Iron promotes protein insolubility and aging in C. elegans.

Many late-onset proteotoxic diseases are accompanied by a disruption in homeostasis of metals (metallostasis) including iron, copper and zinc. Although aging is the most prominent risk factor for these disorders, the impact of aging on metallostasis and its role in proteotoxic disease remain poorly understood. Moreover, it is not clear whether a loss of metallostasis influences normal aging. We have investigated the role of metallostasis in longevity ofCaenorhabditis elegans. We found that calcium, copper, iron, and manganese levels increase as a function of age, while potassium and phosphorus levels tend to decrease. Increased dietary iron significantly accelerated the age-related accumulation of insoluble protein, a molecular pathology of aging. Proteomic analysis revealed widespread effects of dietary iron in multiple organelles and tissues. Pharmacological interventions to block accumulation of specific metals attenuated many models of proteotoxicity and extended normal lifespan. Collectively, these results suggest that a loss of metallostasis with aging contributes to age-related protein aggregation.

Mass Spectrometry. All samples were analyzed by reverse-phase HPLC-ESI-MS/MS using an Eksigent Ultra Plus nano-LC 2D HPLC system (Dublin, CA) connected to a quadrupole time-of-flight TripleTOF 5600 mass spectrometer (AB SCIEX). Typically, mass resolution for MS1 scans and corresponding precursor ions was ~35,000 while resolution for MS2 scans and resulting fragment ions was ~15,000 ('high sensitivity' product ion scan mode).
Data acquisition was performed in data dependent mode (DDA) on the TripleTOF 5600 to obtain MS/MS spectra for the 30 most abundant precursor ions (approx. 50 msec per MS/MS) following each survey MS1 scan (250 msec). Each sample was analyzed in 3 technical injection/MS replicates. Specifically, samples were acquired by reverse-phase HPLC-ESI-MS/MS using an Eksigent Ultra Plus nano-LC 2D HPLC system (Dublin, CA) which was directly connected to a quadrupole time-offlight (QqTOF) TripleTOF 5600 mass spectrometer (AB SCIEX, Concord, CAN). The autosampler was operated in µl-pickup injection mode filling a 3 µl loop with 3 µl analyte per injection. Briefly, after injection, peptide mixtures were transferred onto the analytical C18-nanocapillary HPLC column (C18 Acclaim PepMap100, 75 µm I.D. x 15 cm, 3 µm particle size, 100 Å pore size, Dionex, Sunnyvale, CA) and eluted at a flow rate of 300 nL/min using the following gradient: at 5% solvent B in A (from 0-13 min), 5-35% solvent B in A (from 13-58 min), 35-80% solvent B in A (from 58-63 min), at 80% solvent B in A (from 63-66 min), with a total runtime of 90 min including mobile phase equilibration. Solvents were prepared as follows, mobile phase A: 2% acetonitrile/98% of 0.1% formic acid (v/v) in water, and mobile phase B: 98% acetonitrile/2% of 0.1% formic acid (v/v) in water.
For selected samples, additional data sets were recorded in data-independent mode (DIA) using SWATH MS2 acquisitions for quantitative analysis. In the SWATH-MS2 acquisition, a Q1 window of 25 m/z was selected to cover the mass range of m/z 400-1000 in 24 segments (24 x 100 msec), yielding a cycle time of 3.25 sec, which includes one 250 msec MS1 scan.
Bioinformatic database searches. Mass spectrometric data was searched using the database search engine ProteinPilot (Shilov, 2007 #121) (AB SCIEX Beta 4.5, revision 1656) with the Paragon algorithm (4.5.0.0, 1654). The search parameters were set as follows: trypsin digestion, cysteine alkylation set to iodoacetamide, urea denaturation, and species C. elegans. Trypsin specificity was assumed as C-terminal cleavage at lysine and arginine. Processing parameters were set to "Biological modification" and a thorough ID search effort was used. During the search, Protein Pilot performs an automatic mass recalibration of the data sets based on highly confident peptide spectra. All data files were searched using the SwissProt 2012_07 database) with a total of 6716 C. elegans protein sequences. For Protein Pilot Searches, to assess and restrict rates of false positive peptide/protein identifications, we used the Proteomics System Performance Evaluation Pipeline (PSPEP) tool available in ProteinPilot 4.5 beta, respectively. This tool automatically creates a concatenated forward and reverse decoy database, and provides an Excel output of the experimentally determined false discovery rate at the spectral, peptide and protein levels with standard statistical errors. The discriminating variable for the Paragon Algorithm is the peptide confidence value, which is a 0-99 scaled real number (Shilov, 2007 #121). For database searches, a cut-off peptide confidence value of 99 was chosen with the following justification. For searching the databases the Protein Pilot false discovery rate (FDR) analysis tool (PSPEP) algorithm (Shilov, 2007 #121) provided a global FDR of 1% and a local FDR at 1% in all cases.
Quantitative Skyline MS1 Filtering Analysis. MS1 chromatogram based quantification was performed in Skyline 2.5 an open source software project (http://proteome.gs.washington.edu/software/skyline) as recently described recently in detail by Schilling et al (Schilling, 2012 #161). Briefly, first, comprehensive spectral libraries were generated in Skyline from database searches of the raw data files prior to MS1 Filtering. Second, all raw files acquired in data dependent acquisition mode (DDA), are directly imported into Skyline 2.5 and MS1 precursor ions are extracted for all peptides present in the MS/MS spectral libraries (MS1 resolution setting in Skyline for precursor ion extraction is set at 10,000). Quantitative analysis is based on extracted ion chromatograms www.impactaging.com (XICs) and resulting precursor ion peak areas for M, M+1, and M+2; final quantitative comparisons are typically based on only the highest ranked precursor ion. Precursor peak areas were exported from Skyline into excel sheets using the recently developed statistical program 'MS1Probe' (D'Souza, 2013), a Python-coded bioinformatics program that interfaces with Skyline 'external tools' (http://www.gibsonproteomics.org/ resources/MS1Probe) to calculate ratios per peptide between different experimental conditions (i.e., control vs. iron treated worms). Significance was assessed using two-tailed Student's t-test requiring ρ values of ρ < 0.05.
Raw Data accession and Panorama Spectral Libraries. The raw and processed data (i.e., Skyline files) associated with this manuscript may be downloaded from the Buck Institute ftp site at ftp://ftp.buckinstitute.org:225/Iron_Insoluble_Proteins/. Details for peptide quantification using MS1 Filtering, including peptide peak areas for all MS replicates are provided as excel files as well as summarized in Supplemental Tables S3-S4. A spectral library with MS/MS spectra for all peptides/proteins used for MS1 Filtering and subsequent quantification was transferred and published to the interactive, web-based data sharing Panorama server (Sharma et al, 2012). The spectral viewer can be assessed at https://daily.panoramaweb.org/labkey/project/Gibson/Li thgow_Iron/begin.view?
Funtional Analysis -Protein Ontology. The web-based program DAVID v.6.7 (The Database for Annotation, Visualization and Integrated Discovery), (Huang da, 2009 #120) was used for functional analysis and protein ontology analysis. Analysis was performed for proteins identified in the Insolubelome by mass spectrometry that were significantly and robustly increased in their relative abundance comparing 'control worms' to 'irontreated worms'.

SUPPLEMENTAL TABLES
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