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Bio-orthogonal labeling as a tool to visualize and identify newly synthesized proteins in Caenorhabditis elegans Milena Ullrich1,2, Vanessa Liang1, Yee Lian Chew3, Samuel Banister4, Xiaomin Song5, Thiri Zaw5, Hong Lam3, Slavica Berber3, Michael Kassiou4,6, Hannah R Nicholas3,8 & Jürgen Götz1,7,8 1Brain and Mind Research Institute, University of

Sydney, Camperdown, New South Wales, Australia. 2Institute for Integrative Neuroanatomy, Charité, Universitätsmedizin Berlin, Berlin, Germany. 3School of Molecular Bioscience, University of Sydney, Camperdown, New South Wales, Australia. 4School of Chemistry, University of Sydney, Camperdown, New South Wales, Australia. 5Australian Proteome Analysis Facility, Macquarie University, Sydney, New South Wales, Australia. 6Faculty of Health Sciences, Macquarie University, Sydney, New South Wales, Australia. 7Clem Jones Centre for Ageing Dementia Research (CJCADR), Queensland Brain Institute (QBI), The University of Queensland, Brisbane, Queensland, Australia. 8These authors contributed equally to this work. Correspondence should be addressed to J.G. ([email protected]) or H.R.N. ([email protected]).

© 2014 Nature America, Inc. All rights reserved.

Published online 28 August 2014; doi:10.1038/nprot.2014.150

In this protocol we describe the incorporation of bio-orthogonal amino acids as a versatile method for visualizing and identifying de novo–synthesized proteins in the roundworm Caenorhabditis elegans. This protocol contains directions on implementing three complementary types of analysis: ‘click chemistry’ followed by western blotting, click chemistry followed by immunofluorescence, and isobaric tags for relative and absolute quantification (iTRAQ) quantitative mass spectrometry. The detailed instructions provided herein enable researchers to investigate the de novo proteome, an analysis that is complicated by the fact that protein molecules are chemically identical to each other, regardless of the timing of their synthesis. Our protocol circumvents this limitation by identifying de novo–synthesized proteins via the incorporation of the chemically modifiable azidohomoalanine instead of the natural amino acid methionine in the nascent protein, followed by facilitating the visualization of the resulting labeled proteins in situ. It will therefore be an ideal tool for studying de novo protein synthesis in physiological and pathological processes including learning and memory. The protocol requires 10 d for worm growth, liquid culture and synchronization; 1–2 d for bio-orthogonal labeling; and, with regard to analysis, 3–4 d for western blotting, 5–6 d for immunofluorescence or ~3 weeks for mass spectrometry.

INTRODUCTION Importance of discriminating pre-existing from de novo– synthesized proteins Living organisms respond to both internal and external stimuli in a process that involves the synthesis of proteins and, at some point, their degradation (with the latter process not being investigated by way of this protocol). These vital processes are impaired in physiological aging1–3, but they are also impaired in the context of age-related diseases, such as Alzheimer’s disease4. Numerous conventional methods exist to determine changes in protein levels. Typical examples are the analysis of the signal strength in western blots or of the staining intensity of a histological section. However, with these methods, it is not possible to determine whether any detected protein has been synthesized de novo in response to a particular stimulus or condition or whether it pre-existed the said stimulus or condition. Making this distinction is important because de novo protein synthesis is crucial in many contexts, for example, during learning and memory formation5. More specifically, mRNA translation in the hippocampus, a brain area crucial for learning and memory, which is affected early by the degenerative process in Alzheimer’s disease and related dementias6,7, is spatially controlled8. Long-term synaptic plasticity involves local protein synthesis at both the pre- and postsynapse9,10. Our research group has a long-standing interest in analyzing disease-relevant alterations of the proteome11–14, and the transcriptome assessing both coding (mRNAs)15,16 and noncoding genes (miRNAs)17 in cellular and mouse models of Alzheimer’s disease18.

