a model in which an age-related decline in protein homeostasis results in protein conformational change and a shortened lifespan. It further indicates that reduction in levels of a large number of speciﬁc insoluble proteins can extend lifespan. Results Insoluble proteins accumulate in aged Caenorhabditis elegans Protein aggregation can be deﬁned as an alteration in protein quaternary structure leading to loss of solubility. In some cases, this change in can lead to the formation of aggregates that become insoluble in strong detergents such as SDS. We reasoned that if endogenous proteins were becoming insoluble and aggregating during aging, they could be extracted by a solubility based fractionation. This idea was tested using C. elegans as a model organism where formic acid (FA) has been used pre- viously to solubilize an aggregation-prone form of GFP (Link et al., 2006). To prevent any transgenerational contamination by self-progeny produc- tion, a temperature sensitive sterile strain [TJ1060 spe-9(hc88)I; fer- 15(b26)II] (Fabian & Johnson, 1994) was used. Synchronous populations of eggs were prepared by mild hypochlorite treatment of gravid adults grown at 20 C, counted. and allowed to hatch in the absence of an E. coli food source. The worms were arrested as ﬁrst stage larvae (L1s) and thereby were developmentally synchronized. Samples of about 120 mg (wet weight) of worms were collected at the ﬁrst and eleventh day of adulthood for three biological replicates. We extracted protein by sonication on ice, and postdebris supernatant fractions were normalized for total protein concentration. Samples were then washed several times with an aqueous buffer to remove soluble protein. The water insoluble fraction was resuspended in a similar buffer with 1% SDS and washed sev- eral times. The SDS-insoluble fraction was then resuspended in 70% FA for 1 h with vigorous shaking. The aqueous, SDS-soluble, and SDS-insoluble (FA solubilized) fractions were then analyzed by SDS-PAGE. Although the amount of protein solubilized in the aqueous or detergent buffers were similar between young and old worms, FA treatment of the SDS-insoluble fraction yielded signiﬁcantly more proteins from old worms when com- pared with young adults (Fig. 1). This result conﬁrmed the hypothesis that SDS-insoluble proteins accumulate during aging in C. elegans. To identify the insoluble proteins that accumulate in old worms, we ﬁrst fractionated the protein extracts from three replicate sets of young adults and old worms each as described earlier to obtain a total of six SDS-insoluble fractions. These fractions were then solubilized by FA treat- ment, dried, and resuspended in TEAB buffer for trypsin digestion. After proteolysis, the resulting peptide fractions from the six protein pools were differentially labeled using six different isobaric amine-speciﬁc iTRAQ reagents, mixed, and then fractioned by SCX prior to HPLC separation and mass spectrometry analysis. Under ESI-MS ⁄ MS conditions, the iTRAQ chemical tag yields a distinct chemical reporter in the low mass region that can be used as a measure of relative abundance. Therefore, infor- matic analysis of the tandem spectra from these experiments identiﬁed both the peptides (and proteins) that make up these SDS-insoluble frac- tions, as well as providing a means to determine the relative concentra- tions among the six protein pools. Using this method, we identiﬁed 203 insoluble proteins that are at least 2-fold enriched in aged insoluble frac- tions when compared with the equivalent fraction prepared form young worms (Fig. 2, Table S1). Proteins with functions related to translation, energy production, and protein homeostasis comprised nearly 50% of the proteins in the insoluble fraction (Fig. 2, Table S1). Western blot anal- ysis against aconitase-2 and DAF-21 independently conﬁrmed the mass 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 MW (KDa) 250 160 75 50 30 15 Aging, protein solubility and lifespan in C. elegans, P. Reis-Rodrigues et al. 121 res akr m W M Aqueous 1 11 SDS soluble 1 11 SDS insoluble 1 11 Fraction Age of adulthood Fig. 1 Accumulation of insoluble proteins in aged Caenorhabditis elegans. SDS- PAGE of the aqueous, SDS-soluble and SDS-insoluble fractions of 1 and 11-day-old adult animals. Levels of SDS-insoluble proteins are greatly enhanced in extracts from old animals as compared with extracts from young animals. spectrometry results by showing an accumulation in the insoluble fraction of old animals (Fig. S1). To assess the nature of this SDS-insoluble fraction relative to the total proteome of C. elegans, we carried out gene ontology (GO) analysis to determine which annotation categories were signiﬁcantly enriched among proteins in this fraction. As the list of insoluble proteins was obtained through a biochemical fractionation, we reasoned that we should compare it with a total lysate. To do this, we also carried out GO analysis of a list of around 1200 proteins identiﬁed by iTRAQ in a total lysate (Fig. 2). Although it was clear that proteins of diverse predicted functions were represented in the insoluble fraction, there was notable representation of some important functional groups. We found highly signiﬁcant enrichments of proteins associated with ribonucleoprotein complexes, particularly components of the small ribosomal subunit (Fig. 2). Many proteins that become insoluble during aging also determine lifespan To identify proteins among the 203 proteins in the SDS-insoluble fraction that might inﬂuence lifespan, the top 99 genes with the largest fold- change between young and old worms were selected for reduction in gene expression by RNA interference and survival analysis. We began RNAi treatment only after the animals reached the late L4 stage to avoid developmental effects. Each RNAi treatment that extended lifespan was tested at least three times. Strikingly, reducing the expression of 41% of the insoluble proteins signiﬁcantly extended mean adult hermaphrodite lifespan (Fig. 3, Tables 1 and S2). Lifespan extensions varied between a few percent up to 30%. The lifespan extension is not attributable to delays in development as the worms reached adulthood synchronously with control (RNAi vector only) worms. Notably, only two of the genes tested from this group caused a decrease in lifespan upon knock-down. We then determined whether the insoluble protein cohort was enriched for functions that limit lifespan under our assay conditions. To test this,