Chromatographic behaviour of peptides modified with amine-reacting tags for relative protein quantitation in proteomic applications

Reversed-phase (RP) HPLC separation of peptides labeled with amine-reacting tags for relative protein quantitation (iTRAQ4, iTRAQ8 - isobaric tag for relative and absolute quantitation, TMT - tandem mass tag) has been investigated using large-scale proteomics derived retention datasets. These tags have similar chemistry but use linkers of different length and hydrophobicity, moving the positively charged functional groups further from peptide backbone. Peptide hydrophobicity (RP HPLC retention), on average, increases in the following order: non-labeled < iTRAQ4 < iTRAQ8 < TMT under both low pH (0.1% formic acid) and pH 10 eluent conditions. At the same time, the interplay between hydrophobicity and length of the labeling group drives the deviations from this order. Thus, longer and less hydrophobic iTRAQ8 moiety results in greater retention increase for peptides carrying amphipathic helical structures at the N-terminus. Development of a peptide retention prediction models for these modifications was achieved by predicting correspondent retention shifts Delta HI (hydrophobicity index,% acetonitrile) between unmodified and labelled peptide pairs. (C) 2022 Published by Elsevier B.V.

Automated summary

This study investigated the reversed-phase HPLC separation of peptides labeled with amine-reacting tags for relative protein quantitation. The study found that the hydrophobicity of these labeled peptides varies under different pH conditions, and the hydrophobicity is influenced by the length of the labeling group.