(A) 110 100 90 la 80 iv v 70 r su 60 tn 50 ce 40 re 30 p 20 10 0 (B) 110 100 la 8900 iv v 70 r su 60 tn 50 ce 40 re 30 p 20 10 0 control RNAi rpl-8 RNAi rps-15 RNAi rps-16 RNAi rpa-0 RNAi 0 5 10 15 20 Age of adulthood control RNAi aco-2 RNAi mdh-1 RNAi Aging, protein solubility and lifespan in C. elegans, P. Reis-Rodrigues et al. 123 25 30 0 5 10 15 20 Age of adulthood 25 30 110 100 l 90 va 80 irv 70 su 60 tn 50 ce 40 re 30 p 20 10 0 110 100 l 90 va 80 irv 70 su 60 tn 50 ce 40 rpe 30 20 10 0 control RNAi rps-0 RNAi rps-20 RNAi rps-14 RNAi eft-3 RNAi eft-2 RNAi control RNAi F01G4.6 RNAi atp-2 RNAi Fig. 3 A large fraction of the insoluble proteins reduce Caenorhabditis elegans lifespan. Synchronous populations of adult worms were subjected to RNAi knock-down of transcript levels of various genes encoding proteins identiﬁed in the insoluble fraction of old worms. Kaplan-Meyer survival curves were generated based upon survival at the indicated time points. A) Survival curves while knocking down transcript levels of genes encoding translation-related proteins. B) Survival curves while knocking down transcript levels of genes encoding mitochondrial proteins. 70 60 50 s en40 e G30 % 20 10 0 Total lysate Unchanged Lifespan extension Insoluble fraction Lifespan decrease Fig. 4 The SDS-insoluble fraction is highly enriched for proteins that negatively modulate lifespan. Comparison of survival data after knock-down of genes selected from a total lysate and the insoluble fraction. Percentage of genes with no effect, extending, and decreasing lifespan is shown. Using similar methods, David et al. (2010) reported a wide range of insoluble proteins in aged C. elegans. They indicate that across four independent experiments, 711 proteins were identiﬁed with high conﬁ- dence as being enriched in the insoluble fraction of aged animals. Of these 711 proteins, they identiﬁed 461 proteins that increased in all four inde- pendent experiments. Comparison of our list of proteins that were enriched in the insoluble fraction in aged animals to those generated by David et al. reveals a signiﬁcant overlap in protein identities; over half of the proteins identiﬁed in our study (107 of 203) were present in the Published 2011. This article is a U.S. Government work and is in the public domain in the USA. Aging Cell ª 2011 Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland 0 5 10 15 20 Age of adulthood 25 30 0 5 10 15 Age of adulthood 20 25 age-dependent insoluble set of 461 proteins identiﬁed by David et al., while nearly 80% of the proteins (162 of 203) on our list were seen in at least one of their experiments as being enriched in aged worms. Unlike the report from David et al., we found very little SDS-insoluble protein in young animals as analyzed by PAGE (Fig. 1) or iTRAQ mass spectrometry (Table S1), and nearly every protein that was identiﬁed (203 of 207 pro- teins) increased in aged samples by at least 2-fold. These discrepancies are likely due to slightly different preparation protocols. Our separation proto- col seems to be more stringent in identifying SDS-insoluble proteins that accumulate with age. However, the signiﬁcant overlap between the data sets indicates that both studies puriﬁed a similar fraction of the proteome. Given this signiﬁcant overlap of protein identities, it is not surprising that we ﬁnd similar biological processes enriched after GO analysis [NIH- DAVID gene ontology (Huang da et al., 2009)]. Like David et al., we ﬁnd a signiﬁcant enrichment in the biological processes of embryonic devel- opment, translation, growth, cofactor metabolic process, and determi- nation of adult lifespan. Both studies also identiﬁed a signiﬁcant enrichment in proteins localized to the ribonucleoprotein cellular compo- nent. However, our dataset was not signiﬁcantly enriched in either the chaperone or proteasome cellular component GO categories. Instead, our dataset was signiﬁcantly enriched in proteins localized to the mito- chondrion (Fig. 2). Given the differences in insoluble protein preparation, it is possible that the cellular component GO categories enriched in the David et al. dataset represent a broader spectrum of proteins that become insoluble with age while the enriched cellular component GO categories identiﬁed in our dataset represent the ‘core’ protein compo- nents that become insoluble with age. We went on to evaluate the effects of reducing expression of genes encoding insoluble proteins in lifespan and were struck by the discovery that nearly half of the tested proteins were limiting lifespan in that we observed an increased lifespan when their abundance is reduced. We compared this with a randomly selected gene set where only 18% of the