Figure 1: Species-dependent phosphoglycerate mutase catalytic mechanisms and overview of affinity selection.

(a) Isomerization catalysed by PGMs illustrating the phosphohistidine enzyme/2,3-phosphoglycerate intermediate of human cofactor-dependent PGM (top) and the phosphoserine enzyme intermediate of C. elegans cofactor-independent PGM. (b) Random nonstandard peptide integrated discovery (RaPID) begins with an mRNA library encoding trillions of potential peptides 6–14 amino acids in length. The mRNA library is ligated to an adapter incorporating the amino nucleoside, puromycin. The flexible in vitro translation (FIT) system is used to create the peptide library with an L- or D-N-chloroacetyl tyrosine (dark blue sphere) charged initiator tRNA and 19 proteogenic amino acids (grey spheres), methionine is excluded as its tRNA is charged with the chloroacetyl tyrosine. Incorporation of a cysteine during translation results in macrocyclization via thiolate nucleophilic attack on the chloroacetyl electrophile. After incubating with the library the beads are washed to enrich the bound conjugates which are then reverse transcribed and amplified via PCR. PCR products are transcribed to mRNAs and the process is repeated or PCR products sequenced to reveal the peptide sequences captured. Peptides from these sequences are then produced in milligram quantity using solid-phase peptide synthesis (SPPS).