Novel 1:1 labeling and purification process for C-terminal thioester and single cysteine recombinant proteins using generic peptidic toolbox reagents.

We developed a versatile set of chemical labeling reagents which allow dye ligation to the C-terminus of a protein or a single internal cysteine and target purification in a simple two-step process. This simple process results in a fully 1:1 labeled conjugate suitable for all quantitative fluorescence spectroscopy and imaging experiments. We refer to a "generic labeling toolbox" because of the flexibility to choose one of many available dyes, spacers of different lengths and compositions which increase the target solubility, a variety of affinity purification tags, and different cleavage chemistries to release the 1:1 labeled proteins.

High-throughput physical organic chemistry--Hammett parameter evaluation.

High-throughput analysis techniques were developed to allow the rapid assessment of a range of Hammett parameters utilizing positive electrospray mass spectrometry (ESI+ -MS) as the sole quantitative tool, with the core of the approach being a so-called "analytical construct". Hammett substituent parameters were determined for a range of meta- and para-substituted anilines by high-throughput (HT) assessment of relative reaction rates for competitive amide bond formation reaction with up to five parameters determined in a single pot reaction. Sensitivity of the reaction to substituents' effects (materialized by Hammett's rho parameter) was determined in the first instance, with HT Hammett's sigma substituent parameter assessment then carried out successfully for over 30 anilines, with excellent correlation observed between the HT ESI+ -MS method of determination and literature values.