Amongst structural verification techniques, Nuclear Magnetic Resonance (NMR) offers the unique advantage of non-destructive monitoring of molecules in solution at an atomic level. NMR chemical shifts are unique identifiers that not only provide atomic level identification but also the means to track physicochemical changes in the molecule: conformational change, degradation, aggregation, binding interactions, etc. NMR experiments are designed to yield spectra that correlate NMR signals allowing assignment of signals to their respective atoms, and consequently, structure verification. However, with increasing molecular weight, the process of signal assignment becomes cumbersome when reliant on traditional proton-based methodology alone, due to increasing complexity, low signal dispersion, and overlap. This is especially the case with peptides where repetitions of the same amino acid or multiple occurrences of the same amino acid in the peptide chain further complicate categorical signal assignment. These challenges can be addressed by using 2D (two dimensional) heteronuclear (carbon, nitrogen) connectivity maps which alleviate spectral overlaps to facilitate signal assignment and rapid structure verification. Here, we demonstrate robust strategies for complete signal assignment (1H, 13C, 15N) of peptides ranging from 10 to 39 amino acids ¬- at natural abundance. Further, we demonstrate the benefits of such complete signal assignments in establishing unique peptide fingerprints by NMR: (a) distinguishing between Exenatide peptides (anti-diabetic) that are chemically identical but differ only in the isomerism (d vs l) of the first amino acid, and (b) distinguishing between single amino acid changes and deletions for Bivalirudin (direct thrombin inhibitor).
Session #9: Proteins, Peptides, Genes/Vaccines – With our Magnets Now You’re Seen