- for Cation-Exchange of Peptides-
This strong cation-exchange (SCX) material was developed specifically for HPLC of peptides. At pH 2.7-3.0, peptides lose their (-) charges and have net (+) charge. Thus, PolySULFOETHYL A is a general-purpose alternative to reversed-phase (RPC), fractionating peptides by differences in charge rather than polarity. Compared to other SCX materials based on sulfopropyl- (SP-) groups, PolySULFOETHYL A is unusually hydrophilic. This minimizes hydrophobic interactions with peptides, with high recovery and less peak tailing. Capacity is also high, permitting better retention and fractionation of the weakly basic peptides from tryptic digests. The capacity of an ion-exchange material like this is 4x that of a comparable RPC column. Therefore, ion-exchange should be the initial step of a multi-step purification. Use PolySULFOETHYL A for:
1) Multidimensional HPLC of peptide mixtures, such as tryptic
digests in proteomics analyses (including iTRAQ® and ICAT®* reaction products).
2) Isolation of peptides from natural products.
3) QC and purification of synthetic peptides.
4) Selective isolation of disulfide-linked peptides, phosphopeptides and C-terminal fragments from tryptic digests.
5) Mapping of peptide digests (tryptic, V8, CNBr, etc.).
Proteins can be run on PolySULFOETHYL A columns too; at pH 3, retention is all but guaranteed. An example is the analysis of Lung Surfactant Protein on a PolySULFOETHYL A column operated in the Hydrophilic Interaction (HILIC) mode.
Standard material for peptide applications is 300-Å, in either 3- or 5-µm. The 200-Å material has about 25% greater capacity and is preferred for phosphopeptide isolation and fractionation of iTRAQ® reaction mixtures. For proteins, use 1000-Å material or the 3-µm, 1500-Å material.