Standard Concentration Curve and Absolute Concentration Determination
A standard concentration curve can be generated for absolute quantitation experiments using increasing concentrations of the “heavy peptide” in the presence of a constant amount of the biological sample of interest. The biological sample of interest, termed “matrix” contains the “light” version of the same peptide and therefore acts as a constant internal standard. For cases where we do not have matrix containing endogenous light peptide we synthesize a non-isotopic version termed “light” and use this as the constant internal standard in the curve. Our typical concentration curves contain five heavy peptide serial dilutions, a blank (no heavy peptide with constant light peptide), and double blank (matrix only) and each sample is run in triplicate for a total of 21 runs. Data from all 21 runs are imported into AB Multiquant Software where a linear calibration curve with a weighting of 1/x is used. Once the calibration curve is generated, the biological samples (unknowns) can be processed, measuring the amount of the endogenous ‘light’ peptide present in each sample. The “heavy” peptide is added into each biological sample at a fixed concentration and acts as the internal standard for these samples. Using the calibration curve generated above, the actual concentration of peptide in the biological sample can be computed from the ratio of the endogenous peptide (light) to the added internal standard (heavy).