Strategy for absolute quantification of proteins: CH3Hg+ labeling integrated molecular and elemental mass spectrometry

Currently, molecular mass spectrometry is preferred by many for relative quantification but is not appropriate for absolute quantification of proteins. In this article we demonstrate a proof of concept for the absolute quantitative analysis of proteins via CH3Hg+ labeling and integrated application of molecular and elemental mass spectrometry. The smallest size of CH3Hg+ among monoalkyl mercurials and the specific and covalent interaction with sulfhydryl (-SH) in proteins results in forming a simple complex of CH3Hg+:-SH 1:1 when all -SH are exposed, as confirmed by ESI-MS. Based on the known number of -SH per protein, the absolute protein concentration can be obtained via Hg determination using ICP-MS, in which CH3HgCl could be simply used as an external standard. When bovine pancreatic ribonuclease A, lysozyme and insulin, which have an increasing number of various disulfide linkages in their molecules, were taken as model proteins, their corresponding absolute detection limits (3 sigma) reached 0.6, 1.2 and 0.4 pmol, respectively. These characteristics may be expected to provide an alternative approach for absolute protein quantification, especially specific biomarker determination, in the near future.