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Estimation of absolute protein quantities of unlabeled samples by selected reaction monitoring mass spectrometry.

Ludwig C, Claassen M, Schmidt A, Aebersold R - Mol. Cell Proteomics (2011)

Bottom Line: We found that a linear model based on the two most intense transitions of the three best flying peptides per proteins (TopPep3/TopTra2) generated optimal results with a cross-correlated mean fold error of 1.8 and a squared Pearson coefficient R(2) of 0.88.Applying the optimized model to lysates of the microbe Leptospira interrogans, we detected significant protein abundance changes of 39 target proteins upon antibiotic treatment, which correlate well with literature values.The described method is generally applicable and exploits the inherent performance advantages of SRM, such as high sensitivity, selectivity, reproducibility, and dynamic range, and estimates absolute protein concentrations of selected proteins at minimized costs.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Institute of Molecular Systems Biology, ETH Zürich, 8093 Zürich, Switzerland.

ABSTRACT
For many research questions in modern molecular and systems biology, information about absolute protein quantities is imperative. This information includes, for example, kinetic modeling of processes, protein turnover determinations, stoichiometric investigations of protein complexes, or quantitative comparisons of different proteins within one sample or across samples. To date, the vast majority of proteomic studies are limited to providing relative quantitative comparisons of protein levels between limited numbers of samples. Here we describe and demonstrate the utility of a targeting MS technique for the estimation of absolute protein abundance in unlabeled and nonfractionated cell lysates. The method is based on selected reaction monitoring (SRM) mass spectrometry and the "best flyer" hypothesis, which assumes that the specific MS signal intensity of the most intense tryptic peptides per protein is approximately constant throughout a whole proteome. SRM-targeted best flyer peptides were selected for each protein from the peptide precursor ion signal intensities from directed MS data. The most intense transitions per peptide were selected from full MS/MS scans of crude synthetic analogs. We used Monte Carlo cross-validation to systematically investigate the accuracy of the technique as a function of the number of measured best flyer peptides and the number of SRM transitions per peptide. We found that a linear model based on the two most intense transitions of the three best flying peptides per proteins (TopPep3/TopTra2) generated optimal results with a cross-correlated mean fold error of 1.8 and a squared Pearson coefficient R(2) of 0.88. Applying the optimized model to lysates of the microbe Leptospira interrogans, we detected significant protein abundance changes of 39 target proteins upon antibiotic treatment, which correlate well with literature values. The described method is generally applicable and exploits the inherent performance advantages of SRM, such as high sensitivity, selectivity, reproducibility, and dynamic range, and estimates absolute protein concentrations of selected proteins at minimized costs.

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Biological reproducibility of model selection and calibration curve generation.A and B, to test the reproducibility of the determined mean fold error distributions based on varying peptide and transition combinations, we performed the Monte Carlo cross-validation analysis on three biological L. interrogans samples: a control sample (Fig. 3A), 12 h of treatment with ciprofloxacin (A) and 24 h of treatment with ciprofloxacin (B). C, overlay of linear calibration curves generated for the three different biological samples over a measurement period of 70 h. Each data point represents an averaged value of three technical replicates.
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Figure 4: Biological reproducibility of model selection and calibration curve generation.A and B, to test the reproducibility of the determined mean fold error distributions based on varying peptide and transition combinations, we performed the Monte Carlo cross-validation analysis on three biological L. interrogans samples: a control sample (Fig. 3A), 12 h of treatment with ciprofloxacin (A) and 24 h of treatment with ciprofloxacin (B). C, overlay of linear calibration curves generated for the three different biological samples over a measurement period of 70 h. Each data point represents an averaged value of three technical replicates.

Mentions: To test the biological reproducibility of the obtained mean fold error distribution based on peptide/transition combinations, we performed a Monte Carlo cross-validation analysis on two additional L. interrogans samples. The samples were total cell lysates from cells treated for 12 or 24 h with the antibiotic ciprofloxacin. For each sample, we determined absolute protein abundances (Table I) and generated a label-free best flyer peptide data set using SRM (supplemental Table S2). The above described mean fold error trends were highly similar over all three samples (compare Fig. 3A with Fig. 4, A and B), supporting the universality of the results for the given sample type.


