This chart shows the underlying motif distribution in a PhosphoScan® IMAC LC-MS/MS experiment using 4026 nonredundant tryptic peptides generated from mouse embryo and captured using PTMScan® Phospho-Enrichment IMAC Fe-NTA Magnetic Beads. Within this data set, 84% of the peptides were phosphorylated on serine residues, 13% on threonine, and 3% on tyrosine.
The Motif Logo was generated from a PhosphoScan® IMAC LC-MS/MS experiment using 4026 nonredundant tryptic peptides derived from mouse embryo and captured using PTMScan® Phospho-Enrichment IMAC Fe-NTA Magnetic Beads. The logo demonstrates the relative frequency of amino acids in a given position around the modified site within this data set.
Cells are lysed in a urea-containing buffer, cellular proteins are digested by proteases, and the resulting peptides are purified by reversed-phase solid-phase extraction. Phospho-peptides are then subjected to enrichment using PTMScan® IMAC Beads. Unbound peptides are removed through washing, and the captured phospho-peptides are eluted with basic pH buffer. Reversed-phase purification is performed on microtips to desalt and separate peptides from impurities prior to concentrating the enriched peptides for LC-MS/MS analysis. A detailed protocol is included with the beads.
IMAC Fe-NTA Magnetic Beads are supplied in 20% ethanol. For long term storage, replace the buffer with long term storage buffer (1:1:1 v/v/v of methanol, acetonitrile, and 0.01% acetic acid). See protocol for details. Store at 4ºC. Do not freeze the beads.
PTMScan® Phospho-Enrichment IMAC Fe-NTA Magnetic Beads employ immobilized metal affinity chromatography for capturing phosphorylated peptides. Negatively charged phosphate groups are attracted to the positively charged metal ions on the beads. In conjunction with liquid chromatography tandem mass spectrometry (LC-MS/MS), this approach enables researchers to isolate, identify, and quantitate large numbers of phosphorylated cellular peptides with a high degree of specificity and sensitivity, providing a global overview of phosphorylation in cell and tissue samples. For more information on PTMScan® Proteomics Services, please visit https://www.cellsignal.com/services/index.html.
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 phospho-peptide 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 phospho-peptides. Ga3+ and Fe3+ are comparable with respect to the number of phospho-peptides identified (2). Compared to metal oxide affinity chromatography (MOAC) using TiO2, Fe3+ IMAC performed marginally better with TiO2 having a preference for acidic phospho-peptides (pI > 4) relative to Fe3+ which prefered less acidic peptides (pI < 4) (3). The PTMScan® Fe-NTA Magnetic Beads offer an efficient tool for phospho-peptide 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 PhosphoScan® LC-MS/MS proteomic study.
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