Phosphoprotein Identification

Suitable for simple protein sample. (e.g. gel band cut from 1D/2D gels or immunoprecipitated proteins in solution)

About Phosphoprotein Identification

Phosphoprotein refers to protein carrying at least one phosphorylated amino acids. Phosphorylation is regulated by the complex interplay of protein kinase and phosphatase. Phosphorylation commonly occurs on the hydroxyl groups of serine, threonine and tyrosine (O-phosphate) although also on hydroxy-proline occasionally. N-, S- and acyl-phosphorylation is less prevalent and mostly occurs on histidine, arginine and lysine (N-phosphate), cysteine (S-phosphate), aspartic and glutamic acids (acyl-phosphates) respectively1. Protein with more than one phosphorylation sites can have different biological functions depending on the pattern of phosphorylation. Phosphoamino acids have different stability in different pH environment. For example, O-phosphate is more stable in acidic and neutral milieu.

Phosphorylation is the most prevalent and studied post-translational modification. It governs from as basis as protein folding and degradation to as important as signal transduction, cell proliferation and differentiation. Studies shown that phosphoprotein accounts for more than 50% of total proteins and there are over 100k estimated phosphorylation sites in the human proteome2. Elucidation of the pattern and stoichiometry of phosphorylation could unveil the mechanism behind many biological questions.

Coupled with proper purification and enrichment techniques, mass spectrometer can be used to identify phosphoproteins in a given sample. With the use of high mass accuracy MS and probability scoring algorithm, the correct site of phosphorylation in proteins that carry multiple potential phosphorylation sites could be determined confidently. Nowadays, the identification and quantification of hundreds or thousands of phosphoproteins in a single experiment setup is commonplace. A typical phosphoprotein identification workflow consisted of fractionation, proteolytic digestion and phosphopeptides enrichment as shown in Figure 1. If samples from different sources are labeled with different (combinations of) stable isotopes either in vivo or in vitro, the phosphorylations level of phosphoproteins in different samples could be compared. An example that uses iTRAQ for phosphoprotein quantification is showed in Figure 2.



Figure 1: Raw protein extracts from cells/tissues are too complex for accurate peptide identification and quantification by LC/MS/MS. The complexity of the extract could be reduced either by gel- or solution- based fractionation method. Phosphopetides are enriched by TiO2 to minimize the suppression effect by non-phosphorylated peptides


 Figure 2: Peptides derived from different protein samples could be labelled in vivo by isobaric stable isotope tags, i.e. iTRAQ. The quantification information is revealed in the low mass region of MS2 spectra. To learn more details of iTRAQ, please refer to our iTRAQ service.


Our phosphoprotein identification service:

N-Cell Technology offer phosphoprotein identification service. This service is based on our highly optimized protocol. To name a few, we use:

  • Optimized buffering conditions and mass spectrometer configuration to minimizing the loss of phosphate group through hydrolysis during sample preparation and LC/MS/MS
  • Optimized enrichment protocol to reduce interference from acidic non-phosphorylated peptides
  • Optimized scoring algorithm for the localization of phosphorylation site

Service Package

Phosphoprotein Identification Package

In-solution/-gel digestion

Phosphopeptide Enrichment with TiO2


Raw Data Export and Conversion

Phosphorotein Identification

Generation of Analysis Report

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  1. Require at least 100 ng of proteins.
  2. Sample is analyzed by Thermo LTQ-Orbitraq
  3. Proteins are identified by MASCOT® database search
  4. Intensity weighting, normalization, bias and isotopic overlap correction will be applied when appropriate

Phosphoprotein Identification Service Inquiry

  1. Schlessinger, J., Harvey Lect. 1993, 89, 105-123.
  2. Kalume, D.E.,Molina, H., Pandey, A., Curr. Opin. Chem. Biol. 2003, 7, 64-69