Electrophoresis combined with novel mass spectrometry techniques: powerful tools for the analysis of proteins and proteomes.
Analytical and preparative electrophoresis separation techniques have been essential tools in protein biochemistry and the biological sciences in general. The combination of high resolution electrophoresis techniques with high performance analytical procedures has dramatically enhanced analytical protein biochemistry. In this report we describe the combination of electrophoretic separation techniques with electrospray ionization (ESI) tandem mass spectrometry (MS/MS). A series of different techniques, consisting of automated high performance liquid chromatography (HPLC)-MS/MS, capillary-HPLC-MS/MS, and solid phase extraction (SPE)-capillary zone electrophoresis (CZE)-MS/MS, are described in the context of the identification of high pmol to the low fmol amounts of proteins. Application of these powerful new tools for the analysis of proteins on a large proteome-wide scale is presented. Furthermore, the combination of orthogonal separation techniques, such as immobilized metal affinity chromatography (IMAC) with SPE-CZE-MS/MS, and IMAC followed by HPLC, and by SPE-CZE-MS/MS, are presented for the detailed investigation of post-translational modifications of specific proteins.
Department of Molecular Biotechnology, University of Washington, Seattle, USA. daniel.figeys@nrc.ca
D
D
Figeys
S P
SP
Gygi
Y
Y
Zhang
J
J
Watts
M
M
Gu
R
R
Aebersold
D000595:Amino Acid Sequence; D001331:Automation; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D004586:Electrophoresis / Q000379:methods*; D019075:Electrophoresis, Capillary / Q000379:methods; D007553:Isotope Labeling; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000032:analysis; D010761:Phosphorus Radioisotopes; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000032:analysis*
D010750:Phosphoproteins; D010761:Phosphorus Radioisotopes; D011506:Proteins