Investigating the Impact of Sugar on Protein Stability by High Resolution Mass Spectrometry
Disciplines
Analytical Chemistry | Biochemistry
Abstract (300 words maximum)
Ensuring that protein, antibody, peptide, and enzyme-based medications maintain their native structure during long-term storage is critical to the safety and efficiency of pharmaceuticals. A critical aspect of preservation for protein-based therapeutics is excipients, such as sugars, that can maintain their stability. Sucrose is a widely used stabilizer in the pharmaceutical industry. Various research suggests that sucrose can preserve the conformational stability of proteins during storage conditions. We hypothesize that although sucrose preserves the protein structure, we suspect that sucrose can strongly bind with the protein through glycosylation which may alter the function of the protein. Glycosylation disrupts the native structure of the protein, promotes aggregation, blocks active sites, reduces function, and leads to degradation, all of which decrease the protein’s shelf life. To test this hypothesis, we have employed high resolution mass spectrometry investigations monitoring the change of protein native conformation and glycosylation. Various concentrations of sucrose were prepared, incubated with a model protein Lysozyme, and analysed using mass spectrometry. All mass spectra were obtained using an Orbitrap Exploris 240. In this experiment, the control sample was Lysozyme in water. At lower concentration of 0.1mM sucrose, Lysozyme showed the binding of one sucrose molecule. At an increased concentration of 0.5mM sucrose, seven sucrose molecules are adducted with Lysozyme, while incubation with 1mM sucrose exhibited the formation of nine adducts. Furthermore, Lysozyme incubated with 3 mM sucrose displayed fifteen sucrose adduct formation with Lysozyme. It is evident that despite widespread usage as stabilizing agent of protein-based therapeutics, sucrose is not an effective stabilizing agent because it promotes unintentional glycosylation, which may alter the protein function and stability. In future, tandem mass spectrometry experiments will be performed to locate the glycosylation site of sucrose.
Academic department under which the project should be listed
CSM - Chemistry and Biochemistry
Primary Investigator (PI) Name
Mohammad Halim
Investigating the Impact of Sugar on Protein Stability by High Resolution Mass Spectrometry
Ensuring that protein, antibody, peptide, and enzyme-based medications maintain their native structure during long-term storage is critical to the safety and efficiency of pharmaceuticals. A critical aspect of preservation for protein-based therapeutics is excipients, such as sugars, that can maintain their stability. Sucrose is a widely used stabilizer in the pharmaceutical industry. Various research suggests that sucrose can preserve the conformational stability of proteins during storage conditions. We hypothesize that although sucrose preserves the protein structure, we suspect that sucrose can strongly bind with the protein through glycosylation which may alter the function of the protein. Glycosylation disrupts the native structure of the protein, promotes aggregation, blocks active sites, reduces function, and leads to degradation, all of which decrease the protein’s shelf life. To test this hypothesis, we have employed high resolution mass spectrometry investigations monitoring the change of protein native conformation and glycosylation. Various concentrations of sucrose were prepared, incubated with a model protein Lysozyme, and analysed using mass spectrometry. All mass spectra were obtained using an Orbitrap Exploris 240. In this experiment, the control sample was Lysozyme in water. At lower concentration of 0.1mM sucrose, Lysozyme showed the binding of one sucrose molecule. At an increased concentration of 0.5mM sucrose, seven sucrose molecules are adducted with Lysozyme, while incubation with 1mM sucrose exhibited the formation of nine adducts. Furthermore, Lysozyme incubated with 3 mM sucrose displayed fifteen sucrose adduct formation with Lysozyme. It is evident that despite widespread usage as stabilizing agent of protein-based therapeutics, sucrose is not an effective stabilizing agent because it promotes unintentional glycosylation, which may alter the protein function and stability. In future, tandem mass spectrometry experiments will be performed to locate the glycosylation site of sucrose.