Optimization of solid phase microextraction - capillary zone electrophoresis - mass spectrometry for high sensitivity protein identification.
We have previously described the use of a solid phase extraction (SPE) - capillary zone electrophoresis (CZE) - tandem mass spectrometry (MS/MS) system for protein analysis at the low femtomole to subfemtomole level. Here we describe the systematic optimization of a number of parameters which facilitate the use of the SPE-CZE-MS/MS system and further enhance its performance. Specifically, we describe a robust SPE cartridge design which can be assembled without the use of glue, the evaluation of procedures to chemically modify the inner wall of the fused-silica capillaries used in the system to improve separation and reproducibility, and the comparison of different reverse-phase (RP) resins used for the SPE cartridge. We also explored the effects of transient isotachophoresis with respect to system performance and compatibility with different fused-silica surface coatings, the RP resins used, and MS/MS. The enhanced performance of the optimized system is demonstrated by the analysis of calibrated tryptic digests of bovine serum albumin (BSA).
Figeys
D
D
Department of Molecular Biotechnology, University of Washington, Seattle, USA. daniel.figeys@nrc.ca
Zhang
Y
Y
Aebersold
R
R
D000097:Acetonitriles; D004573:Electrolytes; D019075:Electrophoresis, Capillary / Q000295:instrumentation / Q000379:methods*; D006863:Hydrogen-Ion Concentration; D013058:Mass Spectrometry / Q000379:methods*; D010446:Peptide Fragments / Q000032:analysis / Q000378:metabolism; D011506:Proteins / Q000032:analysis*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012710:Serum Albumin, Bovine / Q000032:analysis / Q000378:metabolism; D014357:Trypsin
D000097:Acetonitriles; D004573:Electrolytes; D010446:Peptide Fragments; D011506:Proteins; D012710:Serum Albumin, Bovine; D014357:Trypsin; C032159:acetonitrile