suggesting a link between the mechanisms that determine lifespan and protein aggregation rates (Morley et al., 2002; Cohen et al., 2006). As age-1 regulates the levels of molecular chaperones (Walker et al., 2001; Hsu et al., 2003), the suppression of protein aggregation and lifespan extension could result of an enhanced protein homeostasis capacity. This model is also consistent with observations that small mole- cules that suppress protein aggregation also extend lifespan (Alavez et al., 2011). We have considered whether other mechanisms are at play in our life- span. Some functions associated with proteins from the insoluble fraction have been previously implicated in limiting lifespan. For example, in a number of model organisms, mutations in genes encoding translation fac- tors result in extended lifespan. It is possible that a reduction in bulk trans- lation rates extends lifespan or that there is an increase in the translation of a subset of mRNAs encoding longevity functions (Zid et al., 2009). In our comparative proteomic analysis, we also found an enrichment for translation functions. Our data also agree with previous studies in that we observed increased lifespan after reducing transcript levels of transla- tion-related genes. Perhaps this is the result of altered translation, although the fact these proteins become insoluble during aging suggests an alternative explanation as to why translation factors limit lifespan. It is possible that the aggregation of components of the translational appara- tus itself limits lifespan by the demands this places on protein homeostatic mechanisms. Here, we have demonstrated that aging results in a range of distinct proteins species entering an insoluble state. We demonstrate further that decreasing the abundance of individual proteins in this insoluble compart- ment results in an increased lifespan, suggesting that these proteins can limit lifespan. The commonality between the behavior of proteins associ- ated with speciﬁc age-related diseases and a range of cellular proteins that inﬂuence lifespan during normal aging provides a novel context for considering the role of aging as a primary risk factor for the most com- mon forms of neurodegeneration. Experimental procedures Caenorhabditis elegans strains and the generation of transgenic strains The Bristol N2 strain was used as the wild-type strain and was obtained from the Caenorhabditis Genetics Center. TJ1060 strain was a gift from Thomas E. Johnson (Univ. of Colorado, Boulder). Insoluble protein extraction TJ1060 [spe-9(hc88)I; fer-15(b26)II] temperature sensitive mutants were grown and aged in synchronous mass cultures (Fabian & Johnson, 1994). After bleaching the worms, L1s were arrested in S-Basal and thereby syn- chronized. Approximately 5000 animals were plated in each enriched peptone plate spotted with E. coli (NA22) and then aged at 25 C (restric- tive temperature). Samples of about 120 mg of worms were collected from several plates at 1 and 11 days of adulthood for three biological rep- licates. Most of the very few dead worms were stuck to the bacterial lawn when collecting the samples. Worms were washed several times with S-basal and resuspended in aqueous lysis buffer (20 mM Tris base, 100 mM NaCl, 1 mM MgCl2, pH = 7.4) with protease inhibitor cocktail COMPLETE from Roche Diagnostics (Mannheim, Germany). The sam- ples were sonicated on ice and then centrifuged at 3000 g to remove carcases. At this point, all samples were normalized for total protein concentration as assessed by BCA assay (Thermo Fisher Scientiﬁc, Aging, protein solubility and lifespan in C. elegans, P. Reis-Rodrigues et al. 125 Rockford, IL, USA). Samples were centrifuged at 16 000 g and washed three times to extract the water-soluble fraction. The pellet was then resuspended and washed three times in the same buffer containing 1% SDS to retain the detergent soluble fraction. The insoluble fraction was then treated for 1 h with 70% FA with vigorous shaking at room temper- ature. The acidic fractions were dried in a Speed-Vac at 25 C. Isobaric tag for absolute and relative quantitation Preparation for mass spectroscopy FA treated protein samples were resuspended in equal volumes of solu- tion consisting of 200 mM TEAB, 0.1% SDS, and 1% NP-40 at pH 8.0, to maintain the same volume to total initial protein ratio from C. elegans. Samples were able to resuspend over 2 h on ice with occasional vor- texing. BCA quantitation was performed, and equal volumes were col- lected from all six samples (three biological repeats of worms at the ﬁrst and eleventh day of adulthood) resulting in 50 lg of total protein being collected. This volume was chosen because it resulted in the maximum amount of protein that can be labeled using aliquoted iTRAQ reagents while utilizing the same volume of each sample. Samples were prepared and labeled with iTRAQ 6-plex reagents according to the manufacturer’s guidelines (Applied Biosystems, Foster City, CA, USA), reduced (5 mM tris(2-carboxyethyl) phosphine at 60 C for 60 min), and blocked (10 mM methyl methanethiosulfonate) for 10 min at RT. Samples were digested at 37 C overnight with 2 lg trypsin (Promega, Madison, WI, USA), with 1 lg added 6 h after digestion began. Samples were then iTRAQ (Applied Biosystems) labeled for 2 h after which all samples were com- bined into strong cation exchange (SCX) loading buffer (Applied Biosys- tems) at pH 3.0 quenching the labeling reaction. Combined samples were cleaned-up using a SCX cartridge (Applied Biosystems) and eluted using a high salt elute of 350 mM KCl and 25% CAN (Applied Biosys- tems). The peptides were then dried in a Speed-Vac at 25 C. Liquid chromatography and mass spectrometry Samples were redissolved in 2% ACN (0.1% FA). An estimated 1.0 lg of labeled sample was loaded and desalted on a Dionex Acclaim LC Packings l-Precolumn Cartridge (5 mm · 300 lm RP-C18 trap column, 5 lm beads, 100 A˚ pore size). Chromatography was performed on a Dionex Acclaim Pepmap column (75 lm · 150 mm RP-C18, 3 lm beads, 100 A˚ pore size) with a 2-80% ACN (0.1% FA) 100 min gradient at a ﬂow rate of 300 lL min)1 (2–35% ACN in 0–70 min, 35–80% ACN 70–100 min). A QSTAR Elite Q-TOF (Applied Biosystems) operated in a positive ion mode (2.3 kV) performed the analysis, with the ﬁve most intense ions between 350 and 1600 m ⁄ z (over 20 counts) and with charge states of 2–5 in the TOF-MS scan were subjected to information-dependent acquisition MS-MS with Smart Exit function employed (setting 8). Ions within ±100 ppm of a former sampled ion were dynamically excluded for 120 s. The sample was analyzed ﬁve times on the mass spectrometer as technical replicates. Data processing The data from ﬁve runs were searched and combined using Protein Pilot software 3.0 (Applied Biosystems) with background correction and bias correction turned off. Searching was conducted with SwissProt Database version 2.0 (Caenorhabditis elegans: 22 983 entries). Only peptides of conﬁdence >50% were used for protein quantiﬁcation, and a 95% conﬁ- dence (unused prot score > 1.3) threshold for protein identiﬁcation as determined by Protein Pilot 3.0 was the criteria for inclusion into the ﬁnal protein report. 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