CaptiVate Services

Identification of protein complexes and protein interactions in mammalian cells using a tagged bait

CaptiVate Shotgun Proteomics Services using  LC-MS/MS

  • Identification of protein complexes and protein interactions in mammalian cells
  • Profiling of signaling pathways
  • analysis of post-translational modifications
Overview

Applications

1. Identify protein interactions and protein complexes

Approach

  • Select protein of interest (bait)
  • Generation of stable cell line expressing tagged bait
  • Purification of protein complex, analysis by affinity purification and LC-MS/MS

Results

  • Identify proteins interacting with your protein of interest
  • Characterize protein complexes around your protein of interest

2. Study protein complex dynamics

Approach

  • Select protein of interest (bait)
  • Generation of stable cell line expressing tagged bait
  • Treatment with drug, ligand, growth factor, etc.
  • Purification of treated and non-treated protein complexes, analysis by affinity purification and LC-MS/MS

Results

  • Identify changes in protein complexes upon drug treatment or stimulation with growth factors
  • Quantitative analysis of treatment-induced changes in protein complexes

Your benefits

  • Full service – from project design to delivery of data
  • Quality – extensive optimization and integrated workflow ensure top quality data
  • Sensitivity – membrane proteins and post-translational modifications accessible
Service Procedure

Example: Analysis of cisplatin-induced changes in p53 complexes in HEK293 cells

Part I: Bait construction and stable cell line generation

Stable-expressio

  • Bait: human full-length p53
  • Stable expression of Strep-tagged p53 in HEK293 cell

Part II: Cisplatin treatment, affinity purification and quantitative LC-MS/MS analysis

  • Treatment of cells with cisplatin, non-treated controlTreatment-of-cells
  • Cell lysisCell-lysis
  • Strep-tag, high-speed affinity purification of p53 complexesStable-expressio
  • Quantitative analysis of 3 replicates eachQuantitative-analysis
  • Data filtering against protein frequency library

Quantitative-description-of-protein

  • Quantitative description of protein complex changes upon cisplatin treatment

Results

  • 92 candidate interactors identifiedp53-complexes-in-HEK293-cells
  • 45 interactors after data filtering (including 19 known interactors)

Known interactors (selected)

TP53BP1, MDM2, CUL7, CUL9, CCT5, HSP90AB1, HSC70, UBR5, SUMO2, CHIP, USP11, SMARCC1

Novel interactors (selected)

OBSL1, FBW6, INTS3, ZFP28, CDC73, RAD50, AKT1

Upregulated proteins

CAPE, SCP1, EDD

Downregulated proteins

ATG13, SUGT1, DNAJ2

Download complete service procedure with more detailed results (73 KB)

Download report datas 53 (Excel file)

Technology

Technological Benefits

Physiological expression levelsPhysiological-expression-levels

  • Single copy integration at defined site ensures physiological expression levels of the bait

Unparalleled sensitivity and specificity

Unparalleled-sensitivity-and-specificity

  • High speed, single-step purification retains transient interactors
  • Analysis on highly sensitive LTQ Orbitrap
  • Quantitative analysis of multiple replicates and data filtering against protein frequency library removes > 95% of all background

High reproducibility

Pearson-correlation-of-intensities-of-identified-peptides

  • Greater than 95% overlap between replicate samples
  • Intensity plot of identified peptides from two replicate experiments (pearson correlation =0.9)

Co-purification-of-membrane-proteinsCo-purification of membrane proteins

  •  Optimized buffer sets ensure excellent coverage of membrane protein baits and interactors
Publications

Publications

Our satisfied customers say...

We were very pleased with the scientific expertise and the project delivery provided by Dualsystems Biotech. The project was planned for three months, all timelines were kept and information was provided on a regular and reliable basis. The final report was delivered in good quality and changes were promptly implemented. Due to their expertise and their efficient service management Dualsystems Biotech was an excellent service partner.

Dr. Robert E. Zoubek, Manager Quality Operations ,Scil Technology GmbH, Germany

We have recently used the expertise of Dualsystems Biotech to identify protein complexes in mammalian cells using their AP-MS/MS service. This turned out to be an extremely successful project, not only because of the exciting results, but also because the people at Dualsystems Biotech turned out to be helpful, competent, were always available for support and delivered the results on time.

Professor Claus Azzalin, Institute of Biochemistry, ETH Zürich, Switzerland

We recently used the Dualsystems protein complex analysis service to identify new cellular interactors for a protein implicated in Parkinson’s disease that has defied many other experimental approaches including Gal4 and LexA two-hybrid systems. The experimental rigor and the quality of the results provided was outstanding. We now have several promising leads for future functional and molecular genetic studies.

Professor Robert J Harvey, Department of Pharmacology, London. UK

The CaptiVate service provided us with the essential protein-protein interaction confirmation that we need to publish our results.  Having it was like having an extra pair of hands in the laboratory!

Prof. Susan J. Baserga, Molecular Biophysics & Biochemistry, Genetics, Therapeutic Radiology, Yale University, USA

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