Absolute and Relative Protein Quantification with the Use of Isotopically Labeled p-Hydroxymercuribenzoic Acid and Complementary MALDI-MS and ICPMS Detection

Chemical labeling with subsequent mass spectrometric detection represents a common approach for protein quantification. Whereas most methods make use of stable isotope labels from natural elements such as D-2, C-13, N-15, or O-18, artificially introduced metals have gained interest as alternative markers. In this work we present the application of p-hydroxymercuribenzoic acid (pHMB) as a labeling reagent for cysteine-containing proteins. As a proof of concept, insulin was chosen as the model protein, and two different workflows were developed to its absolute and relative quantification with the use of complementary MALDI-MS and ICPMS. On the basis of the synthesis of isotopically labeled [Hg-199]pHMB, and thus on the basis of the label-specific isotope dilution concept, a differential labeling procedure can be applied either to the comparative study of two different samples (relative quantification) or to the absolute quantification of insulin. In both cases, final detection by MALDI-MS followed by isotope pattern deconvolution was applied to extract the quantitative data from the mass spectra. Good agreement with the expected values was obtained for the relative insulin quantification, and the recovery for insulin applying the absolute quantification workflow was between 90% and 110% with an RSD of better than 5% in the low picomole range.