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

Phosphopeptide enrichment with 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

  • Thermo Pierce Fe-NTA Phosphopeptide Enrichment Kit enables fast and efficient enrichment of phosphorylated peptides to process protein digests or strong cation-exchange peptide fractions for analysis by mass spectrometry

  • Thermo Pierce TiO2 Phosphopeptide Enrichment and Clean-up Kit enables fast, selective enrichment of phosphorylated peptides for mass spectrometry, it complements the Pierce Fe-NTA IMAC Phosphopeptide Enrichment Kit

  • Sigma PHOS-Select Iron Affinity Gel is an Iron [Fe(III)] chelate matrix based on Sigma's NTA analog chelate ligand. The matrix provides high capacity affinity binding of molecules containing phosphate groups

  • Sigma PHOS-Select Gallium Silica Spin Column Kit is a capture matrix is a Ga3+ chelate silica based on a proprietary nitriloacetic acid (NTA) analog

  • The Pierce Graphite Spin Columns improve phosphopeptide analysis by efficiently binding hydrophilic peptides and efficiently removing urea, salts and other contaminants before MS analysis. The C18 resins and C18 tips that are commonly used to desalt peptides are excellent for use with hydrophobic peptides but do not efficiently capture hydrophilic peptides, like phosphopeptides, resulting in enrichment of only hydrophobic fragments.


  • This review covers several glycoprotein/peptide enrichment strategies:
    Quantitative mass spectrometric analysis of glycoproteins combined with enrichment methods. Ahn YH, Kim JY, Yoo JS. Mass Spectrom Rev. 2015 Mar-Apr;34(2):148-65. doi: 10.1002/mas.21428. Epub 2014 Jun 2. link
  • Solid Phase Extraction of N-linked Glycopeptides Using Hydrazide Tip. Jing Chen, Punit Shah, and Hui Zhang Anal Chem. 2013 Nov 19;85(22):10670-4. doi: 10.1021/ac401812b. Epub 2013 Oct 30. link

  • Thermo aminoxyTMTsixplex Label Reagents
    The carbonyl-reactive Thermo Scientific™ aminoxyTMT™ (Tandem Mass Tag™) Label Reagents enable multiplexed characterization and quantitation of carbonyl-containing biomolecules (carbohydrates, steroids, oxidized proteins) by mass spectrometry (MS).

List of peptide cleanup protocols for LC-MS