1) Sample Preperation (Tissue lysis and Trypsinization):

Will require cell pellet or animal tissue. We Approximatly 1-10 mg of proteins are extracted from the samples using the Guanidine based protein extraction method.Proteins are denatured in 8M urea buffer and digested with trypsin. Peptide are purified using solid phase extraction and quantified using BCS assay for Phophopeptide enrichment.

 2) Phosphopeptide Enrichment:

The Thermo Scientific™ High-Select™ Fe-NTA Phosphopeptide Enrichment Kit enables efficient enrichment of phosphorylated peptides from complex and fractionated protein digests for analysis by mass spectrometry (MS). (This product replaces Cat. No. 88300.)

Features of the Fe-NTA Phosphopeptide Enrichment Kit:
• Convenient—pre-formulated buffers for ease-of-use and spin-column format to enable parallel processing of multiple samples
• High binding capacity—each column enriches up to 150 μg of phosphopeptides
• High specificity—recovers phosphopeptides with > 90% selectivity
• Superior recovery—enriches more total and unique phosphopeptides than other commercially available resins
• High coverage—recovers peptides with single and multiple phosphorylation sites

The High-Select Fe-NTA Phosphopeptide Enrichment Kit enriches phosphopeptides from complex samples using iron-chelate resin in spin columns. Each column can enrich phosphopeptides from 0.5 to 5 mg of total protein digest as starting sample. Together with pre-formulated buffers, the improved and simplified procedure enriches up to 150 µg of phosphopeptides with greater than 90% selectivity in less than 45 minutes.

3) LC-MS Analysis:

 The enriched peptides are run on a optimized 2 hour LC-MS run using a 25 cm C18 (BCH 1.7 micron) column using HCD fragmentation. Additional ETD fragmentation can be requested for additional charges. ETD fragmentation can increase the number of phosphopeptide detected in the samples.