Probing the dynamic nature of signalling pathways by IMAC and SELDI-tof MS.
One major obstacle to the analysis of signalling pathways is the dynamic nature of signalling response to environmental stimuli. To overcome this limitation we applied immobilized metal affinity chromatography (IMAC) in combination with SELDI-tof MS to investigate the temporal variation of protein phosphorylation. We analysed the phospho-proteome variations in our model organism, Leishmania donovani, in response to changes in pH and temperature, which induce differentiation from promastigotes to amastigotes. Investigation of total cell extracts did not allow promastigote and amastigote life cycle stages to be distinguished. However, using IMAC enriched samples, the pattern and intensity of phospho-proteins analysed distinguished both stages reproducibly. Approximately 61% of the phospho-proteins analysed were significantly different in abundance (p<0.02). Of these 61%, 73% showed an increased phosphorylation in promastigotes while 27% showed an increase phosphorylation in amastigotes. The workflow developed is currently being applied to the temporal analysis of environmental stimuli.
Foucher
Aude L
AL
Photeomix, 34 Rue Carnot, 93160 Noisy le Grand, France. afoucher@photeomix.com
Späth
Gerald F
GF
Pemberton
Iain K
IK
D000818:Animals; D002454:Cell Differentiation / Q000502:physiology*; D002846:Chromatography, Affinity / Q000379:methods; D016000:Cluster Analysis; D006863:Hydrogen-Ion Concentration; D047908:Intracellular Signaling Peptides and Proteins / Q000502:physiology*; D007893:Leishmania donovani / Q000502:physiology*; D056004:Molecular Dynamics Simulation; D010750:Phosphoproteins / Q000032:analysis*; D010766:Phosphorylation; D040081:Protein Array Analysis / Q000379:methods; D020543:Proteome / Q000032:analysis; D015800:Protozoan Proteins / Q000032:analysis*; D015203:Reproducibility of Results; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D013057:Spectrum Analysis
D047908:Intracellular Signaling Peptides and Proteins; D010750:Phosphoproteins; D020543:Proteome; D015800:Protozoan Proteins