It appears to be a general belief that stable isotopically labeled (SIL) internal standards yield better assay performance results for quantitative bioanalytical liquid chromatography/mass spectrometry (LC/MS) assays than does any other internal standard. In this article we describe our experiences with structural analogues and SIL internal standards and their merits and demerits. SIL internal standards are the first choice, but deuterium-labeled compounds may demonstrate unexpected behavior, such as different retention times or recoveries, than the analyte. In addition, a SIL internal standard with identical chemical properties as the analyte may cover up assay problems with stability, recovery, and ion suppression. Since SIL internal standards are not always available or are very expensive, structural analogues can be used, however, with consideration of several issues, which are usually displayed during method validation.
The dissection of specific and nonspecific protein complexes in the gas phase is studied by collisionally activated decomposition. In particular, the gas phase dissection of multiple protonated homodimeric Human Galectin I, E. Coli Glyoxalase I, horse heart cytochrome c, and Hen egg Lysozyme have been investigated. Both the Human Galectin I and E. Coli Glyoxalase I enzymes are biologically active as a dimer, exhibiting molecular weights of approximately 30 kDa. Cytochrome c and Lysozyme are monomers, but may aggregate to some extent at high protein concentrations. The gas phase dissociation of these multiple protonated dimer assemblies does lead to the formation of monomers. The charge distribution over the two concomitant monomers following the dissociation of these multiple protonated dimers is found to be highly dissimilar. There is no evident correlation between the solution phase stability of the dimeric proteins and their gas-phase dissociation pattern. Additionally, in the collisionally activated decomposition spectra diffuse ion signals are observed, which are attributed to monomer ions formed via slow decay of the collisionally activated dimer ions inside the reflectron time-of-flight. Although, the formation of these diffuse metastable ions may complicate the interpretation of collisionally activated decomposition mass spectra, especially when studying noncovalent protein complexes, a simple mathematical equation may be used to reveal their origin and pathway of formation.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.