Why Is Phosphopeptide Enrichment Important? - Ylva Laurin, Ann-Marie Nissfolk, Johan Öhman, Ulrika Meyer, Helena Hedlund and Marianne Alben

Studying Cancer Cell Signalling Using TiO₂ Mag Sepharose™ Magnetic Beads

Phosphorylation is a common reversible post-translational modification involved in the regulation of many essential biological processes, for example cell signalling, which is of prime importance for the study of various disease states such as cancer. The phosphoproteins and phosphopeptides resulting from these processes are important to the understanding of tumour progression, however, there are various barriers to their study. Within the cellular environment, phosphopeptides are usually transient and found in very low concentrations. In addition when proteins are phosphorylated they attain a negative charge and, compared to their nonphosphorylated counterparts, they are poorly ionised which further complicates their measurement using mass spectrometry. Therefore, determination of their relative levels as a function of disease progression is an ongoing challenge for researchers. One approach is to use a simple enrichment technique that ‘concentrates’ the phosphopeptides in samples to aid detection using standard mass spectroscopy approaches. This paper outlines how such a simple enrichment process using TiO₂ Mag Sepharose™ magnetic beads was developed, validated using a model sample, and tested in a complex sample derived from a leukaemia cell line.

About the TiO₂ Mag Sepharose magnetic beads TiO₂ Mag Sepharose magnetic beads use titanium dioxide (TiO₂)-based chromatography to simplify the capture and enrichment of phosphopeptides. TiO₂ has a high affinity for phosphopeptides and provides efficient enrichment of these from complex samples. When the tubes are placed in MagRack, the magnetic beads are attracted to the magnet within a few seconds. This allows fast removal of the supernatant whilst the beads remain in the tubes. The easy visibility of the beads ensures reliable collection of all the targeted peptides