Proteome analysis of Escherichia coli using high-performance liquid chromatography and Fourier transform ion cyclotron resonance mass spectrometry.
The basic problem of complexity poses a significant challenge for proteomic studies. To date two-dimensional gel electrophoresis (2-DE) followed by enzymatic in-gel digestion of the peptides, and subsequent identification by mass spectrometry (MS) is the most commonly used method to analyze complex protein mixtures. However, 2-DE is a slow and labor-intensive technique, which is not able to resolve all proteins of a proteome. To overcome these limitations gel-free approaches are developed based on high performance liquid chromatography (HPLC) and Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS). The high resolution and excellent mass accuracy of FT-ICR MS provides a basis for simultaneous analysis of numerous compounds. In the present study, a small protein subfraction of an Escherichia coli cell lysate was prepared by size-exclusion chromatography and proteins were analyzed using C4 reversed phase (RP)-HPLC for pre-separation followed by C18 RP nanoHPLC/nanoESI FT-ICR MS for analysis of the peptide mixtures after tryptic digestion of the protein fractions. We identified 231 proteins and thus demonstrated that a combination of two RP separation steps - one on the protein and one on the peptide level - in combination with high-resolution FT-ICR MS has the potential to become a powerful method for global proteomics studies.
Ihling
Christian
C
Biotechnological-Biomedical Center, Faculty of Chemistry and Mineralogy, University of Leipzig, Germany.
Sinz
Andrea
A
D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D017357:Cyclotrons; D029968:Escherichia coli Proteins / Q000032:analysis*; D005583:Fourier Analysis; D013058:Mass Spectrometry / Q000379:methods*; D020543:Proteome / Q000032:analysis*
D029968:Escherichia coli Proteins; D020543:Proteome