Strong cation exchange chromatography in analysis of posttranslational modifications: innovations and perspectives

Abstract

Strong cation exchange (SCX) chromatography has been utilized as an excellent separation technique that can be combined with reversed-phase (RP) chromatography, which is frequently used in peptide mass spectrometry. Although SCX is valuable as the second component of such two-dimensional separation methods, its application goes far beyond efficient fractionation of complex peptide mixtures. Here I describe how SCX facilitates mapping of the protein posttranslational modifications (PTMs), specifically phosphorylation and N-terminal acetylation. The SCX chromatography has been mainly used for enrichment of these two PTMs, but it might also be beneficial for high-throughput analysis of other modifications that alter the net charge of a peptide.

Figure 1

Phosphopeptide enrichment capabilities for various SCX resins. 50 μg casein was digested with trypsin and loaded in triplicates onto SCX-1, SCX-2, SCX-3 cartridges, and TiO₂-packed tips, followed by peptide elution. In case of the SCX cartridges, the sequential peptide elution was done using four buffers (A–D) characterized by increasing molarity of ammonium acetate (50, 100, 200, or 500 nM ammonium acetate). The TiO₂ tips were preconditioned with 100% acetonitrile, conditioned with 0.2 M phosphate buffer pH7, and equilibrated with solvent containing 50% acetonitrile and 0.1% formic acid. The peptide sample was then loaded onto TiO₂ resin, washed six times with buffer containing 50% acetonitrile, 0.1% formic acid, and 0.1 M KCl, and eluted with 0.2 M phosphate buffer pH 7 and 0.5% aqueous ammonia; both TiO₂ eluates were combined. The volume of each eluate was concentrated, and triplicate samples were analyzed by LC-MS/MS (Esquire HCTplus mass spectrometer, Bruker Daltonics). The displayed percentage of phosphorylated peptides was calculated for three forms of casein (UniProt accession numbers P02663, P02662, and P02663). Standard deviation was calculated using experimental triplicates.