Absolute protein quantification - to obtain absolute measurements of protein concentrations in complex samples we used a liquid chromatography / tandem mass spectrometry (LC-MS/MS) method that is a modification of previous work in absolute quantification making use of either
... external or internal standards with or without isotopically labelled synthetic peptides as standard [1-3]. The main characteristics of the current approach are that it combines mass spectrometric analysis in the multiple reaction monitoring mode (MRM), for specific and sensitive quantification of target proteins, with the use of peptides derived from purified recombinant proteins as external calibrants.
To first optimize the MS settings and select MRM parameters, LC-MS/MS analysis of Lys-C (Wako, Osaka, Japan) digests of each purified recombinant target protein was performed essentially as previously described [4]. Completion of digestion of both purified proteins and crude E. coli extracts was confirmed by measuring peptide yields with time and using different concentrations of Lys-C. LC-MS/MS analysis of peptides was performed on a QSTAR XL hybrid quadrupole time-of-flight instrument (AB/MDS-Sciex, Toronto, Canada) as previously described [4]. Different fragmentation voltage settings were tested to optimize marker peptide detection. Peptides that were readily observable (excluding N-terminal and C-terminal peptides which contain extra amino acids originating from the recombinant protein expression vector) and whose precursor and product ion pair was computationally found to be unique in the E. coli proteome were used as protein specific signals to quantify the corresponding endogenous protein target.
E. coli samples were cooled immediately on ice, washed twice by centrifugation with wash buffer (50 mM Tris-HCl, 50 mM NaCl, 1 mM EDTA, pH 7.0) and split into 1 ml aliquots. These were then centrifuged for 5 min at 5,000 g to collect cells, and pellets were stored at -80 ºC. To quantify target proteins in experimental E. coli samples, two tubes of frozen cell pellets collected from a single sampling of a continuous culture were used. Peptide mixtures derived from Lys-C digestion of recombinant standard proteins or from total E. coli cell extracts were similarly separated by LC-MS/MS on a 1.5 mm × 50 mm C18 reversed-phase column at a flow rate of 0.2 ml/min. Peptides were eluted with a twostep gradient combination (1% to 40% acetonitrile over 50 min and up to 50% over the next 5 min) and the column eluent electrosprayed into the API 3000 (AB/MDS-Sciex) triplequadrupole instrument for simultaneous monitoring of specific marker peptides for 68 different protein targets. MRM analysis was performed in positive mode with an ion-spray voltage of 5.5 kV and a monitoring time of 10 ms per peptide. To quantify proteins accurately, the peak areas in extracted ion current chromatograms corresponding to target specific peptides were integrated automatically using the Analyst software (ver.1.4.1, AB/MDS-Sciex), followed by manual verification, and the results were used to calculate the absolute amount of protein in the cell extracts. Absolute intracellular concentrations of proteins were calculated using the measured dry cell weight per 1 ml of culture.
-----------------References--------------------
[1] S. A. Gerber, J. Rush, O. Stemman, M. W. Kirschner, S. P. Gygi, Proc. Natl. Acad. Sci. U. S. A. 100, 6940 (2003). http://doi.org/d2ztfh [2] W. Wang et al., Anal. Chem. 75, 4818 (2003). http://doi.org/bmzr3v [3] L. Anderson, C. L. Hunter, Mol. Cell Proteomics 5, 573 (2006). http://doi.org/c8f5g7 [4] K. Shinoda et al., J. Proteome Res. 5, 3312 (2006). http://doi.org/dqdj7x
Measurement method list: LC-MS/MS and 2D gel-MALDI-TOF
Methods description - Notes
Absolute protein quantification - to obtain absolute measurements of protein concentrations in complex samples we used a liquid chromatography / tandem mass spectrometry (LC-MS/MS) method that is a modification of previous work in absolute quantification making use of either external or internal standards with or without isotopically labelled synthetic peptides as standard [1-3]. The main characteristics of the current approach are that it combines mass spectrometric analysis in the multiple reaction monitoring mode (MRM), for specific and sensitive quantification of target proteins, with the use of peptides derived from purified recombinant proteins as external calibrants.
To first optimize the MS settings and select MRM parameters, LC-MS/MS analysis of Lys-C (Wako, Osaka, Japan) digests of each purified recombinant target protein was performed essentially as previously described [4]. Completion of digestion of both purified proteins and crude E. coli extracts was confirmed by measuring peptide yields with time and using different concentrations of Lys-C. LC-MS/MS analysis of peptides was performed on a QSTAR XL hybrid quadrupole time-of-flight instrument (AB/MDS-Sciex, Toronto, Canada) as previously described [4]. Different fragmentation voltage settings were tested to optimize marker peptide detection. Peptides that were readily observable (excluding N-terminal and C-terminal peptides which contain extra amino acids originating from the recombinant protein expression vector) and whose precursor and product ion pair was computationally found to be unique in the E. coli proteome were used as protein specific signals to quantify the corresponding endogenous protein target.
E. coli samples were cooled immediately on ice, washed twice by centrifugation with wash buffer (50 mM Tris-HCl, 50 mM NaCl, 1 mM EDTA, pH 7.0) and split into 1 ml aliquots. These were then centrifuged for 5 min at 5,000 g to collect cells, and pellets were stored at -80 ºC. To quantify target proteins in experimental E. coli samples, two tubes of frozen cell pellets collected from a single sampling of a continuous culture were used. Peptide mixtures derived from Lys-C digestion of recombinant standard proteins or from total E. coli cell extracts were similarly separated by LC-MS/MS on a 1.5 mm × 50 mm C18 reversed-phase column at a flow rate of 0.2 ml/min. Peptides were eluted with a twostep gradient combination (1% to 40% acetonitrile over 50 min and up to 50% over the next 5 min) and the column eluent electrosprayed into the API 3000 (AB/MDS-Sciex) triplequadrupole instrument for simultaneous monitoring of specific marker peptides for 68 different protein targets. MRM analysis was performed in positive mode with an ion-spray voltage of 5.5 kV and a monitoring time of 10 ms per peptide. To quantify proteins accurately, the peak areas in extracted ion current chromatograms corresponding to target specific peptides were integrated automatically using the Analyst software (ver.1.4.1, AB/MDS-Sciex), followed by manual verification, and the results were used to calculate the absolute amount of protein in the cell extracts. Absolute intracellular concentrations of proteins were calculated using the measured dry cell weight per 1 ml of culture.
-----------------References--------------------
[1] S. A. Gerber, J. Rush, O. Stemman, M. W. Kirschner, S. P. Gygi, Proc. Natl. Acad. Sci. U. S. A. 100, 6940 (2003).
[2] W. Wang et al., Anal. Chem. 75, 4818 (2003).
[3] L. Anderson, C. L. Hunter, Mol. Cell Proteomics 5, 573 (2006).
[4] K. Shinoda et al., J. Proteome Res. 5, 3312 (2006).
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