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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?
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