Investigating the Gas Phase Ubiquitin Oligomers Formation by High Resolution Orbitrap Exploris 240 Mass Spectrometer
Disciplines
Analytical Chemistry | Biochemistry
Abstract (300 words maximum)
Ubiquitin is a 76 amino-acid residue protein which covalently binds to substrates and modifies them using a class of posttranslational modification called Ubiquitination. Ubiquitination is regulated by three types of enzymes, namely, ubiquitin-activating enzymes (E1), ubiquitin conjugating enzymes (E2), and ubiquitin ligases (E3). E1 activates ubiquitin so it can be transferred to its active site, cysteine, by E2 which can bond with both E1 and ubiquitin. Then, E3 recognizes and binds the target substrate using an isopeptide bond between the last amino acid, glycine 76 of the ubiquitin molecule to a lysine on the substrate protein. This enzymatic cascade is repeated until a small chain of several ubiquitin molecules marks the target protein for degradation in the proteasome. To understand how ubiquitin functions in the removal of unwanted or damaged proteins, it is critical to know its quaternary organization and conformational changes. In this research, we investigate the gas phase oligomeric structure of Ubiquitin in neutral and basic environments using high resolution mass spectrometry. In neutral conditions, the measured charged states ranged from 2+ to 11+ for the monomer, with the highest peak of 8+ at 1057.20 m/z. For the dimer, the charges ranged from 5+ to 9+, with 7+ being the most intense peak located at 2415.30 m/z, and for the trimer, 7+ to 11+ states were observed, with the highest peak at 10+ at 2535.96 m/z. Moreover, we observed a single tetramer charged state of 13+ at 2605.78 m/z. In basic condition, similar pattern is observed for monomer, dimer, and trimer, however, no tetramer is noticed. Overall, the peaks observed in neutral conditions were more intense than the peaks observed in basic conditions. This study provides experimental evidence Ubiquitin can form more oligomers in neutral condition compared to the basic (native like) environment.
Academic department under which the project should be listed
CSM - Chemistry and Biochemistry
Primary Investigator (PI) Name
Mohammad A. Halim
Investigating the Gas Phase Ubiquitin Oligomers Formation by High Resolution Orbitrap Exploris 240 Mass Spectrometer
Ubiquitin is a 76 amino-acid residue protein which covalently binds to substrates and modifies them using a class of posttranslational modification called Ubiquitination. Ubiquitination is regulated by three types of enzymes, namely, ubiquitin-activating enzymes (E1), ubiquitin conjugating enzymes (E2), and ubiquitin ligases (E3). E1 activates ubiquitin so it can be transferred to its active site, cysteine, by E2 which can bond with both E1 and ubiquitin. Then, E3 recognizes and binds the target substrate using an isopeptide bond between the last amino acid, glycine 76 of the ubiquitin molecule to a lysine on the substrate protein. This enzymatic cascade is repeated until a small chain of several ubiquitin molecules marks the target protein for degradation in the proteasome. To understand how ubiquitin functions in the removal of unwanted or damaged proteins, it is critical to know its quaternary organization and conformational changes. In this research, we investigate the gas phase oligomeric structure of Ubiquitin in neutral and basic environments using high resolution mass spectrometry. In neutral conditions, the measured charged states ranged from 2+ to 11+ for the monomer, with the highest peak of 8+ at 1057.20 m/z. For the dimer, the charges ranged from 5+ to 9+, with 7+ being the most intense peak located at 2415.30 m/z, and for the trimer, 7+ to 11+ states were observed, with the highest peak at 10+ at 2535.96 m/z. Moreover, we observed a single tetramer charged state of 13+ at 2605.78 m/z. In basic condition, similar pattern is observed for monomer, dimer, and trimer, however, no tetramer is noticed. Overall, the peaks observed in neutral conditions were more intense than the peaks observed in basic conditions. This study provides experimental evidence Ubiquitin can form more oligomers in neutral condition compared to the basic (native like) environment.