry / Q000379:methods*; D011506:Proteins / Q000032:analysis*; D014357:Trypsin
D011506:Proteins; D014357:Trypsin