Retention prediction of small peptides in reversed-phase liquid chromatography

The value of reversed-phase high-performance liquid chromatography (RP-HPLC) and the field of proteomics would be greatly enhanced by accurate prediction of retention times of peptides of known composition. The present study investigates the hydrophilicity/hydrophobicity of amino acid sidechains at the N-and C-termini of peptides while varying the functional end-groups at the termini. We substituted all 20 naturally occurring amino acids at the N-and C-termini of a model peptide sequence, where the functional end-groups were N α -acetyl-X-and N α -amino-X-at the N-terminus and -X-C α -carboxyl and -X-C α -amide at the C-terminus. Amino acid coefficients were subsequently derived from the RP-HPLC retention behaviour of these peptides and compared to each other as well as to coefficients determined in the centre of the peptide chain (internal coefficients). Coefficients generated from residues substituted at the C-terminus differed most (> 2.5 min between the -X-C α -carboxyl and -X-C α -amide peptide series) for hydrophobic side-chains. A similar result was seen for the N α -acetyl-X-and N α -amino-X-peptide series, where the largest differences in coefficient values (> 2 min) were observed for hydrophobic peptides. Coefficients derived from substitutions at the Cterminus for hydrophobic amino acids were dramatically different compared to internal coefficients for hydrophobic side-chains, ranging from 17.1 min for Trp to 4.8 min for Cys. In contrast, coefficients derived from substitutions at the N-terminus showed relatively small differences from the internal coefficients. Subsequent prediction of peptide retention time, within an error of just 0.4 min, was achieved by a predictive algorithm using a combination of internal coefficients and a weighted coefficient for the C-terminal residue.

Section: (I) Amino Acid Compositionmentioning

confidence: 99%

Requirements for prediction of peptide retention time in reversed-phase high-performance liquid chromatography: Hydrophilicity/hydrophobicity of side-chains at the N- and C-termini of peptides are dramatically affected by the end-groups and location

Tripet

Cepeniene

Kovacs

et al. 2007

“…4B) where, despite slightly anomalous behaviour of the His value in 50 mM NaCl, the Δt R values were essentially independent of the effectiveness of the anion (Cl -< ClO -4 ) in the mobile phase (a correlation of 0.995). [40,[55][56][57][58][59][60][61][62][63][64][65] or synthetic model peptides [39,42,54,66] have been published over the past 25 years. Unlike the scales reported by Kovacs et al [39] which were determined in the absence of nearest-neighbor effects (i.e., intrinsic side-chain hydrophilicity/ hydrophobicity was measured), these earlier scales were, amongst other factors, influenced by nearest-neighbour effects such as those described in the present study.…”

Section: An Interesting Anomaly Frommentioning

confidence: 99%

Quantitation of the nearest-neighbour effects of amino acid side-chains that restrict conformational freedom of the polypeptide chain using reversed-phase liquid chromatography of synthetic model peptides with l- and d-amino acid substitutions

Kovacs

Mant

Kwok

et al. 2006

Side-chain backbone interactions (or "effects") between nearest neighbours may severely restrict the conformations accessible to a polypeptide chain and thus represent the first step in protein folding. We have quantified nearest-neighbour effects (i to i+1) in peptides through reversed-phase liquid chromatography (RP-HPLC) of model synthetic peptides, where L- and D-amino acids were substituted at the N-terminal end of the peptide sequence, adjacent to a L-Leu residue. These nearest-neighbour effects (expressed as the difference in retention times of L- and D-peptide diastereomers at pHs 2 and 7) were frequently dramatic, depending on the type of side-chain adjacent to the L-Leu residue, albeit such effects were independent of mobile phase conditions. No nearest-neighbour effects were observed when residue i is adjacent to a Gly residue. Calculation of minimum energy conformations of selected peptides supported the view that, whether a L- or D-amino acid is substituted adjacent to L-Leu, its orientation relative to this bulky Leu side-chain represents the most energetically favourable configuration. We believe that such energetically favourable, and different, configurations of L- and D-peptide diastereomers affect their respective interactions with a hydrophobic stationary phase, which are thus quantified by different RP-HPLC retention times. Side-chain hydrophilicity/hydrophobicity coefficients were generated in the presence of these nearest-neighbour effects and, despite the relative difference in such coefficients generated from peptides substituted with L- or D-amino acids, the relative difference in hydrophilicity/hydrophobicity between different amino acids in the L- or D-series is maintained. Overall, our results demonstrate that such nearest-neighbour effects can clearly restrict conformational space of an amino acid side-chain in a polypeptide chain.

“…1 is the relative hydrophobicities of the residues within peptides 1-6 in the presence of different ion-pairing reagents. Data obtained from retention behaviour of peptides during RP-HPLC have frequently been employed to derive relative hydrophilicity/hydrophobicity values or "coefficients" of amino acid sidechains [13,[31][32][33][34][35][36][37][38][39][40][41][42]. Values obtained in this manner may then be used for such purposes as prediction of peptide retention behaviour, e.g., for narrowing down the position of a peptide of interest following RP-HPLC of a complex peptide mixture such as a protein digest.…”

Section: Effect Of Concentration Of Ion-pairing Reagent On Elution Bementioning

confidence: 99%

Effect of anionic ion-pairing reagent concentration (1–60mM) on reversed-phase liquid chromatography elution behaviour of peptides

Shibue

Mant

Hodges

2005

The homologous series of volatile perfluorinated acids-trifluoroacetic acid (TFA), pentafluoropropionic acid (PFPA) and heptafluorobutyric acid (HFBA)--continue to be excellent anionic ion-pairing reagents for reversed-phase high-performance liquid chromatography (RP-HPLC) after more than two decades since their introduction to this field. It was felt that a thorough, step-by-step re-examination of the effects of anionic ion-pairing reagents over a wide concentration range on RP-HPLC peptide elution behaviour is now due, particularly considering the continuing dominance of such reagents for peptide applications. Thus, RP-HPLC was applied over a range of 1-60 mM phosphoric acid, TFA, PFPA and HFBA to two mixtures of 18-residue synthetic peptides containing either the same net positive charge (+4) or varying positive charge (+1, +2, +3, +4). Peptides with the same charge are resolved very similarly independent of the ion-pairing reagent used, although the overall retention times of the peptides increase with increasing hydrophobicity of the anion: phosphate < TFA- < PFPA- < HFBA-. Peptides of differing charge move at differing rates relative to each other depending on concentration of ion-pairing reagents. All four ion-pairing reagents increased peptide retention time with increasing concentration, albeit to different extents, again based on hydrophobicity of the anion, i.e., the more hydrophobic the anion, the greater the increase in peptide retention time at the same reagent concentration. Interestingly, phosphoric acid produced the best separation of the four-peptide mixture (+1 to +4 net charge). In addition, concentrations above 10 mM HFBA produced a reversal of the elution order of the four peptides (+1 < + 2 < + 3 < + 4) compared to the elution order produced by the other three reagents over the entire concentration range (+4 < + 3 < + 2 < + 1).