The benefits of mixed-mode chromatography columns for separation of peptides and protein digests

Abstract

In this work, the evaluation and comparison of mixed-mode chromatography and reversed-phase chromatography for separation of peptides and protein digests have been performed. The effects of pH of aqueous part of mobile phase as well as the effects of organic modifier on retention, resolution, and peak shape were investigated on several columns including three mixed-mode columns possessing reversed-phase/anion-exchange mechanism, two reversed-phase octadecyl columns, and one column with mixed-mode reversed-phase/anion-exchange character only in defined pH range. The set of peptides varying in their polarity, length, amino acid sequence, and charge state, namely dipeptides, N-blocked dipeptides, and oligopeptides, was selected to describe the chromatographic behavior under different conditions properly. These measurements showed the potential of mixed-mode chromatography columns for analysis of differently charged peptides in a single run. The applicability of the tested conditions has been verified by the analysis of cytochrome C digested fragments. Two types of samples were analyzed and compared, i.e., commercial cytochrome C digested standard and cytochrome C digested via trypsin spin columns. The obtained results point to the necessity of using mass spectrometry detection because of large number of unknown peaks in cytochrome C digested standard, probably originating from chymotryptic and miscleavage activities. Graphical abstract

Advances in Therapeutic Peptides Separation and Purification

“…Aebischer et al developed theoretical models to predict the effectiveness of dilution and determine the minimum dilution value required, if applicable [60]. The theoretical Kadlecová and co-workers demonstrated the benefits of MMC columns for the separation of peptides [54]. They evaluated three MMC columns with RP/anion-exchange mechanisms: two RP octadecyl columns and one column with MMC RP/anion-exchange characteristics only within a defined pH range [54].…”

“…The theoretical Kadlecová and co-workers demonstrated the benefits of MMC columns for the separation of peptides [54]. They evaluated three MMC columns with RP/anion-exchange mechanisms: two RP octadecyl columns and one column with MMC RP/anion-exchange characteristics only within a defined pH range [54]. They selected a set of peptides varying in polarity, length, amino acid sequence, and charge state, which included dipeptides, N-blocked dipeptides, and oligopeptides [54].…”

“…They evaluated three MMC columns with RP/anion-exchange mechanisms: two RP octadecyl columns and one column with MMC RP/anion-exchange characteristics only within a defined pH range [54]. They selected a set of peptides varying in polarity, length, amino acid sequence, and charge state, which included dipeptides, N-blocked dipeptides, and oligopeptides [54]. They demonstrated the potential of MMC columns for analyzing differently charged peptides in a single run [54].…”

“…They selected a set of peptides varying in polarity, length, amino acid sequence, and charge state, which included dipeptides, N-blocked dipeptides, and oligopeptides [54]. They demonstrated the potential of MMC columns for analyzing differently charged peptides in a single run [54].…”

“…Kadlecová and co-workers demonstrated the benefits of MMC columns for the separation of peptides [54]. They evaluated three MMC columns with RP/anion-exchange mechanisms: two RP octadecyl columns and one column with MMC RP/anion-exchange characteristics only within a defined pH range [54].…”