Three dimensional structure is the key to understanding how proteins function, or malfunction. A complete understanding of protein structure will allow us to gain a greater understanding of biology and disease. Mass spectrometry (MS) is sensitive and fast. Although not fully appreciated, MS is also an excellent method for probing biomolecular structure. The Julian lab has developed a unique method combining MS with photochemistry to determine distance constraints between specific side chains in peptides and proteins. This information can be used to evaluate and even guide calculations, allowing us to solve structures for peptides in a single day. Currently, we are improving our instrumentation to enable experiments with whole proteins.
Post-translational modifications (PTMs) occur after proteins have been synthesized and have important structural consequences. Most PTMs change the mass of the affected protein and are easily detected by MS. However, a small number of subtle modifications, such as conversion of chirality from the L to D configuration, lead to no change in mass and are difficult to detect. We have developed several methods for probing biomolecular structure at this finer level of detail. For example, atom-specific generation of a radical created by photodissociation of a specific bond leads to radical directed dissociation of the biomolecule. Fragmentation is sufficiently sensitive to structure to identify epimers (molecules with multiple chiral centers where all but one have identical stereoconfigurations). We are currently using this method to identify peptide epimers in proteins extracted from human eye lenses in an effort to unravel the connection between epimerization, cataract formation, and aging in general.
Lipids are small molecules most well-known for energy storage and as the building blocks of cell membranes, but lipids can also function in signaling and are an important class of biomarkers for disease. Lipid structure typically consists of a hydrophilic headgroup with hydrophobic tail(s). The structural diversity available to create different headgroup/tail motifs, including potential isomers, is truly vast. Analytical methods for characterizing lipid structure must be capable of handling this chemical diversity and simultaneously distinguishing very subtle structural variations (such as differences in the orientation of a single OH group or the location of a single double bond). We utilize radical chemistry to investigate lipid structure. Importantly, radical directed dissociation yields fragmentation throughout lipid structures (i.e. both the headgroup and tail are fragmented), yielding complete structural characterization, whereas collision induced dissociation, a more common method, frequently leads to minimal fragmentation of the headgroup alone.
Redox homeostasis is incredibly important in biology and is achieved when oxidant and antioxidant forces a cell are properly balanced. Unchecked oxidation can lead to oxidative stress and numerous diseases such as cancer, Amyotrophic lateral sclerosis, Parkinson’s disease, Alzheimer’s disease, and aging itself. However, free radicals and other reactive species also have important roles within cells. For example, reactive species are a primary weapon employed by the immune system. One of the difficulties associated with greater understanding of redox homeostasis is identification and quantification of the redox contributions from all relevant molecular species. We have developed a rapid mass spectrometry based assay for identification of antioxidant peptides. Examination of numerous proteins reveals that many contain regions that are inherently antioxidant. The molecular mechanisms and conditions that yield antioxidant peptide sequences are currently under investigation.