Table 1.

Summary of the strategies to assign the dimethylamino NMR peaks of reductively ¹³C-methylated hen egg white lysozyme.

Name	Assignment Strategy	Advantages	Disadvantages
Bradbury [25]	Compare experimental pKa values to model compounds and predicted electrostatic interactions from the X-ray crystal structure	Assignment of the α-amine and ε-amines involved in electrostatic interactions	Relies on the 3D protein structure; pKa values were not sufficient to assign all ε- amines
	Compare selective broadening effects of Gd3+ to predictions from the X-ray crystal structure	Assignment of amines within 24 Å of the paramagnetic ion	Relies on the 3D protein structure; relies on a known paramagnetic ion-binding site
Gerken [37]	Compare experimental pKa values and chemical shift to model compounds and predicted electrostatic interactions from the X-ray crystal structure	Assignment of the α-amine and ε-amines involved in electrostatic interactions	Relies on the 3D protein structure; pKa values were not sufficient to assign all ε- amines
Macnaughtan [38]	Correlates isotope- incorporation measurements from NMR peaks and MS isotopic profiles of tryptic peptides	Does not require a 3D protein structure	Degenerate isotope- incorporation limits absolute assignments; the α- and ε- amines must be separated to measure the MS isotopeincorporation
Roberson [39]	Uses pH to alter the relative rates of the α-amine reaction compared to the ε-amines	Does not require a 3D protein structure; absolute assignment of the α-amine; the MS isotope-incorporation of an ε-amine near the N- terminus can be measured	Limited to assigning the α- amine and one ε-amine near the N-terminus
	Uses aminopeptidase to assign the α-amine and an ε- amine near the N-terminus	Does not require a 3D protein structure	Limited to assigning the α- amine and one ε-amine near the N-terminus; relies on aminopeptidase activity toward the protein of interest
Larda [2]	Compares spin-label-induced line broadening with predictions from the X-ray crystal structure	Assignment of amines affected differently by soluble spin-labels	Relies on the 3D protein structure and data from another method