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Phosphoproteomics

Our 6-step Phosphoproteomics Profiling process

Schematic of phosphoproteomics process

Phosphoproteomics workflows generally require around 1 mg of sample lysate, 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.

  1. Sample collection – Performed in your lab
  2. Extraction of proteins  Proteins are extracted and denatured
  3. Generation of peptides – Proteins are reduced, alkylated and digested into peptides
  4. Enrichment of phosphopeptides – Using TiO2 enrichment
  5. LC-MS/MS analysis  Peptides are analysed by LC-MS/MS on Orbitrap-based mass spectrometers
  6. Automated data analysis – Phosphopeptides are identified and phosphorylation sites are assigned to proteins

We’re always trying to provide the highest quality proteomics data in the field by using state-of-the-art Mass Spectrometers to the most advanced platforms and constantly optimizing our sample preparation methods based on the latest development of technologies.

MS Equipment available:

  • Thermo Fisher Orbitrap Fusion Lumos
  • Thermo Fisher Orbitrap Fusion
  • Thermo Fisher Orbitrap Q Exactive HF instruments

Workflow

Step 1 -> Please get in touch to discuss experimental setup and sample preparation

Step 2 -> We send you the sample preparation protocol and checklist

Step 3 -> You prepare and ship the samples to PEPSCOPE

>Shipping instructions

Step 4 -> We process your samples

Step 5 -> We analyze the results and prepare a comprehensive report

Step 6 -> You get your results

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Measuring Target Phosphorylation through Mass Spectrometry

PEPSCOPE’s comprehensive phosphoproteomics services to our clients include mass spectrometry-based proteomics solutions for analyses of cell lines and tissue. This service permits the identification and quantification of proteins, phosphopeptides (abundance change and phosphorylation status) and kinases in a single experiment.

Depending on the research question we:

  • Maximize the total number of proteins in a proteome
  • Identify the total number of different peptides in a proteome

Our solutions are focused on the customer’s needs and enable insights into the mode of action of post-translational phosphorylation modifications. Through our strong and long term collaboration with the Erasmus Medical Center in Rotterdam (group of Theo Luider) we have access to their thorough knowledge/experience and skills.

Our services include:

  • Contract and custom research
  • Consultancy
  • Assay development and validation

PEPSCOPE offers multiplexing up to 10 different samples through Tandem-Mass-Target (TMT) labeling. Through the Erasmus MC, Pepscope has many years experience using TMT labeling.