Cell lysis was accomplished in urea buffer (8 m urea, 50 mm Hepes, pH 7.6) using 2 ml buffer for 1 × 108 cells. Ten milligrams of total cell lysate of each sample (light, medium and heavy labeled) were mixed to have a final amount of 30 mg SILAC sample for peptide preparation. Phosphotyrosine peptides were enriched using a mixture of two phosphotyrosine antibodies: The pY1000 antibody that is part of the PTMScan Phospho-Tyrosine Rabbit mAb (P-Tyr-1000) Kit (Cell Signaling Technologies, Danvers, MA, USA; catalog number 8803), in addition to 50 μl NHS-coupled 4G10 antibody. For the pY eluates, a second step of phosphopeptide enrichment was performed on TiO2 tips as described23 (link) prior to mass spectrometry (MS) analysis.
Protocol detail
Phosphotyrosine Peptide Enrichment for SILAC Proteomics
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Antibodies
Antibody
Buffer
Cell
Hepes
Light
Mass spectrometry
Peptides
Phosphopeptide
Phosphotyrosine
Rabbit
Urea
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Variable analysis
independent variables
- Cell lysis method (urea buffer)
- Amount of cell lysate (10 mg) for each sample (light, medium, and heavy labeled)
- Phosphotyrosine peptides enriched using a mixture of two phosphotyrosine antibodies (pY1000 and 4G10)
- Phosphopeptides further enriched on TiO2 tips prior to mass spectrometry (MS) analysis
- PH of urea buffer (7.6)
- Amount of urea buffer (2 ml) used for 1 × 10^8 cells
- Total SILAC sample amount (30 mg) for peptide preparation
- Positive control: Not explicitly mentioned.
- Negative control: Not explicitly mentioned.
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