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|Author(s):||Eric L. Kilpatrick; Wei-Li Liao; Johanna Camara; Illarion V. Turko; David M. Bunk;|
|Title:||Expression and characterization of 15N-labeled human C-reactive protein in Escherichia coli and Pichia pastoris for use in isotope-dilution mass spectrometry|
|Published:||September 01, 2012|
|Abstract:||Isotope dilution mass spectrometry using stable isotope labeled internal standards is unsurpassed in its ability to quantify analytes with accuracy and low measurement uncertainty. The extension of this technique to proteins relies upon the expression of recombinant proteins generated with stable isotope incorporation. The current study expressed soluble 15N-labeled human C-reactive protein (15N-rCRP) in both Escherichia coli and Pichia pastoris. Soluble protein yield from E. coli was only 20 g/L but was performed without signal sequences or coexpression of accessory proteins. Yields from P. pastoris were more than fifty-fold over that from the bacteria. The fitness of the expressed proteins to be used as internal standards for isotope dilution mass spectrometry was evaluated using affinity-binding techniques, tryptic digestion equivalency and incorporation of 15N. The 15N-rCRP was shown to bind to p-aminophenylphosphorylcholine resin which also served as a means of purification. Additionally, the protein was immunoprecipitated by monoclonal antibody to CRP to reflect a second affinity function. Co-digestion with trypsin of varying amounts of purified human CRP with a constant level of 15N-rCRP demonstrated the high degree of linearity in the ratio analysis (r2 = 0.998) required of an internal standard Custom software was used to determine that the percentage of incorporation of 15N into the protein was 98.2 % (CV = 0.3 %) based on the matching of theoretical isotopic distributions with experimental spectra in order to maximize the Pearson correlation coefficient. In this report we confirm the suitability of 15N-rCRP as an internal standard in isotope-dilution mass spectrometry.|
|Citation:||Protein Expression and Purification|
|Keywords:||C-reactive protein, isotope dilution, mass spectrometry, recombinant, internal standard|
|Research Areas:||Mass Spectrometry|