Proteomic characterization of the human centrosome by protein correlation profilling Andersen JS et...
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Transcript of Proteomic characterization of the human centrosome by protein correlation profilling Andersen JS et...
Proteomic characterization of the human centrosome by
protein correlation profilling
Andersen JS et al.
Nature 426:570-574
Introduction• Centrosome: microtubule-
organizing center
• Centrosome hard to isolate b/c:
– Tiny, single-copy organelle– Lack delineating membranes– Tightly associated with nucleus
• Mass-spectrometry: highly sensitive tool
How can we do proteomic analysis of poorly purified samples?
Goal
• Isolate interphase centrosomal proteins
• Mass spectrometric analysis
• Discriminate between novel genuine centrosomal proteins and contaminating proteins
Methods
Aebersold R. & Mann M. (2003) Nature 422:198-207
Density-gradient centrifugation
LC MS/MS
Results
Elution time (min)
Total ion chromatogram
Mass Spectrum
Fragmentation Spectrum -tubulin
Results
• Identification of 2,000 peptides, i.e. more than 500 proteins
• 47/60 known centrosomal proteins were recovered
• 60 proteins are uncharacterized and could be now centrosomal proteins
• How to determine if they are genuine centrosomal proteins?
Confirming novel centrosomal components
• 23 uncharacterized proteins were cloned and fused to GFP and Myc.
• Expression of reporter-constructs in human osteosarcoma cells U2OS
• 19/23 proteins localized to the centrosome
Nocodazole
-Tu
bu
lin
-T
ub
ulin
Cep152 Cep63
Cep135ALMS1
Protein correlation profiling
Extracted Ion current
Protein correlation profiling
• Graph the relative abundance of proteins in 5 elution fractions
• Centrosomal proteins abundance pattern is close to the consensus
• Goodness of fit chi-square test
– Centrosomal: low 2
– Contaminating: high 2
• 41 candidate centrosomal proteins with 2< 0.05
Novel centrosomal proteins
• 19 proteins validated by both (immuno)localization and protein correlation profiling
• Average mass of 130kDa ; 75% with coiled-coil regions
• Both salt-soluble and salt-insoluble proteins
• Several proteins linked to human diseases
Conclusion
• Protein correlation profiling has great potential for the study of multiprotein complexes
• Avoids the trade-off between a rigorous purification and the inclusion of loosely associated proteins
• Could be used along with :
– siRNA to understand the function of centrosomal proteins– Isotope-labelling to study spatial and temporal
differences in proteins composition
Critique
• Figures not annotated sufficiently
• Do not discuss false negatives and false positives
• List as novel centrosomal components proteins that were identified as such in 1995.
Discussion
• Could we confirm that the Protein Correlation Profiling method really works by testing it on a complex where all the components are known?
• Do members of large protein complexes always have the same elution pattern?
• Is PCP alone sufficient to associate a protein to a complex/organelle
• Would this method be appropriate for the characterization of other organelles?