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?