Reversed-phase chromatography of peptides carrying non-canonical proline analogues, with special focus on 4R/4S-fluoroproline

We investigated the reversed-phase chromatographic behaviour of peptides carrying nine non-canonical proline analogues using LC-MS/MS bottom-up proteomics analysis of Escherichia coli (E.coli) following co-translational incorporation. Of particular interest were the epimers 4R- and 4S-fluoroproline. On average, peptide interactions with the hydrophobic C18 phase increased in the order: 4R-Flp < Pro < 4S-Flp. The mean retention shifts were found to be -0.11 and +0.06% acetonitrile for 4R- and 4S-Flp, respectively (0.1% formic acid eluent additive). This diminutive average difference in hydrophobic properties belies the unpredictable chromatographic behaviour of modified peptides. The incorporation of either fluorinated epimer, each relatively amphipathic, can make certain peptides more hydrophobic, while for others, the peptide becomes more hydrophilic. Incorporation of a single 4R-fluoroproline produces a shift ranging from (2.18 to +2.55) %ACN, while 4S-fluoroproline produces shifts ranging from (-4.82 to +4.67) %ACN on the RP HPLC elution scale. In addition to common composition-dependent factors affecting peptide retention, such as peptide length, we identified several sequence-specific features that explain deviations from the average retention characteristics. These include nearest neighbour effects and amphipathic helicity. Other analogues investigated encompassed diverse chemical features, including hydroxylation (4R-hydroxyproline), dehydrogenation (3,4-dehydroproline), ring expansion (pipercolic acid), heteroatom inclusion (3-thia-proline & 4-thia-proline) and bridge ring structures (4,5-trans-methanoproline & 4,5-cis-methanoproline).