ausblenden:
Schlagwörter:
Absolute protein quantification; Escherichia coli proteome; FASP; Filter-aided sample preparation; Protein copy number; Total Protein Approach
Zusammenfassung:
UNLABELLED: We describe a proteomic approach combining the multi-enzyme digestion FASP-sample processing strategy and the 'Total Protein Approach' applied to absolute quantification of proteins in Escherichia coli. Consecutive digestion of whole cell lysates with LysC and trypsin allowed the generation of two populations of peptides at a yield of 76%. Subsequent two 4-hour LC-MS/MS analyses allowed the identification of 19,000 unique peptides per sample. Notably, only 1.2 and 2.4% of the identified peptides were found to be incompletely cleaved by the LysC and trypsin, respectively. The analysis resulted in the identification of 2200 proteins per sample. We show high reproducibility of the approach, allowing the accurate estimation of cellular protein concentrations. Quantitative analysis of the DNA content per sample enabled the calculation of the protein content per bacterial cell and, as a result, estimation of protein copy numbers. The accuracy of these estimations was confirmed by analyzing protein complexes with known subunit stoichiometry and cellular abundances. In stationary culture, a single bacterium contains about 6500 copies of ribosomes, 300 molecules of RNA polymerase and 10 DNA polymerase assembles. The here presented experimental and computational workflow offers an easy way to analyze proteomes quantitatively.; BIOLOGICAL SIGNIFICANCE: We demonstrate a proteomic workflow for in-depth analysis of small proteomes with minimal fractionation extent and mass spectrometry measuring time. For the first time we provide the quantitative picture of the Escherichia coli proteome at protein copy number. Copyright 2014. Published by Elsevier B.V.
