Gas-Phase Ion/Ion Reaction of Biomolecules
thesisposted on 06.12.2019, 15:48 by Mack ShihMack Shih
Mass spectrometry is a versatile, powerful analytical tool for chemical and biomolecule identification, quantitation, and structural analysis. Tandem mass spectrometry is key component in expanding the capabilities of mass spectrometry beyond just a molecular weight detector. Another key component is the discovery of electrospray ionization allowing not only liquid samples to be ionized but also generation of multiply charged ions enabling mass spectrometry analysis of large biomolecules. The fragmentation pathway of ion during tandem mass spectrometry is highly dependent on the nature of the ion as well as the form of dissociation technique employed. To-date, no single form of ion or dissociation method can provide all the structure information needed; therefore, it is common to use multiple forms of ions, different charge carrier or modifications, with a variety of other dissociation techniques to generate complimentary information. Practically, it is not always easy to generate the desired form of ion via ionization methods and is one of the limitations. Gas-phase ion/ion reactions provide an easy approach in manipulation of ions, either through changing the ion type or covalent modifications, in the gas-phase with the goal of enhancing the capabilities of mass spectrometers for either molecular weight or structural analysis. In this dissertation, studies of new gas-phase ion/ion chemistry for biomolecules such as carbohydrates and phopho/sulfopeptides were performed, and exploration into mass spectrometry analysis of IgGs is discussed.
Ion/ion reactions with carbohydrates were investigated with the goal of finding a charge-transfer or covalent modification reaction which can increase the structural information of carbohydrates upon tandem mass spectrometry. No luck was achieved with charge transfer ion/ion reactions which increased the overall fragmentation information in tandem mass spectrometry. Novel gas-phase covalent chemistry was discovered where alkoxides were found to form ester and ethers. It was also discovered the aldehyde functional group at the reducing end of carbohydrates are susceptible to Schiff-base modifications. Schiff-base has been previously reported in peptides and this is the first time it has been discovered for carbohydrates.
In the next project a gas-phase approach for the rapid screening of polypeptide anions for phosphorylation or sulfonation based on binding strengths to guanidinium-containing reagent ions was developed. The approach relies on the generation of a complex via reaction of mixtures of deprotonated polypeptide anions with dicationic guanidinium-containing reagent ions and subsequent dipolar DC collisional activation of the complexes. The relative strengths of the electrostatic interactions of guanidinium with deprotonated acidic sites follows the order carboxylate
Hyaluronic acid, a linear carbohydrate polymer with repeating units of D-glucuronic acid and N-acetyl D-glucosamine, was found to exhibit unique properties in its electrospray ionization mass spectrum that was never seen before. Electrospray of hyaluronic acid in aqueous solution in the negative polarity presented an incredibly intriguing mass spectrum, which we termed “emerald city” consisting of max charge or max charge-1 anions of hyaluronic acid. This is the first biomolecule observed to have the capability to deprotonate at every acid site that is possible. These set of highly charge anions exhibits unique characteristics upon use as a charge inversion reagent to charged invert multiply protonated proteins. A max of thirty-three protons was transferred when myoglobin 24+ was charge-inverted to a max charge state of 9- in the negative mode. Further research should be conducted to fully understand this phenomenal and its possible utilities.
Lastly, mass spectrometry analysis of monoclonal antibodies was performed. Monoclonal antibodies are 150 kDa sized protein complexes and is a major area of interest for pharmaceutical industry. Mass spectrometry analysis of big proteins is an emerging area for mass spectrometry and is quite different compared to small and medium molecule analysis on the mass spectrometer. Detailed in the last chapter are methods developed for sample cleanup of immunoglobulin G as well as the application of q2 DDC for removal of loosely bound adducts to achieve sharper peaks in the mass spectrum. Studies of protein denaturation was also conducted with methods such as circular dichroism and differential ion mobility also employed. And finally, a photochemical reaction setup was shown to cleave twelve out of sixteen total disulfide bonds in the immunoglobulin G within seconds compared to traditional solution phase reactions which can take hours.