Monday: |
Welcome |
Introduction to post translational modifications (Martin R. Larsen) |
Introduction to mass spectrometry (Martin R. Larsen/Frank Kjeldsen/Thomas J.D. Jørgensen) |
Introduction to phosphoproteomics and its relevance in biology (TBA) |
| |
Evening: Laboratory exercise |
• Introduction to the laboratory |
• I ntroduction to enrichment of phosphopeptides by IMAC |
• Analysis of phosphopeptides by MALDI MS |
• Initiate PAC derivatization protocol |
• Precipitation of proteins from SILAC labeled HeLa cells |
|
Tuesday: |
Morning: Lectures |
|
- Immobilized metal affinity chromatography (IMAC) |
- Titanium dioxide (TiO2) chromatography |
- Sequential Elution from IMAC (SIMAC) |
- Phosphoramidate chemistry (PAC) |
- Other enrichment methods |
|
|
| |
Evening: Laboratory exercise |
Introduction to enrichment of phosphopeptides by titanium dioxide and SIMAC
|
MALDI MS analysis of phosphopeptides
|
Continue PAC enrichment procedure
|
Digestion of HeLa cell lysates
|
In-gel digestion of unknown phosphoprotein
|
|
Wednesday |
Morning: Lectures |
Mass spectrometric analysis of phosphorylated peptides (Martin R. Larsen/Tine Thingholm)
|
Calcium induced phosphorylation signaling in nerve terminals (Phillip J. Robinson)
|
Other biological applications of phosphoproteomics (TBA)
|
| |
Evening: Laboratory exercise |
Analysis of purified phosphopeptides from unknown protein by TiO2 chromatography and MALDI MS-MSMS
|
Advanced fragmentation of phosphopeptides on orbitrap XL instrument (CID, HCD, ETD)
|
Enrichment of phosphopeptides from HeLa cell lysate
|
|
Thursday |
Morning: Lectures |
Bioinformatics in Phosphoproteomics (Karin Hjernø/Giuseppe Palmisano)
|
- Tools
|
- Pathway analysis
|
- Prediction analysis
|
Examples of characterization of phosphorylation in biological systems using quantitative proteomics (TBA)
|
| |
Evening: Laboratory exercise |
|
- Interpretation of phosphopeptide spectra
|
- Phosphopeptide identification
|
Data processing using quantitative software’s
|
|
Evening event: Dinner and Bowling |
|
Friday |
Morning: Lectures: |
Biological applications to phosphoproteomics
|
Kinomics and chemical proteomics in phosphoproteomics (Bernard Kuster)
|
| |
| |
Laboratory exercise:
The participants will be divided into smaller groups of 3-4 people. As shown above, the laboratory course will be executed with as much hands on for the participants as possible. However, operation of expensive mass spectrometers will be performed by skilled operators. The participants will be performing a large variety of phosphopeptide enrichment methods, including IMAC, TiO2, SIMAC and PAC on standard peptide samples originating from 12 proteins. The purified phosphopeptides will be analyzed by the participants on our MALDI MS instruments. The participants will be performing a large scale phosphoproteomics experiments where they will purify, identify and quantify phosphopeptides from un-stimulated HeLa cells and HeLa cells after 5 min stimulation with EGF. These cells will be labeled with SILAC for quantitation using various software’s. The participants will become familiar with bioinformatics tools for phosphopeptide identification and quantitation and in addition they will be testing various software’s for pathway analysis and kinase prediction.
|
| |
