Inhaltspezifische Aktionen

Kinomics

Scientific Manager

Dr. phil. nat. Astrid Weiß

UGMLC Pulmonary Pharmacotherapy

Chair: Prof. Dr. Ralph Schermuly
Biomedical Research Center Seltersberg (BFS)
Justus-Liebig-Universität Giessen
Schubertstrase 81
35392 Giessen
Tel.: +49-(0)641-99-39882
Email: astrid.weiss@innere.med.uni-giessen.de

Technical Manager

Julia Baldauf

UGMLC Pulmonary Pharmacotherapy
 
 
 
 

Kinomics is a sub-category of Proteomics which aims to investigate the activity of a particular set of proteins such as protein kinases as well as signaling pathways that are regulated by those enzymes. In addition to protein kinases, their counterparts, i.e., protein phosphatases, are also of interest in this field of research. Kinases and phosphatases are two important families of key molecules as they control the function of almost a third of all proteins. This is carried out by a transfer (or removal, respectively) of a phosphate group from ATP to the corresponding protein kinase´s substrate. This type of post-translational protein modification is a fundamental process in all mammalian cells but also in other species.
The technology module Kinomics offers the analysis of protein kinase activity (i.e., the Kinotype) in biological specimens including human biopsies or tissue from experimental animal models as well as cell culture samples. The basis of the methodology is a microarray chip (PamChip) which is spotted with short peptides that contain tyrosine, serine or threonine residues. These amino acids serve as substrate molecules for a variety of different protein kinases. During incubation of the sample containing active protein kinases substrate recognition and the subsequent phosphorylation of the immobilized peptide at the respective amino acid takes place. This event is detected by antibodies which are fluorescently labeled and thereby can be monitored by a CCD camera. Based on the signature of phosphorylation of the various peptides on the PTK-PamChip (196 peptides with Tyr residues) and the STK-PamChip (144 peptides with Ser/Thr residues), the differential activity of 380 upstream protein kinases out of the total of 538 members of the human kinome can be predicted for two experimental conditions, e.g., diseased versus healthy, or treated versus control. With this technology several basic scientific objectives can be addressed including also the analyses of the mode of action for inhibitors or activators including pharmacological substances directly on the PamChip, e.g. off- and on-Chip treatment.

The technology module Kinomics is open for collaboration with all groups of the JLU and external institutions. For detailed information on the services provided by the technology module and on the design of the respective experiments including sample preparation protocols, etc., the collaborators are encouraged to contact any member of this technology module via email or phone.

Instrumentation/Software

  • Automated workstation for processing flow-through PamChips: PamStation®12
    • Manufacturer: PamGene International BV, ‘s-Hertogenbosch, The Netherlands
    • Intended for simultaneous processing of up to 3 PamChips each containing 4 microarrays
    • Run time for 1 run with 3 PamChips including 12 samples is 2 to 3 hours
    • 3 runs can be performed on 1 working day which allows for the investigation of 36 samples
    • Samples must run once on a PTK-PamChip and once also on a STK-PamChip, separately
  • PamChips containing peptide microarrays: PTK-PamChip® with 196 peptides, STK-PamChip® with 144 peptides (PamGene International BV, ‘s-Hertogenbosch, The Netherlands)
  • Software for data acquisition as well as for the creation and running of protocols: Evolve3 (version 3.1.0.5) and PamChip Annotator (version 1.0) (both PamGene International BV, ‘s-Hertogenbosch, The Netherlands, commercially available)
  • Software for image quantification and statistical analysis of data: IrfanView (version 4.70 - 64 bit) (Irfan Skiljan, Vienna University of Technology, freely available), R script (version 3.3.1, freely available), BioNavigator63 (version 6.3.67.0, PamGene International BV, ‘s-Hertogenbosch, The Netherlands, commercially available), Tercen data analysis platform (provided by PamGene International BV, ‘s-Hertogenbosch, The Netherlands, commercially available)
  • Software for mathematical processing and further statistical analyses: MS Office 365 Excel, GraphPad Prism Version 10 (both commercially available)
  • Web-based tools for further analyses (all freely available):
  • Software for graphical presentation: MS Office 365 Powerpoint and GraphPad Prism Version 10 (both commercially available)

The manager of the technology module should best be approached already at the beginning of the project to assist in the planning and design of the study. All steps of the cooperation are discussed and the details of the experimental workflow as well as the sample preparation protocols are shared with the collaborators. Data acquisition and analyses are part of the service and future support in preparing manuscripts and grant proposals is also guaranteed by the members of the technology module as there is special software needed. Furthermore, certain requirements and standards should be fulfilled to present the kinomics data according to good scientific practice. Therefore, the technology module offers a service which can be subdivided into the following service steps as part of a collaboration:

  • Request for a meeting via scientific manager
  • Meeting #1 to clarify aims and expectations as well as methodological questions such as:
    • What is the research question? What is the hypothesis? Are preliminary data available?
    • How many experimental groups should be investigated?
    • Which conditions will be chosen for a two-group comparison?
    • How many samples are planned? How many biological or technical replicates are possible?
    • Which PamChips should be used – only PTK (tyrosine) or STK (serine/threonine) or both?
    • How many PamChips are required and how many runs need to be performed?
    • Are there any positive controls, i.e., kinases which are known to be altered in their activity?
    • Would it make sense to perform a pilot study with different time points/concentrations?
    • Are there data existing and hypotheses about certain kinases and experimental setups?
    • What is the species and the genetic engineering safety level?
    • Is the lysate derived from a cell line, tissue or primary cells? Purified or sorted cells?
    • Are these lysates from animal tissue (animal proposal) or clinical specimens (ethic vote)?
    • Do you have experience in protein lysate preparation, e.g., for Western blot analyses?
  • Meeting #2
    • Detailed planning of the experiment, timeline, costs, responsible contact person
    • Final agreement on the design of the PamStation experiment including explanation of the lysate preparation protocol and transfer of the respective reagents if needed
  • Shipping of samples or assistance in lysate preparation
  • Lysate check by protein concentration measurement and total phospho-tyrosine Western blot
  • Ordering of PamChips
  • Pilot experiment to ensure the feasibility of the method for the respective research question
  • Meeting #3 to discuss the results from the pilot study and to decide how to continue
  • Performance of PamStation experiments by the members of the technology module
    • Data acquisition and basic quality control
    • Raw data analyses including PCA and heatmaps to identify outliers
  • Meeting #4 to decide on the upcoming analyses and figures
  • Detailed analyses using bioinformatic tools and kinomic profiling, full set of analyses
  • Meeting #5
    • To discuss results and define further aims for graphical representation of the results
    • To decide on potential protein expression analyses by JESS Simple Western system
  • Transfer to collaborators:
    • All data including raw data and image files
    • Remaining lysates for subsequent standard WB analyses
  • Meeting #6 with discussion of the results and on the outcome of the collaboration
  • Support for grant proposal or manuscript writing
  • General aspects which will be discussed on an individual basis:

As there are continuous updates of such documents to improve service and protocols, the files will only be available directly from the members of the technology module and will be immediately provided upon request.

  • Specific terms of use for core facilities and technology modules: Allgemeine Nutzungsordnung der Central Platform for Deep Phenotyping in Medicine (JLU cDeeP)
  • Collaboration agreement
  • Sample submission sheet
  • Sample description table
  • Lysate preparation protocol