Strong Cation Exchange Chromatography in Analysis of Posttranslational Modifications: Innovations and Perspectives
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.
