The PTMScan^® Control Peptides Phospho-Enrichment IMAC enable quality control of enrichment performance using PTMScan^® workflows.  These synthetic peptides contain a specific post-translational modification (PTM) that can be enriched by the associated PTMScan^® beads, as well as a stable heavy isotope that can be distinguished from endogenous peptides by the mass spectrometer.

Immobilized metal affinity chromatography, or IMAC, has been widely used to enrich proteins and peptides from biological samples by binding to clusters of negative charge. Divalent transition metal ions Co²⁺, Cu²⁺, Ni²⁺, and Zn²⁺ are often used to purify proteins rich in poly-Histidine or Cysteine as well as proteins with metal affinity. Trivalent metal ions, Fe³⁺, Ga³⁺, Al³⁺, as well as Ti⁴⁺ and Zr⁴⁺ are commonly used for phosphopeptide enrichment for proteomic studies (1). Iminodiacetic acid (IDA) or nitrilotriacetic acid (NTA) are used for chelating the metal ions to agarose-coated beads. In comparison studies, NTA has been shown to perform better than IDA at selectively capturing and identifying more phosphopeptides. Ga³⁺ and Fe³⁺ are comparable with respect to the number of phosphopeptides identified (2). Compared to metal oxide affinity chromatography (MOAC) using TiO₂, Fe³⁺ IMAC performed marginally better with TiO₂ having a preference for acidic phosphopeptides (pI > 4) relative to Fe³⁺ which preferred less acidic peptides (pI < 4) (3). The PTMScan^® Phospho-Enrichment IMAC Fe-NTA Magnetic Beads #20432 offer an efficient tool for phosphopeptide enrichment with little or no bias for phospho-residue context. They can be employed independently or in conjunction with immunoaffinity based enrichment to complement any PTMScan^® LC-MS/MS proteomic study.

Use with Cell Signaling Technology’s PTMScan^® Phospho-Enrichment IMAC Fe-NTA Magnetic Beads #20432 kit protocol from the incubation step. Because the optimal amount of PTMScan^® Control Peptides Phospho-Enrichment IMAC for each user’s experiments will depend on unique factors, such as mass spectrometer sensitivity, users may dilute these control peptides as needed in the appropriate solvent.

1. Aliquot PTMScan^® Control Peptides Phospho-Enrichment IMAC for storage as single-use units at -20°C or proceed to immediate usage.
2. Perform secondary trypsin digest of sample peptides in the appropriate buffer and volume for two hours at 37°C, e.g., 50 µL of buffer containing 5% acetonitrile, 50 mM ammonium bicarbonate, and 100 ng/µL trypsin.
3. Add 0.95 mL Loading Buffer (0.1% trifluoroacetic acid, 85% acetonitrile) to the digested solution and vortex.
4. Clear sample peptides by centrifugation.
5. Transfer clarified sample peptides to tubes containing washed IMAC magnetic beads.
6. Add 10 µL of PTMScan^® Control Peptides Phospho-Enrichment IMAC to tubes containing beads and sample peptides and mix well.
7. Continue with IMAC workflow at the 30 minute incubation step.
8. Detect PTMScan^® Control Peptides Phospho-Enrichment IMAC in the LCMS data file.