Implementing mass spectrometry for the structural and compositional analysis of proteins

Abstract

Lectins are sugar-binding proteins that perform various biological functions such as cellular recognition attachments etc. Pulses the dried edible seeds of certain plants are great sources of lectins and a proteomics approach directed at lectins identification would bring substantial improvement in the lectin identification process. Lectins from nine different plant species were analyzed through SDS-PAGE and proteomics analysis. After LC-MS/MS analysis proteins were identified with protein database searches using Uniprot. Functionally uncharacterized proteins were identified with database searches were annotated with the Pfam and NCBI-CCD databases. In-vitro pharmacological screening will be carried out to assess the pharmacological effects of these lectins. To understand the functions of proteins at a molecular level it is often necessary to determine their three-dimensional structure. Fast Photochemical Oxidation of Protein (FPOP) is a hydroxyl-radical-based protein footprinting (HRPF) technique that utilizes a pulsed KrF laser (248 nm) to trigger photolysis of hydrogen peroxide to produce hydroxyl radicals which subsequently modify the solvent exposed surface area of proteins. However this technique has some disadvantages: being time-consuming especially when dealing with a large sample size adjustment of flow rate and difficulty with membrane protein oxidation. To address these issues we developed a platform to perform FPOP in microtiter plates instead of the traditional capillary set up. To ensure reliability and reproducibility of microtiter FPOP and evaluate microtiter FPOP against traditional flow FPOP we used three systems: adenine-based hydroxyl radical dosimetry; oxidation of the model peptide [Glu]1-Fibrinopeptide B (GluB); and HRPF analysis of the model protein myoglobin. We demonstrated that microtiter based FPOP can provide comparable oxidation of model peptide and model protein as compared to traditional capillary FPOP. Automation of the system substantially reduces experimental time and minimize human errors