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Cited by 222

The DOI matches its Crossref record; 10 of the 10 references it cites were found in a registry or an index.

Multi-enzyme digestion FASP and the ‘Total Protein Approach’-based absolute quantification of the Escherichia coli proteome

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Wiśniewski, Jacek R., Rakus, Dariusz·2014·Journal of Proteomics
DOI 10.1016/j.jprot.2014.07.012
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Abstract

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.

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Show all 10 references

Research works printed but not linked: 24

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Metadata

Authors
Wiśniewski, J. R., & Rakus, D.
Venue
Journal of Proteomics
Publisher
Elsevier BV
Published
2014-09
Volume
109
Pages
322-331
ISSN
1874-3919 (ISSN-L)
DOI
10.1016/j.jprot.2014.07.012
OpenAlex
W2031070745
Cited by
222
References
10
FWCI
6.80
Type
journal article
Language
en
Open access
hybrid
Licence

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Wiśniewski, J. R., & Rakus, D. (2014). Multi-enzyme digestion FASP and the ‘Total Protein Approach’-based absolute quantification of the Escherichia coli proteome. Journal of Proteomics. https://doi.org/10.1016/j.jprot.2014.07.012

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