Amidination
 
Amidination takes place when an amino group is treated with an imide ester.
The reaction proceeds with reasonable yield when conducted under alkaline conditions (pH around 10) . Side reactions, such as cross-linking take place at lower pH. Both a- and  -amino groups can be amidinated, and the basicity of the modified residue increases. The amidinyl groups incorporated are stable in acidic media. The role of amino groups in protein function can be investigated by amidination. Proteins can be readily radiolabeled by amidinating with ¹³C- or ³H-labeled imide ester. Amidination of the  -amino group on lysine residues is particularly useful in peptide mapping and in determining the primary structure of a protein (see Protein Sequencing). The proteolytic enzyme trypsin does not cleave at the modified lysine residues, thereby limiting cleavage to arginine residues. Moreover, amidinyl groups are removed by aminolysis, and the resulting deprotected peptides are cleaved further with trypsin.
 1. Acetamidination of Proteins
After reduction and alkylation of the protein (100–700 nmol), it is dissolved in a few mL of 0.2 M triethylamine-HCl buffer, pH 10.3, containing 5.0 M guanidinium chloride (GdmCl) (1). Ethyl (or methyl) acetamide hydrochloride is dissolved in an equivalent amount of NaOH solution to maintain a final acetamide concentration of 0.1 to 0.15 M (100-fold molar excess of acetamide over amino groups). The reaction mixture is incubated for 1 hr at 25°C, dialyzed against 0.05 M NH₄HCO₃ containing 2.5 M GdmCl, and then against 0.05 M NH₄HCO₃. The protein is finally lyophilized.
 2. Deamidination by Methylaminolysis
 Acetamidinated protein or peptide (2.7 mg) is dissolved in 1.6 mL of 6 to 9 M urea. Then 0.9 mL of methylamine-formic acid buffer (9.6 M methylamine adjusted with HCOOH to pH 11.5) is added,  and the reaction mixture is held for 4 h at 25°C. The final concentration of methylamine is 3.5 M.  The reaction mixture is exhaustively dialyzed against deionized water at 4°C or, for peptides,
isolated by gel filtration on Sephadex G-10, equilibrated and eluted with 0.1 M NH₄HCO₃.
References
1. G. C. DuBois et al. (1981) Biochem. J. 199, 335–340. 
2. J. K. Inman et al. (1983) Methods Enzymol. 91, 559–569.