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Phosphoproteomics

High-end phosphoproteomics by mass spectrometry

High-end phosphoproteomics schemaExperimental design

Protein phosphorylation on serine, threonine and tyrosine residues is the prevalent post-translational modification (PTM) and occurs on over one-third of all cellular proteins. In humans 518 protein kinases have been identified that regulate phosphorylation networks and control biological processes such as proliferation, differentiation and apoptosis. Phosphorylation takes place mainly on serine residues (86.4%), followed by threonine residues (11.8%) and tyrosine residues (1.8%).

Workflow

PEPSCOPE’s phosphoproteomics workflows generally require around 1 mg of sample lysate (see Experimental design), use urea-based protein digestion, followed by peptide desalting and lyophilization, peptide fractionation using high-pH reversed-phase chromatography. Phosphopeptides are enriched by TiO2, sometimes multiple times, resulting in numerous LC-MS/MS measurements per biological sample to be analyzed (PEPSCOPE can do this up to 10 different samples).

At PEPSCOPE, we have the ability to multiplex samples in order to study proteome-wide phosphorylation events by means of different quantitative mass spectrometry approaches, such as Tandem-Mass-Targets (TMT) labeling. Chemical labeling TMT is a chemical derivatization technique used to identify, compare and quantify proteins from multiple samples in one single experiment by mass spectrometry.

Example of our in-depth phosphoproteomics analysis

As an example we have analyzed U87 which is a glioma cell line using our phosphoproteomics workflow.

Results:

Total identified proteins 6323
Total identified protein kinases 204
Total identified phospho-peptides 8402

The following conclusions could be drawn:

  • Mass spectrometry profiling can be used to identify large numbers proteins and phospho-peptides in a glioma cell line
  • A large number of proteins could be identified with and without phosphorylation
  • A very high coverage (80%) is obtained for the proteins and kinases involved in glioma related pathways

Besides delivering high quality proteomics data to individual customers PEPSCOPE is also focusing on partnerships with Pharmaceutical companies and Academic/Research Institutes

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