Proteomics and Validation

PFA-Proteomics Contract Research Services

The Proteome Factory is specialised in contract research for differential proteome analysis (proteomics, differential protein display) studies, identification and validation of regulated proteins, biomarkers and targets from all kind of protein containing samples. Only proteome analysis gives new insights in quantitative regulation of proteins caused by internal and external factors like diseases and drugs. The identification of regulated protein species (name, amount and modification) is the prerequisite to understand functional protein networks and their correlations. Biomarkers, biomarker profiles and targets can be identified. The proteomics information can be used to get better insights in in-vivo and in-vitro pharmacology or for optimization of fermentation processes and plant breeding.
For this purpose Proteome Factory has developed a comprehensive PFA-Proteomics Platform which opens new opportunities in biomarker and protein target discovery, quantification, identification and validation as well as pharmacokinetics of protein drugs. The proteomics platform consists of sophisticated gel-based and gel-free proteomics methods like iTRAQ™* proteomics and proprietary proteomics technologies ile MeCAT. In each proteomics study Proteome Factory decides in close cooperation with its partners and clients which proteomics technologies of the PFA-Proteomics Platform should be used to meet the scientific requirements. The study design, optimized sampling of biological material, sample prefractionation (e.g. affinity depletion of abundant plasma/CSF proteins) as well as statistical requirements are very important to ensure a high quality and valuable proteome study.
Picture: 2DE proteomics -

Application Areas of the PFA-Proteomics Platform

• Biomarker discovery and identification
• Biomarker profile discovery and identification
• Protein and peptide target discovery and identification
• Identification of regulated proteins and protein species
• Optimization of fermentation processes
• Optimization of plant breeding
• Pharmaco proteomics
• Identification of vaccine candidates
• Immuo proteomics
• Phospho proteomics
• Membrane proteomics

PFA-Proteomics Plattform

Proteome Factory performs gel-based and gel-free proteomics methods, which provide complementary information about the proteome.

• Biomarker- and Target Finding by extreme high resolution 2DE proteomics
• MudPIT (LC-ESI-MSMS based multi-dimensional protein identification technique)
• Label-free LC-ESI-MSMS proteomics
• ITRAQ based LC-ESI-MSMS proteomics
• Plasma Proteomics
• Immuno Proteomics
• Pharmako Proteomics
• Membrane Proteomics
• Pharmacokinetics by MeCAT (absolute protein quantification down to attomol range)
• Validation of biomarkers and target proteins by SRM / MRM analysis (mass western)
• Validation of biomarkers and targets by 2DE western blot and 1D western blot

Extreme High Resolution 2D Electrophoresis

Proteome Factory’s extreme high resolution two dimensional gel electrophoresis (2DE) based on the 2DE development of Professor Klose (Klose and Kobalz, Electrophoresis, 1995, 16, 1034-59) is still the most powerful separation technique for protein species worldwide allowing the best and most reliable proteome analysis. Several thousand protein spots (theoretically up to 10,000 protein spots depending on sample and gel size, up to 40x30 cm) can be separated and relatively quantified.
Even Proteome Factory’s 2DE gels with a size of 23x30 cm are much more powerful in protein separation than commercially available IPG-based 2DE because Proteome Factory’s 2DE technique has several advantages:
1. significant better protein separation resolution,
2. higher sensitivity with less protein amount,
3. less artificial streaks,
4. separation of basic protein up to pH 10.5 (up to pH 11.5 in 60x30 cm gels) and
5. high compatibility to protein identification by MS.

Workflow of Extreme High Resolution 2DE

The 2DE proteomics platform of Proteome Factory is very robust and highly standardized allowing reproducible analysis of all kind of protein containing samples using SOPs (standard operating procedures) without the need of time and cost intensive method development. Depending on scientific problem and customer request the 2DE procedure can be specifically optimized if required.
Proteome Factory’s 2DE approach comprises the following steps:

• Protein extraction
• Protein concentration determination
• 2DE protein separation (high resolution IEF and SDS-PAGE)
• Protein gel staining (MS compatible silver staining, Fluorescent staining)
• Digitalisation of 2DE gels
• Differential 2DE gel image analysis with differential protein display
• Evaluation of regulated protein spots with intensities, regulation factors and statistics
• Spot picking of differential protein spots by spotXpress
• Automated protein identification analysis
• Protein identification by database searching or de novo peptide sequencing

Advantages for Our Customer

• High quality and challenging proteome analysis for research & development
• Analysis results belong to customer
• Confidentiality
• Close collaboration
• Pilot studies to multi-year research projects
• High sensitive protein identification and quantification
• Highly qualified experts for protein and proteome analysis at Proteome Factory

*iTRAQ™ is a trademark of Applera Corporation or its subsidiaries.