PTMScan® Control Peptides Phospho-Enrichment IMAC #59524
Product Information
Product Usage Information
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.
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.
Storage
This product is stable for 24 months when stored at -20°C. Aliquot to avoid multiple freeze/thaw cycles.
Product Description
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.
Background
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.
限制使用
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