Collision-Induced Dissociation Of Dipeptides In Negative Ion Electrospray Ionization Tandem Mass Spectrometry

This purpose of this project was to investigate the collision-induced dissociation of dipeptides in negative ion electrospray ionization tandem mass spectrometry, with a focus on the mechanism of the production of imidazole-type fragments not previously reported from the fragmentation of the dipeptides being studied. The majority of the dipeptides studied were alanine N-terminal or serine C-terminal dipeptides. All dipeptides were dissolved in 50:50 methanol:water, 3 mM ammonium formate. Collision-induced dissociation in the collision cell of a triple quadrupole mass spectrometer was used to fragment [M-H]- precursor ions. Accurate mass measurements confirmed the molecular formula of the imidazole-type fragments. Further MS/MS studies were performed to provide information about the fragmentation mechanism for the formation of the imidazole-type fragments. The m/z values of intermediate ions in the formation of the imidazole-type fragments were confirmed through second-generation product ion scans and precursor ion scans. More sophisticated instrumentation will be required to further probe the structure of the intermediate ions.

A Computational Analysis of the Collision-Induced Dissociation Mechanisms of the Dipeptide Glycine-Serine Under Mass Spectrometry

Collision-induced dissociation (CID) of peptides using tandem mass spectrometry (MS) has been used to determine the identity of peptides and other large biological molecules. Mass spectrometry (MS) is a useful tool for determining the identity of molecules based on their interaction with electromagnetic fields. If coupled with another method like infrared (IR) vibrational spectroscopy, MS can provide structural information, but in its own right, MS can only provide the mass-to-charge (m/z) ratio of the fragments produced, which may not be enough information to determine the mechanism of the collision-induced dissociation (CID) of the molecule. In this case, theoretical calculations provide a useful companion for MS data and yield clues about the energetics of the dissociation. In this study, negative ion electrospray tandem MS was used to study the CID of the deprotonated dipeptide glycine-serine (Gly-Ser). Though negative ion MS is not as popular a choice as positive ion MS, studies by Bowie et al. show that it yields unique clues about molecular structure which complement positive ion spectroscopy, such as characteristic fragmentations like the loss of formaldehyde from the serine residue.2 The increase in the collision energy in the mass spectrometer alters the flexibility of the dipeptide backbone, enabling isomerizations (reactions not resulting in a fragment loss) and dissociations to take place. The mechanism of the CID of Gly-Ser was studied using two computational methods, B3LYP/6-311+G* and M06-2X/6-311++G**. The main pathway for molecular dissociation was analyzed in 5 conformers in an attempt to verify the initial mechanism proposed by Dr. James Swan after examination of the MS data. The results suggest that the loss of formaldehyde from serine, which Bowie et al. indicates is a characteristic of the presence of serine in a protein residue, is an endothermic reaction that is made possible by the conversion of the translational energy of the ion into internal energy as the ion collides with the inert collision gas. It has also been determined that the M06-2X functional¿s improved description of medium and long-range correlation makes it more effective than the B3LYP functional at finding elusive transition states. M06-2X also more accurately predicts the energy of those transition states than does B3LYP. A second CID mechanism, which passes through intermediates with the same m/z ratio as the main pathway for molecular dissociation, but different structures, including a diketopiperazine intermediate, was also studied. This pathway for molecular dissociation was analyzed with 3 conformers and the M06-2X functional, due to its previously determined effectiveness. The results suggest that the latter pathway, which meets the same intermediate masses as the first mechanism, is lower in overall energy and therefore a more likely pathway of dissociation than the first mechanism.

Maximizing Product Formation and Minimizing Degradation: A Look at Buffering Conditions and Post-Reaction Degradation for the In-Line Jaffe Reaction with Capillary Electrophoresis

Creatinine levels in blood serum are typically used to assess renal function. Clinical determination of creatinine is often based on the Jaffe reaction, in which creatinine in the serum reacts with sodium picrate, resulting in a spectrophotometrically quantifiable product. Previous work from our lab has introduced an electrophoretically mediated initiation of this reaction, in which nanoliter plugs of individual reagent solutions can be added to the capillary and then mixed and reacted. Following electrophoretic separation of the product from excess reactant(s), the product can be directly determined on column. This work aims to gain a detailed understanding of the in-capillary reagent mixing dynamics, in-line reaction yield, and product degradation during electrophoresis, with an overall goal of improving assay sensitivity. One set of experiments focuses on maximizing product formation through manipulation of various conditions such as pH, voltage applied, and timing of the applied voltage, in addition to manipulations in the identity, concentration, and pH of the background electrolyte. Through this work, it was determined that dramatic changes in local voltage fields within the various reagent zones lead to ineffective reagent overlapping. Use of the software simulation program Simul 5 enabled visualization of the reaction dynamics within the capillary, specifically the wide variance between the electric field intensities within the creatinine and picrate zones. Because of this simulation work, the experimental method was modified to increase the ionic strength of the creatinine reagent zone to lower the local voltage field, thus producing more predictable and effective overlap conditions for the reagents and allowing the formation of more Jaffe product. As second set of experiments focuses on controlling the post-reaction product degradation. In that vein, we have systematically explored the importance of the identity, concentration, and pH of the background electrolyte on the post-reaction degradation rate of the product. Although prior work with borate background electrolytes indicated that product degradation was probably a function of the ionic strength of the background electrolyte, this work with a glycine background electrolyte demonstrates that degradation is in fact not a function of ionic strength of the background electrolyte. As the concentration and pH of the glycine background increased, the rate of degradation of product did not change dramatically, whereas in borate-buffered systems, the rate of Jaffe product degradation increased linearly with background electrolyte concentration above 100.0 mM borate. Similarly, increasing pH of the glycine background electrolyte did not result in a corresponding increase in product degradation, as it had with the borate background electrolyte. Other general trends that were observed include: increasing background electrolyte concentration increases peak efficiency and higher pH favors product formation; thus, it appears that use of a background electrolyte other than borate, such as glycine, the rate of degradation of the Jaffe product can be slowed, increasing the sensitivity of this in-line assay.