Development and validation of proteomic technologies towards absolute quantitation of Pseudomonas aeruginosa membrane proteins
Quantification of proteins by liquid chromatography mass spectrometry (LC-MS) has become an established field. Many strategies have been designed to enable relative or absolute protein quantification for both global or targeted studies. The work presented reveals the limitations of the approaches cu...
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University of Cambridge
2011
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| Online Access: | http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.596757 |
| Summary: | Quantification of proteins by liquid chromatography mass spectrometry (LC-MS) has become an established field. Many strategies have been designed to enable relative or absolute protein quantification for both global or targeted studies. The work presented reveals the limitations of the approaches currently employed and suggests solutions to some of the challenges encountered. Methods were developed to that seek to improve current proteomic approaches both in terms of analytical throughput and proteomic coverage. A Quantitative concatamer (QconCAT) was designed to enable the quantification of the RND family of efflux proteins in <i>Pseudomonas aeruginosa</i>. A high throughput LC-MS method that enabled quantification of RND proteins within 10 minutes was developed. This approach revealed that biological variation was greater than the subsequent steps in quantification. This approach was then used to successfully quantify RND proteins within clinically isolated strains and compare their expression in biofilm and planktonic cultures. Comparison with AQUA type peptides revealed that Q-peptides can not be assumed to be present in stoichiometric amounts. Quantification of pure recombinant proteins also revealed that recovery of the surrogate peptide can not be assumed. The accuracy of quantification was variable and dependent upon the surrogate peptide used. An emerging non-isotope dilution strategy that makes quantitative measurements across multiple peptides was shown to quantify proteins from both simple and complex mixtures at similar levels to the isotope dilution strategies. When employed to quantify the membrane proteome of Pseudomonas aeruginosa it quantified 1092 proteins across three strains, spanning 3 orders of magnitude. |
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