Study of posttranslational modifications in proteins and noncovalent interactions in small models by mass spectrometry

Posttranslational modifications are a major part of chemistry, specifically biochemistry and proteomics. Posttranslational modifications (PTMs) come in a wide variety, such as glycosylation, phosphorylation, alkylation, methylation, oxidation, nitration, and nitrosylation. Each one varies in how it modifies a protein, and many in vivo conditions are hard to replicate in vitro, making them difficult to identify. With rapidly increasing technology analysis of PTMs has become a major focus because each one has a specific biological function, and previous technologies limited the information gained from analyzing PTMs. Scientists can now determine structural availability, adverse effects on protein structure and function, as well as many other biological concerns that are more important now than ever.;The goal of this project was to utilize mass spectrometry and some method development procedures to allow two major proteins to undergo posttranslational modifications to see if the modification can be identified, specifically where the modification occurred and in what quantities.;Using the eNOS protein, we were able to nitrate the protein and detect the sites of nitration utilizing a tryptic digest followed by LC/MS and LC/MS-MS on an ion trap mass spectrometer using standard proteomics procedures. Out of 30 total tyrosine residues that are available in the eNOS sequence, 25 of them were sequenced in combined six tryptic digests. Out of the 25 residues that were sequenced, only 6 showed no modification. The other 19 of the 25 sequenced tyrosines had the addition of one or two NO2 groups. The second protein used was CrAT protein. Out of 22 total tyrosine residues that are available in the CrAT sequence, 12 of them were sequenced in combined four digests. Out of the 12 residues that were sequenced, 2 showed no modification. The other 10 of the 12 sequenced tyrosines had the addition of one or two NO2 groups. For both protein studies, all the tyrosine residues identified to have modification occurred on the surface of the tertiary structure of the protein. This is expected because of accessibility of the nitrating reagent to the surface.;The second major study done utilized the extended kinetic method and was done to determine thermochemical data for a set of crown ether complexes. Our lab specializes in determining thermochemical data using gas-phase ion chemistry. Results show a binding energy ladder for seven crown ether complexes analyzed with 1,10-diaminodecane as well as with a methylated derivative N,N'-dimethyl-1,8-octanediamine. This study shows our lab's ability to determine valid thermochemical data utilizing an ion trap mass spectrometer with collision-induced dissociation as the main source of fragmentation.