SIMAC: Enrichment of Phosphopeptides

The ability to identify proteins post-translationally modified by phosphorylation is the goal of many proteomic studies. Immobilized metalion affinity chromatography (IMAC; UNIT 10.11B) has been shown to have a strong binding affinity for phosphorylated amino acid residues (see reviews by Porath, 1992 (link); Smith and Figeys, 2008 (link); Macek et al., 2009 (link)). However, it has been shown that IMAC has an equally strong affinity for peptides with low overall pI (peptides with a high content of Asp and Glu), and its relatively low binding capacity can be a limitation for complex samples (Arrell et al., 2006 (link)). An alternative to the usual metals used in IMAC (e.g., Fe³⁺, Ga³⁺, and Al³⁺) is the use of titanium dioxide (TiO₂) beads, which have a very high affinity for phosphopeptides and are particularly efficient at enriching phosphopeptides from complex samples. By binding samples to the TiO₂ beads in highly acidic buffers, the selectivity of TiO₂ beads for negatively charged phosphopeptides is further improved (Larsen et al., 2005 (link); Thingholm et al., 2006 (link); Thingholm and Larsen, 2009 (link)). Complementarity between IMAC and TiO₂ stems from the fact that, while both will bind both mono- and multiply phosphorylated peptides, TiO₂ binds multiply phosphorylated peptides so tightly that their elution from TiO₂ is nearly impossible. Therefore, the phosphoproteome sequence coverage can be increased when the two methods are combined, in an approach termed SIMAC (Sequential elution from IMAC; Thingholm et al., 2008 (link), 2009 (link); see Fig. 10.25.2). This protocol outlines the steps involved in performing SIMAC for the enrichment of mono- and multiply phosphorylated peptides, although either IMAC or TiO₂ can be used independently following the same protocol.
NOTE: Use only HPLC- and/or mass spectrometry–grade reagents throughout the protocol.