A study of peptide-peptide interaction by matrix-assisted laser desorption/ionization

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry was u sed to study peptide-peptide interaction. The interaction was seen when 6-a za-2-thiothymine was used as a matrix (pH 5.4), but was disrupted with a mo re acidic matrix, alpha -cyano-4-hydroxycinnamic acid (pH 2.0). In the pres ent study, we show that dynorphin, an opioid peptide, and five of its fragm ents that contain two adjacent basic residues (Arg(6)-Arg(7)), all interact noncovalently with peptides that contain two to five adjacent acidic resid ues (Asp or Glu). Two other nonrelated peptides containing two (Arg(6)Arg(7 )) or three (Arg(1)-Lys(2)-Arg(3)) adjacent basic amino acid residues were studied and exhibited the same behavior. However, peptides containing adjac ent Lys or His did not form noncovalent complexes with acidic peptides. The noncovalent bonding was sufficiently stable that digestion with trypsin on ly cleaved Arg and Lys residues that were not involved in hydrogen bonding with the acidic residues. In an equimolar mixture of dynorphin, dynorphin f ragments (containing the motif RR), and an acidic peptide (minigastrin), th e acidic peptide preferentially complexed with dynorphin. If the concentrat ion of minigastrin was increased 10 fold, noncovalent interaction was seen with dynorphin and all its fragments containing the motif RR. In the absenc e of dynorphin, minigastrin formed noncovalent complexes with all dynorphin fragments. These findings suggest that conformation, equilibrium, and conc entration do play a role in the occurrence of peptide-peptide interaction. Observations from this study include: (1) ionic bonds were not disrupted by enzymatic digests, (2) conformation and concentration influenced complex f ormation, and (3) the complex did not form with fragments of dynorphin or u nrelated peptides that did not contain the motifs RR or RKR, nor with a fra gment of dynorphin where Arg(7) was mutated to a phenylalanine residue. The se findings strongly suggest that peptide-peptide interaction does occur, a nd can be studied by MALDI if near physiologic pH is maintained. (C) 2001 A merican Society for Mass Spectrometry.