Original

Development of Large-Scale Protein Identification Technology Using High Resolution LC-MS

Masato Taoka

Laboratory of Biochemistry, Department of Chemistry, Graduate School of Science, Tokyo Metropolitan University, 1-1 Minami-osawa, Hachioji-shi, Tokyo, Japan

Abstract:

The author demonstrated a newly developed fully integrated online two-dimensional (2 D) LC-MS/MS system that allows non-gel based mass identification of components in a complex protein mixture by analyzing its proteolytic digests. The system was composed of a 2 DLC, which consists of two independent HPLC one with an ion exchange and the other with a reversed phase capillary columns, a hybrid type MS spectrometer with an electrospray ionization source, and a software program for retrieval of mass spectral information. The system successfully showed that proteins present in standard mixtures could be identified at fmol levels over a 300-fold difference in molar quantity with significant reproducibly. Approximately 4,000 peptides derived from 1,000 proteins, which include the proteins rarely seen in 2 D-PAGE coupled mass spectrometric analysis, were identified in a single analytical run from E. coli K 12 lysate by using the system. This 2 DLC-MS/MS system can be used for large-scale protein and peptide analysis in strategy of proteomics, in particular, for mass.spectrometric identification of protein components in crude biological sources.

Keywords: proteomics, twodimensional, liquid chromatography, tandem mass spectrometry, Escherichia coli

[Full Text PDF]