Thermo Scientific Pierce Graphite Spin Columns improve phosphopeptide analysis by efficiently binding hydrophilic peptides and efficiently removing urea, salts and other contaminants before mass spectrometry analysis.
Pierce Graphite Spin Columns enable fast and efficient capture, concentration, desalting and elution of hydrophilic peptides in less than 10 minutes. These columns are ideal for improving mass spectrometric (MS) analyses of samples from protein digests), strong-cation exchange fractions, and enriched phosphopeptides eluted from titanium dioxide and immobilized metal affinity chromatography (IMAC) columns and tips.
Highlights:
- Convenient spin format for parallel processing of multiple samples
- High-binding capacity with excellent recovery of up to 100µg of hydrophilic peptides per column
- Porous graphite resin for cleaning up phosphopeptide samples before MS analysis
Product Details:
The Pierce Graphite Spin Columns improve phosphopeptide analysis by efficiently binding hydrophilic peptides and efficiently removing urea, salts and other contaminants before MS analysis. The C18 resins and C18 tips that are commonly used to desalt peptides are excellent for use with hydrophobic peptides but do not efficiently capture hydrophilic peptides, like phosphopeptides, resulting in enrichment of only hydrophobic fragments. The Pierce Graphite Spin Columns address this issue and are ideal for matrix-assisted laser desorption ionization (MALDI) or nanoelectrospray ionization techniques.
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| C18 resin is ineffecive for recovery of hydrophilic peptides. Hydrophilic peptides, including many phosphopeptides, bind poorly to C18 media. Phoshopeptides and other hydrophilic peptides do bind efficiently to porous graphite resin, providing a better method for rapid desalting and concentration prior MS analysis. |
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| Graphite clean-up enables phosphopeptide identification. U2OS human osteosarcoma cells synchronized at the G2/M boundary with nocodazole (200ng/mL, 36 hours) were lysed with 6M guanidine•HCl. After enzymatic protein digestion (100µg), phosphopeptides were enriched with IMAC and desalted with Pierce Graphite Spin Columns or C18 tips before LC-MS/MS analysis on a Thermo Scientific Orbitrap XL Mass Spectrometer. Two representative spectra are shown for two phosphopeptides not observed after C18 cleanup. Panel A: A novel doubly-phosphorylated peptide was identified within the putative ATP binding site of cyclin dependent kinase cdc2 (CDK1). This phosphopeptide is not present in Phospho.ELM version 8.2 database. Panel B: Dual specificity mitogen-activated protein kinase kinase 2 (MP2K2) phosphopeptide. |
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Improved recovery of representative hydrophilic phosphopeptides using graphite spin columns. Stable isotope-labeled A3 and B9 peptides (10pmol) were acidified with 1% trifluoroacetic acid, processed according to instructions for C18 tips or the Pierce Graphite Spin Columns and eluted with 50% acetonitrile with 0.1% formic acid. The corresponding heavy isotope labeled peptides (5pmol) were spiked in the eluate, dried and resuspended in 0.1% formic acid. Samples were analyzed by targeted LC-MS/MS with the Thermo Scientific Orbitrap XL Mass Spectrometer to quantitate percent recovery.
Peptides:
A3 = RPRAApTFPFR¹
B9 = RTPKDpSPGIPPFR¹ |
| ¹Position of heavy isotope labeled amino acid used for absolute MS quantitation. |
Related Resources:
Review of sample preparation for mass spectrometry
Related Products:
Pierce Magnetic Phosphopeptide Enrichment Kit
Phosphoprotein Enrichment kit
Halt Phosphatase Inhibitor
MS-grade Endoproteinases
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