Integrated Capillary Zone Electrophoresis–Electrospray Ionization Tandem Mass Spectrometry System with an Immobilized Trypsin Microreactor for Online Digestion and Analysis of Picogram Amounts of RAW 264.7 Cell Lysate

Abstract

A capillary zone electrophoresis (CZE) electrospray ionization (ESI) tandem mass spectrometry (MS/MS) system was integrated with an immobilized trypsin microreactor. The system was evaluated and then applied for online digestion and analysis of picogram loadings of RAW 264.7 cell lysate. Protein samples were dissolved in a buffer containing 50% (v/v) acetonitrile (ACN), and then directly loaded into the capillary for digestion, followed by CZE separation and MS/MS identification. The organic solvent (50% (v/v) ACN) assisted the immobilized trypsin digestion and simplified the protein sample preparation protocol. Neither protein reduction nor alkylation steps were employed, which minimized sample loss and contamination. The integrated CZE–ESI-MS/MS system generated confident identification of bovine serum albumin (BSA) with 19% sequence coverage and 14 peptide identifications (IDs) when 20 fmol was loaded. When only 1 fmol of BSA was injected, one BSA peptide was consistently detected. For the analysis of a standard protein mixture, the integrated system produced efficient protein digestion and confident identification for proteins with different molecular weights and isoelectric points when a low-femtomole amount was loaded for each protein. We further applied the system for triplicate analysis of a RAW 264.7 cell lysate; 2 ± 1 and 7 ± 2 protein groups were confidently identified from only 300 pg and 3 ng loadings, respectively. The 300 pg sample loading corresponds to the protein content of three RAW 264.7 cells. In addition to high-sensitivity analysis, the integrated CZE–ESI-MS/MS system produces good reproducibility in terms of peptide and protein IDs, peptide migration time, and peptide intensity