Cucurbit[n]uril (n = 6 and 7)-Based High Affinity Enrichment of Basic Peptides Containing Multiple K/R Residues via...

Host-guest interactions by artificially synthesized macrocyclic molecules, such as cucurbit[n]uril (CB[n], n refers to the number of glycoluril units), exhibited comparable affinity to natural biological interactions, offering orthogonal selectivities. These unique high-affinity interactions are crucial in bioseparation, particularly in the separation of complex biomacromolecules. However, to date, the application of supramolecular systems in bioseparation remains insufficient and challenging. In this work, a site-specific host-guest interaction-based method by CB[6] and CB[7] was developed for the selective enrichment of basic peptides, and their recognition for basic lysine/arginine (K/R) residues exhibited distinct selectivity. Although they both exhibited affinity to basic peptides, CB[6] tended to bind to peptides containing multiple K/R residues, while CB[7] preferred to adsorb peptides containing both phenylalanine residues and basic K/R residues. This difference was attributed to their distinct cavity sizes. Furthermore, CB[6]-containing monolith was employed for the enrichment of basic peptides from mouse liver sample digested with Staphylococcus aureus protease V8 (Glu-C). It is noteworthy that basic peptides containing 3-4 K/R residues can be successfully enriched, whereas those with more than 5 K/R residues cannot be effectively eluted. It was also observed that 1043 unique peptides were newly identified from 2.5 μg of mouse liver after the capture of basic peptides, with a markable increase in the identification coverage (31%). These newly identified peptides are generally more acidic and shorter than those depleted by CB[6], with median isoelectric point (pI) values of 5.8 and 7.0 and median lengths of 10 and 16 amino acids, respectively.