Chemical derivatization of biomolecules and biomolecular ions to access new dissociation pathways in the gas phase
Since the development of mass spectrometry over a century ago, it has dramatically advanced numerous fields of science, including chemistry, biology, medicine, geology, astrophysics, and several others while mass spectrometric methods have, themselves, rapidly matured. The ability to generate ions and subsequently control not only the stability and trajectory of them, but also the extent to which they may be activated or reacted with other ions, allows nearly limitless experimental possibilities. The potential for this analytical method has only increased with new developments in instrumentation and gas-phase ion chemistry. In this dissertation, gas-phase ion/ion reactions and intramolecular ion dissociation reactions are discussed. In both cases, the reactivity of a nucleophile is exploited to create new covalent bonds while breaking other ones. The reactivity of N-hydroxysuccinimide (NHS) and N-hydroxysulfosuccinimide (sulfo-NHS) esters with different nucleophiles has been studied extensively. It has been previously shown that unprotonated amine functional groups are reactive towards these esters in the gas phase via ion/ion reactions, in which a nucleophilic attack is initiated by the amine on the electrophilic carbonyl carbon of the ester reagent. This results in the formation of an amide bond with concomitant loss of the neutral (sulfo-)NHS molecule. This reaction phenomenology has been used to show the ability to ligate either individual amino acids or peptides to an existing peptide, effectively extending the original peptide to create a new peptide. This is shown to be a highly efficient, highly controlled, and rapid method for generating new peptides in the gas phase. Ion/ion reactions have also shown that guanidines are highly reactive towards NHS and sulfo-NHS esters in the gas phase. Because it is nearly impossible for reactions between guanidines and esters in the solution phase to occur, these are a new type of reaction. Unprotonated arginine residues, which have the highly basic guanidine functionality in the side chain, are always protonated, and therefore not nucleophilic/reactive in the presence of these esters. As the basicity of a solution containing both esters and guanidines increases, the esters become unstable and dissociate well before the guanidines become unprotonated. The reaction of a guanidine with an ester is similar to amines, in that an amide-like bond is formed; however, the dissociation pathways are significantly different for modified arginine. One of those dissociation pathways results in the dissociation of the guanidine, leaving an amine. This effect is actually observed as the conversion of arginine into ornithine, which is an amino acid that provides unique reaction properties. Intramolecular ion dissociation reactions have been studies by focusing on a selective cleavage known as the ornithine effect. This was discovered following the conversion of arginine to ornithine in peptides with subsequent activation of the ornithine-containing peptides. This activation led to the intramolecular dissociation of that peptide at the C-terminal amide bond of the ornithine residue. The mechanism proposed for this process involves a nucleophilic attack of the amine of ornithine on the carbonyl carbon of its C-terminal amide bond (or carboxylic acid, if it is the C-terminal residue), forming a cyclic structure termed a lactam. It is likely of little coincidence that ornithine is not coded for in DNA due to its spontaneous lactam formation. This cleavage has been found to be the most selective cleavage observed, requiring the least activation energy, compared to other known selective cleavages, including the aspartic acid effect, the proline effect, and loss of post-translational modifications, such as phosphorylations. The introduction of this "weak-spot" into protein backbones is demonstrated as a means for significantly altering, and simplifying, protein fragmentation behavior, ideally to be used for rapid protein identification.
McLuckey, Purdue University.
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