Performance of combinatorial peptide libraries in capturing the low-abundance proteome of red blood cells. 2. Behaviour of resins containing individual amino acids

Sixteen different amino acids (Arg, Asn, Asp, Gln, Glu, Gly, His, Ile, Lys, Phe, Pro, Ser, Thr, Trp, Tyr, Val) have been separately linked to chromatographic beads and used for studying the mechanism of binding of such baits to proteins, as represented by the cytoplasmic proteome of the human red blood cell (RBC). The 16 different amino acid columns were confronted with equal amounts of RBC lysate, washed to remove unbound material, and eluted with denaturing agents. All eluates were analyzed by nanoLC-MS/MS. The results: there appears to be a dichotomy between a class of "Grand Catchers" (Arg, His, Ile, Lys, Phe, Trp, Tyr, Val), all able to bind from 330 up to 441 unique gene products, and the "Petite Catchers" (Asn, Asp, Gln, Glu, Gly, Pro, Ser, Thr), that bind in general half as much, with the notable exception of Glu that under the described conditions seems to bind only traces of proteins. By comparing homogeneous classes of amino acids (e.g., the basic, the hydrophobic aromatic, the neutral hydrophilic, etc.), it is found that, in general, more than half as many proteins are held in common among the members of each family. In a 16-way comparison, 72 proteins (less than 10% of the total amount, which amounts to 800 unique, nonredundant, identified proteins) appear to be the common catch of all 16 amino acids, suggesting that such proteins might have either multiple binding sites or general motifs recognized by any generic bait. By far, it would appear that the strongest interactions and thus the strongest catches occur with the three aromatic moieties of Phe, Trp, and Tyr, all able to capture a practically identical number of proteins. Ionic interactions, which in principle should be the strongest ones, appear to behave in a peculiar way: they are quite strong with the three basic amino acids (Arg, His, Lys) but almost inexistent with their acidic counterparts. This suggests a peculiar mechanism of interaction: upon formation of the ion pair, the linkage between the protein and the bait is stabilized by the hydrophobicity of the underlying chain (e.g., a butyl in the case of Lys).