Proteomic characterization of the human centrosome by protein correlation profilling Andersen JS et al. Nature 426:

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: 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 -Tubulin -Tubulin Cep152Cep63 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?