Estimation of absolute protein quantities of unlabeled samples by selected reaction monitoring mass spectrometry.

Ludwig C, Claassen M, Schmidt A, Aebersold R - Mol. Cell Proteomics (2011)

Biological reproducibility of model selection and calibration curve generation.A and B, to test the reproducibility of the determined mean fold error distributions based on varying peptide and transition combinations, we performed the Monte Carlo cross-validation analysis on three biological L. interrogans samples: a control sample (Fig. 3A), 12 h of treatment with ciprofloxacin (A) and 24 h of treatment with ciprofloxacin (B). C, overlay of linear calibration curves generated for the three different biological samples over a measurement period of 70 h. Each data point represents an averaged value of three technical replicates.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3316728&req=5

Figure 4: Biological reproducibility of model selection and calibration curve generation.A and B, to test the reproducibility of the determined mean fold error distributions based on varying peptide and transition combinations, we performed the Monte Carlo cross-validation analysis on three biological L. interrogans samples: a control sample (Fig. 3A), 12 h of treatment with ciprofloxacin (A) and 24 h of treatment with ciprofloxacin (B). C, overlay of linear calibration curves generated for the three different biological samples over a measurement period of 70 h. Each data point represents an averaged value of three technical replicates.
Mentions: To test the biological reproducibility of the obtained mean fold error distribution based on peptide/transition combinations, we performed a Monte Carlo cross-validation analysis on two additional L. interrogans samples. The samples were total cell lysates from cells treated for 12 or 24 h with the antibiotic ciprofloxacin. For each sample, we determined absolute protein abundances (Table I) and generated a label-free best flyer peptide data set using SRM (supplemental Table S2). The above described mean fold error trends were highly similar over all three samples (compare Fig. 3A with Fig. 4, A and B), supporting the universality of the results for the given sample type.

Bottom Line: We found that a linear model based on the two most intense transitions of the three best flying peptides per proteins (TopPep3/TopTra2) generated optimal results with a cross-correlated mean fold error of 1.8 and a squared Pearson coefficient R(2) of 0.88.Applying the optimized model to lysates of the microbe Leptospira interrogans, we detected significant protein abundance changes of 39 target proteins upon antibiotic treatment, which correlate well with literature values.The described method is generally applicable and exploits the inherent performance advantages of SRM, such as high sensitivity, selectivity, reproducibility, and dynamic range, and estimates absolute protein concentrations of selected proteins at minimized costs.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Institute of Molecular Systems Biology, ETH Zürich, 8093 Zürich, Switzerland.

ABSTRACT
For many research questions in modern molecular and systems biology, information about absolute protein quantities is imperative. This information includes, for example, kinetic modeling of processes, protein turnover determinations, stoichiometric investigations of protein complexes, or quantitative comparisons of different proteins within one sample or across samples. To date, the vast majority of proteomic studies are limited to providing relative quantitative comparisons of protein levels between limited numbers of samples. Here we describe and demonstrate the utility of a targeting MS technique for the estimation of absolute protein abundance in unlabeled and nonfractionated cell lysates. The method is based on selected reaction monitoring (SRM) mass spectrometry and the "best flyer" hypothesis, which assumes that the specific MS signal intensity of the most intense tryptic peptides per protein is approximately constant throughout a whole proteome. SRM-targeted best flyer peptides were selected for each protein from the peptide precursor ion signal intensities from directed MS data. The most intense transitions per peptide were selected from full MS/MS scans of crude synthetic analogs. We used Monte Carlo cross-validation to systematically investigate the accuracy of the technique as a function of the number of measured best flyer peptides and the number of SRM transitions per peptide. We found that a linear model based on the two most intense transitions of the three best flying peptides per proteins (TopPep3/TopTra2) generated optimal results with a cross-correlated mean fold error of 1.8 and a squared Pearson coefficient R(2) of 0.88. Applying the optimized model to lysates of the microbe Leptospira interrogans, we detected significant protein abundance changes of 39 target proteins upon antibiotic treatment, which correlate well with literature values. The described method is generally applicable and exploits the inherent performance advantages of SRM, such as high sensitivity, selectivity, reproducibility, and dynamic range, and estimates absolute protein concentrations of selected proteins at minimized costs.

Show MeSH