Molecules and methods for iterative polypeptide analysis and processing

A composition comprising:

• (a) a substrate, wherein the substrate comprises a plurality of polypeptide molecules covalently attached to the substrate;
• and
• (b) a non-naturally occurring N-terminal amino acid binding (NAAB) protein bound to a N-terminal amino acid residue of a polypeptide molecule of the plurality of polypeptide molecules, wherein the NAAB protein (i) selectively binds the N-terminal amino acid residue with at least about a 1.5:1 ratio of specific to non-specific binding, and (ii) is derived from a tRNA synthetase and comprises one or more mutations in an amino acid-binding domain of the tRNA synthetase, wherein both the NAAB protein and the tRNA synthetase have specificity towards a first amino acid, and wherein the tRNA synthetase has specificity towards said first amino acid in a free form, whereas the NAAB protein has specificity towards said first amino acid incorporated into an N-terminus of a polypeptide, forming the N-terminal amino acid residue.