Proline and Methionine as Charge Reducing Agents in Native Mass Spectrometry
Disciplines
Analytical Chemistry | Biochemistry
Abstract (300 words maximum)
Native mass spectrometry (MS) is an emerging technique that examines large protein complexes, revealing their structure, stoichiometry, and masses. Transferring weakly associated complexes from aqueous solution to a gaseous state using electrospray ionization (ESI) highlights the complexity of protein structure. With the addition of an amino acid as a charge-reducing agent, native MS allows for the observation of the tendency of protein and amino acids to compete for protein for protons during ESI, lowering the overall charged state of the protein. The chief objective of this research is to perform a comparative analysis of the effects of several amino acids on the structure of a protein. Several amino acids solutions were prepared at varied concentrations (mM) with the model protein, lysozyme, to distinguish each solution by the changes of the charge distribution of folded and unfolded protein. The control, lysozyme in water, exhibited m/z peaks ranging from 8+ to 11+, with a dominant peak at 10+. When lysozyme was incubated at various concentrations of L-proline, the charge state distribution of the proteins shifted significantly favoring lower charge states from 5+ to 12+, where the most prominent peak at 8+. As the concentration of L-proline increased, a notable shift in the mass spectra was observed, continuously moving towards lower charged states. This shift suggests a correlation between the increased stability and adherence to the proteins’ native structure. Importantly, proline was not adducted with lysozyme even at higher concentrations. However, for L-methionine, various charges states are observed from ranging from 9+ to 13+, lower concentrations (0.1 mM – 0.5 mM) were characterized by elevated spectral noise and the formation of adduct peaks in the protein native structure, indicating protein denaturation. However, in higher concentrations (0.8 –5.0 mM), the solutions were more stable and mimicked a similar trend seen in the concentrations of L-proline, maintaining the proteins’ native structure. Further attempts are required to repeat these experiments to produce more reliable results.
Academic department under which the project should be listed
CSM - Chemistry and Biochemistry
Primary Investigator (PI) Name
Mohammad Halim
Proline and Methionine as Charge Reducing Agents in Native Mass Spectrometry
Native mass spectrometry (MS) is an emerging technique that examines large protein complexes, revealing their structure, stoichiometry, and masses. Transferring weakly associated complexes from aqueous solution to a gaseous state using electrospray ionization (ESI) highlights the complexity of protein structure. With the addition of an amino acid as a charge-reducing agent, native MS allows for the observation of the tendency of protein and amino acids to compete for protein for protons during ESI, lowering the overall charged state of the protein. The chief objective of this research is to perform a comparative analysis of the effects of several amino acids on the structure of a protein. Several amino acids solutions were prepared at varied concentrations (mM) with the model protein, lysozyme, to distinguish each solution by the changes of the charge distribution of folded and unfolded protein. The control, lysozyme in water, exhibited m/z peaks ranging from 8+ to 11+, with a dominant peak at 10+. When lysozyme was incubated at various concentrations of L-proline, the charge state distribution of the proteins shifted significantly favoring lower charge states from 5+ to 12+, where the most prominent peak at 8+. As the concentration of L-proline increased, a notable shift in the mass spectra was observed, continuously moving towards lower charged states. This shift suggests a correlation between the increased stability and adherence to the proteins’ native structure. Importantly, proline was not adducted with lysozyme even at higher concentrations. However, for L-methionine, various charges states are observed from ranging from 9+ to 13+, lower concentrations (0.1 mM – 0.5 mM) were characterized by elevated spectral noise and the formation of adduct peaks in the protein native structure, indicating protein denaturation. However, in higher concentrations (0.8 –5.0 mM), the solutions were more stable and mimicked a similar trend seen in the concentrations of L-proline, maintaining the proteins’ native structure. Further attempts are required to repeat these experiments to produce more reliable results.