Sharel Cornelius / Chemistry & Biochemistry / Faculty Mentor: Saiful M Chowdhury
“Bioconjugation of proteins is a crucial technique for probing cellular systems and developing biotherapeutic constructs which includes chemical crosslinkers. Crosslinking mass spectrometry has emerged as an effective technique for investigating determining structural characteristics and examining protein-protein interactions. We evaluated the site-specific labeling of tyrosine residues employing 4-phenyl-3H-1,2,4-triazole-3,5(4H)-dione (PTAD) as the bioconjugation reagent. Additionally, we devised a novel PTAD-based tyrosine reactive crosslinker (TYCL) to examine the structural topology of SARSCOV2 spike protein.
We first tested the method of bioconjugation of tyrosine residues with PTAD in neurotensin peptide. The resulting mass of the labeled peptide appeared at 675.32 m/z [M+2PTAD+3H]3+.
Cross-linking of the tyrosine residues using the TYCL was studied in various tyrosine-containing proteins like myoglobin, BSA, and SARSCoV-2 S1 spike protein. The inter-crosslinked peptides with residues Y146 and Y146 in myoglobin were observed at 785.11 m/z [M+3H]3+. This crosslinking corroborated the formation of dimer in myoglobin. In the case of BSA, we observed one dead-end-crosslinked peptide at 870.80 m/z [M+3H]3+ and two inter-crosslinked peptides at 1146.47 m/z [M+3H]3+ and 886.52 m/z [M+3H]3+. We also observed multiple crosslinking in the SARSCoV-2 S1 spike protein.
We developed effective PTAD-based reagents for tyrosine bioconjugation and cross-linking for mapping vital structural information and protein-protein interactions.”
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