Volatile and Non-volatile Buffers as Charge Reducing Agents in Native Mass Spectrometry
Disciplines
Biochemistry
Abstract (300 words maximum)
Native mass spectrometry (MS) is an advanced technique that enables the analysis of the structure, stoichiometry, and masses of large protein complexes. By transferring weakly associated complexes from solution into the gas phase of a mass spectrometer, this method provides valuable insights into complex stoichiometry and protein structure. Utilizing buffers as charge-reducing agents in native mass spectrometry (MS) is crucial for maintaining the native structure of proteins and protein complexes. The main objective of this research is to perform a comparative analysis of charge reducing capacity of various volatile ammonium and non-volatile sodium-based buffers on a model protein employing native mass spectrometry. Different concentrations of various buffers were prepared and combined with a lysozyme solution. These solutions were then analyzed using a mass spectrometer and differentiated with respect to mass-to-charge (m/z) ratio. The control, lysozyme in water, exhibited m/z peaks ranging from 8+ to 11+, with the strongest peak at 10+. Upon incubation of different concentration of volatile ammonium-based buffers (1.0 mM to 50.0 mM), m/z peaks ranging from 5+ to 12+ were observed, with a dominant peak at 7+. The spectra data indicated that the model protein remain folded at all concentration of the ammonium buffer. Similarly, various concentrations of non-volatile sodium-based buffers demonstrated m/z peaks ranging from 6+ to 13+, with a dominant peak at 8+. Lower concentrations (0.1 mM – 1.0 mM) of non-volatile sodium-based buffers allowed the model protein to remain folded. At higher concentrations (5.0-10.0 mM), lysozyme began to unfold, and the mass spectra showed elevated spectral noise and adducts.
Academic department under which the project should be listed
CSM - Chemistry and Biochemistry
Primary Investigator (PI) Name
Mohammad A. Halim
Volatile and Non-volatile Buffers as Charge Reducing Agents in Native Mass Spectrometry
Native mass spectrometry (MS) is an advanced technique that enables the analysis of the structure, stoichiometry, and masses of large protein complexes. By transferring weakly associated complexes from solution into the gas phase of a mass spectrometer, this method provides valuable insights into complex stoichiometry and protein structure. Utilizing buffers as charge-reducing agents in native mass spectrometry (MS) is crucial for maintaining the native structure of proteins and protein complexes. The main objective of this research is to perform a comparative analysis of charge reducing capacity of various volatile ammonium and non-volatile sodium-based buffers on a model protein employing native mass spectrometry. Different concentrations of various buffers were prepared and combined with a lysozyme solution. These solutions were then analyzed using a mass spectrometer and differentiated with respect to mass-to-charge (m/z) ratio. The control, lysozyme in water, exhibited m/z peaks ranging from 8+ to 11+, with the strongest peak at 10+. Upon incubation of different concentration of volatile ammonium-based buffers (1.0 mM to 50.0 mM), m/z peaks ranging from 5+ to 12+ were observed, with a dominant peak at 7+. The spectra data indicated that the model protein remain folded at all concentration of the ammonium buffer. Similarly, various concentrations of non-volatile sodium-based buffers demonstrated m/z peaks ranging from 6+ to 13+, with a dominant peak at 8+. Lower concentrations (0.1 mM – 1.0 mM) of non-volatile sodium-based buffers allowed the model protein to remain folded. At higher concentrations (5.0-10.0 mM), lysozyme began to unfold, and the mass spectra showed elevated spectral noise and adducts.