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.

Figure 1

ESI-FTMS spectra of amine methylated proteins (A) ubiquitin, (B) myoglobin, (C) RNase A, and (D) lysozyme.

Figure 2

(A) Active ester of 4-(trimethylamino)-3-butyric acid prepared for acylation of protein amines. (B) ESI-FTMS of ubiquitin acylated with compound 1.

Figure 3

ESI-FTMS spectra of thiol alkylated (with iodoacetamide, if thiol present), amine methylated, acid amidated (with glycine methyl ester) proteins (A) ubiquitin, (B) myoglobin, (C) RNase A, and (D) lysozyme.

Figure 4

ESI-FTMS spectra of thiol alkylated (iodoacetamide), amine methylated, acid amidated lysozyme. The acid amidation used four separate amines, as indicated (A–D).

Figure 5

Relative quantitation of two protein samples and determination of protein amines and acids. (A) Mass spectrum of 15+ ions of 1:1 mixture of thiol-alkylated lysozyme methylated with either D₀ or D₂ formaldehyde. (B) Mass spectrum of 16+ ions of 1:1 mixure of thiol-alkyated, methylated lysozyme amidated with either D₀ or D₃ glycine methyl ester.

Figure 6

Percentage of unique proteins in the yeast proteome as a function of accuracy of the mass determination and the number of thiol (A) or thiol, amine, and acid (B) functional groups. Percent uniqueness was calculated from the number of proteins with unique numbers of functional groups within a certain mass accuracy (50, 100, or 200 ppm) divided by the total number of proteins within a 1000 Da mass window from 5 to 50 kDa in the yeast proteome.

Scheme 1

Protein Modification Strategy