title: Global amine and acid functional group modification of proteins.
abstract: A sequential reaction methodology is employed for the complete derivatization of protein thiols, amines, and acids in high purity under denaturing conditions. Following standard thiol alkylation, protein amines are modified via reductive methylation with formaldehyde and pyridine-borane. Protein acids are subsequently amidated under buffered conditions in DMSO using the coupling reagent (7-azabenzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate. The generality of the approach is demonstrated with four proteins and with several amines yielding near-quantitative transformations as characterized by high-resolution Fourier transform mass spectrometry. The developed approach has numerous implications for protein characterization and general protein chemistry. Applications in mass spectrometry (MS) based proteomics of intact proteins (top-down MS) are explored, including the addition of stable isotopes for relative quantitation and protein identification through functional group counting. The methodology can be used for altering the physical and chemical properties of proteins, as demonstrated with amidation to modify protein isoelectric point and through derivatization with quaternary amines. Additionally, the chemistry has applications in the semisynthesis of monodisperse polymers based on protein scaffolds. We prepare proteins modified with azides and alkynes to enable further functionalization via copper(I)-catalyzed 1,3-dipolar Huisgen cycloaddition ("click") chemistry.
authors.affiliation: Departments of Biochemistry and Chemistry, University of Wisconsin-Madison, Madison, Wisconsin 53706, USA.
authors.forename: Casey J
authors.initials: CJ
authors.lastname: Krusemark
authors.forename: Jonathan T
authors.initials: JT
authors.lastname: Ferguson
authors.forename: Craig D
authors.initials: CD
authors.lastname: Wenger
authors.forename: Neil L
authors.initials: NL
authors.lastname: Kelleher
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Belshaw
mesh_terms: D000143:Acids / Q000737:chemistry*; D000478:Alkylation; D000480:Alkynes / Q000737:chemistry; D000588:Amines / Q000737:chemistry*; D001386:Azides / Q000737:chemistry; D001896:Boron Compounds / Q000737:chemistry; D002384:Catalysis; D003300:Copper / Q000737:chemistry; D003432:Cross-Linking Reagents / Q000737:chemistry; D004121:Dimethyl Sulfoxide / Q000737:chemistry; D005557:Formaldehyde / Q000737:chemistry; D013058:Mass Spectrometry / Q000379:methods; D008745:Methylation; D010449:Peptide Mapping / Q000379:methods*; D040901:Proteomics / Q000379:methods; D011725:Pyridines / Q000737:chemistry; D012680:Sensitivity and Specificity; D020539:Sequence Analysis, Protein / Q000379:methods*; D013438:Sulfhydryl Compounds / Q000737:chemistry
chemical_list: D000143:Acids; D000480:Alkynes; D000588:Amines; D001386:Azides; D001896:Boron Compounds; D003432:Cross-Linking Reagents; D011725:Pyridines; D013438:Sulfhydryl Compounds; C041928:pyridine borane; D005557:Formaldehyde; D003300:Copper; D004121:Dimethyl Sulfoxide