ABSTRACT
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 10âªtrifluoroacetic acidSubject(s)
Cysteine/chemistry
, Glutamine/chemistry
, Peptides/chemistry
, Peptides/isolation & purification
, Chromatography, High Pressure Liquid/methods
, Chromatography, Reverse-Phase
, Cyclization
, Cysteine/analogs & derivatives
ABSTRACT
Separation selectivity of C18 reversed-phase columns from different manufacturers has been compared to evaluate the applicability of our sequence-specific retention calculator (SSRCalc) peptide retention prediction algorithms. Three different versions of SSRCalc are currently in use: 300-A pore size sorbents (TFA as ion-pairing modifier, pH 2), 100 A (TFA, pH 2), and 100 A (pH 10), which have been applied for the separation of randomly chosen mixture of tryptic peptides. The major factor affecting separation selectivity of C18 sorbents was found to be apparent pore size, while differences in end-capping chemistry do not introduce a significant impact. The introduction of embedded polar groups to the C18 functionality increases the retention of peptides containing hydrophobic amino acid residues with polar groups: Tyr and Trp. We also demonstrate that changing the ion-pairing modifier to formic/acetic acid significantly reduces the algorithm's predictive ability, so models developed for different eluent conditions cannot be compared directly to each other.