Mass Spectrometry:
The heart of our experiment lies in the LC-MS/MS. we recently made use of native mass spectrometry (MS), sometimes called “native electrospray ionization MS”, which allows proteins in their native or near-native states in solution to be introduced into the gas phase and interrogated by MS. the protein’s ion mobility (IM) can also be evaluated. IM not only separates protein ions, but also provides valuable information on their size and conformation. Although native-MS with IM analysis is a fast growing field, we have learned some important lessons by using native-MS and IM-MS techniques in photosynthesis research and have contributed to the development of the subject.
Figure 1 Native MS characterization of OCP and FRP, two proteins involved in photoprotection
Another new approach that we have implemented is the application of protein chemical cross-linking coupled with MS. Certain types of amino acid side chains, such as lysine, can be covalently linked by chemical cross-linking reagents in a large protein complex, like PSII, The cross-linked sample is proteolytically digested and is submitted to LC-MS/MS to identify the cross-linked species. With increasing high sensitivity, XL-MS has become a robust tool to study protein-protein interactions in complexes. Recently, we used this method to isolate and characterize a megacomplex containing the PBS and both photosystems, demonstrating convincingly its value in photosynthesis research.
Figure 2 LC-MS/MS interrogation of chemical crosslinking product of photosystem II, a machine that produces all the oxygen on Earth
MS-based footprinting has gained increased power for the analysis of higher-order protein structure. In most cases, footprinting reports on the solvent accessibility of amino acids (AA) side chains which becomes altered upon ligand binding or changes in conformation. This takes advantage of irreversible covalent modification of those solvent accessible AAs such as site-specific amino acid labeling and hydroxyl radical footprinting. One lone exception is HDX-MS, which utilizes reversible hydrogen/deuterium exchange where alterations in the hydrogen bonding network on the protein backbone give differential footprints.
Figure 3 Protein footprinting probed by LC-MS
Optical Spectrophotometry
Biochemical Preparation
Molecular Biology
Cell Culture
