Peptide Separation

Strong cation-exchange (SCX) chromatography
SCX has been used extensively for the fractionation of proteins and peptides based on charge. The SCX stationary phase usually contains aliphatic sulfonic acid groups that are negatively charged in aqueous solution, therefore tightly binding any strongly basic analytes. To recover the analyte, the resin is then washed with a solvent neutralizing this ionic interaction. Most tryptic peptides in acidic pH are characterized by a net charge of +2 and above, and they can be therefore separated by SCX from peptides possessing a net charge of +1, such as trypsin-generated phosphopeptides, C-terminal peptides, or peptides with blocked N-termini (i.e., peptides with blocked N-terminal free amine group, for instance, by N-acetylation), as well as from peptides containing higher charges, including ones containing missed cleavages and therefore more arginine and lysine residues. SCX fractionation can also be performed in a solid-phase extraction cartridge format for a rapid but lower resolution fractionation. SCX can also be conducted in microscale solid-phase extraction format (i.e. after IMAC) by utilizing a stage tip packed with an SCX disk.

  • Fractionation of peptides by strong cation-exchange liquid chromatography. Chan KC, Issaq HJ. Methods Mol Biol. 2013;1002:311-5. link
  • Strong cation exchange chromatography in analysis of posttranslational modifications: innovations and perspectives. Edelmann MJ. J Biomed Biotechnol. 2011;2011:936508. link
  • A solid phase extraction-based platform for rapid phosphoproteomic analysis. Dephoure N, Gygi SP. Methods. 2011 Aug;54(4):379-86 link
  • Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips. Rappsilber J, Mann M, Ishihama Y. Nat Protoc. 2007;2(8):1896-906 link

for biotin labeled peptides

  • Check out this biotinylation overview at Pierce link

IMAC (Immobilized Metal Affinity Chromatography)
To achieve robust MS results, enrichment of phosphopeptide samples is essential because of low abundance and poor ionization relative to non-phosphorylated peptides. Phosphopeptide enrichment reduces sample complexity and enables effective identification and characterization of phosphorylated peptides by MS.

A practical recipe to survey phosphoproteomes.
Edelman WC, Haas KM, Hsu JI, Lawrence RT, Villén J. Methods Mol Biol. 2014;1156:389-405. doi: 10.1007/978-1-4939-0685-7_26. link

N-glycosylated peptides

Peptide separation techniques