Identify Phosphorylation Site

Each experiment covers identifying ALL Phosphorylation sites (Ser, Thr and Tyr) in a single protein per sample. This service works the best with purified proteins or IP samples with target proteins enriched. Customers can send samples in-gel or in-solution, and provide the amino acid sequence or accession number of the target protein.

Identification of Phosphorylation-site involves enriching phosphorylated peptides followed by mass spectrometry. The enrichment is critical since phosphorylated proteins often represent 1-2% of the total protein population. In each run, a positive control consisting of a peptide with a known phosphosite is subjected to the same experimental conditions as the test samples. The presence of phospho-sites is confirmed by the presence of a MSMS peak showing the loss of phosphate.

Phosphorylation Site Identification Procedure

Gel Bands/Spots or In-Solution Samples

Procedure: step 1: cleaning and buffer exchange of in-gel or in-solution samples

Reduction/Alkylation & Trypsin Digestion

Procedure: step 2: Reduction, alkylation & trypsin digestion

Phospho-Peptide Enrichment

Phosphorylation Site Identification Procedure: step 3 - Phospho-Peptide Enrichment

MS

Phosphorylation Site Identification Procedure: step 4 - MS showing the intact phospho-peptide peak

MS/MS: Phosphorylation Site

Phosphorylation Site Identification Procedure: step 5 - MSMS showing the phospho-peptide peak with loss of phosphophate

Database Search for Phosphorylation Sites

Sample Info

Sample type: We accept in-solution, in-gel samples, or IP beads for this service.

Required information: Amino acid sequence or accession number of the target protein.

Protein amount: 2-20 µg of purified protein or protein mixture with target proteins enriched (such as IP).

Protein concentration: 5-20 mg/ml is preferred for in-solution samples.

Buffer: Please feel free to submit samples in whichever buffer that you would normally use.  Please inform our scientists about the buffer composition. Additional charge may apply on samples that require extensive amount of the extra work such as protein eluting, concentrating, buffer exchange or serum abundant protein depletion.

Biohazardous Materials: We do NOT accept samples containing any biohazardous materials. If your samples contain any biohazardous materials, please make sure to deactivate them completely using the appropriate procedure and declare them in the order form. In addition, we do NOT accept any samples containing Level 4 biohazardous materials even if they are deactivated. Please refer to this page for the rank of biohazardous materials.

Pricing

Services DescriptionAcademic/Governmental LabsIndustrial/Commercial Labs
CodePriceCodePrice
Phospho-Peptide Enrichment321A$245321N$295
Identification of Phosphorylation-Site322A$769322N$899
Manual inspection of MS/MS spectrum202A$115202N$135

Each order should have a minimum of 2 samples. Turnaround time is 5-7 business days. Price covers the identification of all phosphorylation sites for 1 protein per sample; following are the major steps:

  • Reduction and alkylation
  • Trypsin digestion
  • Peptide extraction
  • Desalting
  • Phospho-peptide enrichment (if you order item # 321)
  • NanoLCMSMS on peptides matching the mass of phosphorylated peptides from virtual digest
  • Standard database search against one species in NCBI or SwissProt database using MASCOT
  • Data report:
    • Top 10 hits via MASCOT search
    • List of precursors with predicated phosphorylation site
    • MSMS spectrums on precursors showing loss of phosphate

Price does NOT cover:

  • Protein identification
  • Sample preparation
  • Data request in addition to the standard report

If customer does not provide protein sequence or accession number, an additional $150 will be charged for protein identification