Effect of cyclization of N-terminal glutamine and carbamidomethyl-cysteine (residues) on the chromatographic behavior of peptides in reversed-phase chromatography.

N-terminal loss of ammonia is a typical peptide modification chemical artifact observed in bottom-up proteomics experiments. It occurs both in vivo for N-terminal glutamine and in vitro following enzymatic cleavage for both N-terminal glutamine and cysteine alkylated with iodoacetamide. In addition to a mass change of -17.03 Da, modified peptides exhibit increased chromatographic retention in reversed-phase (RP) HPLC systems. The magnitude of this increase varies significantly depending on the peptide sequence and the chromatographic condition used. We have monitored these changes for extensive sets (more than 200 each) of tryptic Gln and Cys N-terminated species. Peptides were separated on 100 Å pore size C18 phases using identical acetonitrile gradient slopes with 3 different eluent compositions: 0.1% trifluoroacetic acid; 0.1% formic acid and 20 mM ammonium formate at pH 10 as ion-pairing modifiers. The observed effect of this modification on RP retention is the product of increased intrinsic hydrophobicity of the modified N-terminal residue, lowering or removing the effect of ion-pairing formation on the hydrophobicity of adjacent residues at acidic pHs; and possibly the increased formation of amphipathic helical structures when the positive charge is removed. Larger retention shifts were observed for Cys terminated peptides compared to Gln, and for smaller peptides. Also the size of the retention increase depends on the eluent conditions: pH 10≪trifluoroacetic acid<formic acid. Different approaches for incorporation these findings in the peptide retention prediction models are discussed.