Characterization Of The Higher Order Structure Of Proteins Using LC-MS Assisted Assignments Of NMR Resonances

Biological macromolecular drugs have incomparable advantages over other types of drugs in terms of effectiveness and safety,and have been widely used in the treatment of tumors and autoimmune diseases.The higher order structure is one of the critical quality attributes of biological macromolecular drugs because it is related to the safety,efficacy and pharmacokinetic properties.At present,the methods for the study of the higher order structure in proteins mainly include X-ray crystal diffraction(XRD),nuclear magnetic resonance spectroscopy(NMR),cryo-electron microscopy(cryo-EM),circular dichroism spectroscopy(CD),and hydrogen deuterium exchange method(HDX).XRD has extremely high resolution and can provide more refined high-level structural information,but it cannot be widely used because most proteins are not easy to crystallize,and the results measured by XRD are based on the solid phase structure of crystals,which cannot accurately reflect the protein in the liquid phase.The advantage of CD is that it is quick and easy to detect.The disadvantage is that it has low resolution and can only provide information about secondary structure.The advantage of HDX is that it can provide site-specific higher order structural information.However,due to poor experimental precision,its application is not widely.NMR spectroscopy is a powerful technique for probing protein structure,interaction and dynamic studies in solution.However,its application in biopharmaceuticals is mitigated by the difficulty in NMR resonance assignment as the isotopically labeled biopharmaceuticals are not always available.This paper shows a new MS-assisted strategy for the NMR assignment of naturalabundance samples.The strategy involves selective modification of the target amino acid and quantification of the modification level by both NMR and MS.The differential nature of protein modification is exploited wherein various levels of modification across target residues on the protein can be achieved depending on their solvent accessibility and generate partially modified protein materials.The NMR assignment of the side-chain resonances is made by correlating the quantitative results obtained from both NMR and MS.Arginine residues are essential in a variety of biological processes.Structurally,the guanidino group forms multiple hydrogen bonds in the maintenance of protein tertiary structure.Functionally,through the formation of salt bridges,arginine residues play critical roles in protein–protein interactions,in enzyme active sites,and in protein–nucleic acid recognition.Therefore,the paper shows a new MS-assisted strategy for the NMR assignment of ?-NH of arginine side chains.By correlating the rank order of the modification extent of the arginine residues from NMR and LC–MS,the arginine resonances of the ubiquitin can be assigned,that is,signals A,B,and C correspond to R54 ?-NH,R72 ?-NH,R42 ?-NH,respectively.The method is further applied to serve the development of a new CGRP antibody.Based on the results of epitope identification by ELISA and NMR methods,the epitopes of CGRP-BOAN-Ig G2 and CGRP-TEVA-Ig G2 are located in the 25-37 segment of CGRP,and the C-terminal amide is necessary for the binding of the two antibodies;the arginine R11 and R18 are also part of the epitope of the reference antibody,but not part of the epitope of the new antibody.LC-MS had been used to assist the assignment of the NMR signals of two arginines in ?-CGRP protein.The NMR signal was obtained by the direct ranking comparison of the modification rate,that is,the signal A corresponds to R11 ?-NH,and the signal B corresponds to R18 ?-NH.In summary,the paper has successfully established MS-based methodology to assign the NMR resonances of the arginine ?-NH.This method can be extended to somewhat larger proteins for the investigation of protein structure,in particular with regard to the biological importance of arginine or to the assignment of NMR resonances of the side chains of other amino acids in proteins.This study is also useful for the application of NMR-based approach as a probe to characterize protein structure,protein–ligand binding interaction,and protein–protein complexes.