The model organism C. elegans Historically, the mouse has been the preferred model system for investigating memory functions under physiological and pathological conditions, and for dissecting pathomechanisms in Alzheimer’s disease and related neurodegenerative diseases. Increasingly, however, invertebrates, such as C. elegans19 or Drosophila melanogaster, are being used in the field20,21, establishing a fruitful research cycle that goes from invertebrates to the mouse and back again. The protocol we detail herein has been developed in order to monitor de novo protein synthesis in vivo using C. elegans22. This organism has several features that make it a powerful research tool, complementing, but not replacing, the mouse as a model organism: (i) it is easy to culture as it feeds on bacteria; (ii) it reproduces and develops rapidly: within 3 d it develops from an egg to an adult worm, with about 300 progeny originating from one self-fertilized hermaphrodite; (iii) its small size enables assays to be performed in a microtiter format; (iv) the worm is transparent, an ideal characteristic when using fluorescent markers in vivo; (v) although it is a complex multi­cellular animal, an adult hermaphrodite has only 959 somatic cells that form all the organs, including 302 neurons that form the nervous system; (vi) it has a short life span of 2–3 weeks, which enables researchers to conduct aging studies within a reasonable time frame; and (vii) in C. elegans, genetic modifications, such as transgenic expression or RNAi-mediated gene knockdown, are relatively easy to perform compared with other experimental organisms. Furthermore, most human genes implicated in ­ disease have nature protocols | VOL.9 NO.9 | 2014 | 2237

protocol

© 2014 Nature America, Inc. All rights reserved.

homologs in C. elegans, and signal transduction pathways with a role in disease are conserved between C. elegans and humans23. Methods to visualize proteins in vivo To visualize proteins in vivo and to define their proper localization and translocation in response to various stimuli, several methods have become available recently. These methods include the fusion of proteins with SNAP or CLIP protein tags that can be specifically labeled with synthetic probes24. The SNAP-tag, with a size of 20 kDa, is highly versatile, because it rapidly reacts with a wide variety of synthetic probes attached to benzylguanine. The CLIPtag is of the same size, and it has been engineered to react with another group of derivatives. The two labeling methods can be combined to perform pulse-chase experiments and to investigate protein turnover over time25. Drawbacks of these approaches include the relatively large size of the tag and the requirement for manipulation of the target protein, which may cause substantial perturbations to protein structure and/or stability. Another disadvantage is the small number of proteins that can be investigated at any time. A complementary method to these approaches is the recently developed fluorescein arsenical hairpin (FlAsH/tetracysteine) technology26. The combination of a small genetically encoded peptide tag with a smallmolecule detection reagent makes this technology particularly suitable for the investigation of biochemical changes in living cells that are difficult to study with large fluorescent proteins as molecular tags. Neither of these methods, however, is versatile enough to both visualize the site of protein synthesis and decipher the identity of the proteins involved. Protein analysis by western blotting and mass spectrometry Western blot analyses are routinely performed to identify distinct proteins within a biological sample, such as a tissue extract, coupling gel electrophoresis with the transfer of the proteins according to size to nylon or nitrocellulose membranes and the incubation of these membranes with antibodies. A greater separation of proteins is achieved by 2D gel electrophoresis, in which proteins are separated in a first dimension by isoelectric point (pI) and in a second dimension by mass11. To detect a larger number of proteins than with ‘classic’ western blotting, some forms of multiplexing are available, as well as novel methods such as microfluidics western blotting27. However, for an unbiased approach to identifying large numbers of proteins, mass spectrometry should be considered the method of choice, often combined with chromatography28. As mass spectro­ metry is not inherently quantitative, several methods have been developed that enable researchers to determine the relative levels of proteins in, for example, an experimental compared with a control sample. Frequently used methods are isotope-coded affinity tags29; labeling with isobaric tags, such as iTRAQ14; label-free quantification30; and stable isotope labeling with amino acids in cell culture (SILAC)31,32. Methods such as SILAC have been successfully used to determine protein synthesis, degradation and turnover33,34. De novo protein synthesis investigated in various experimental systems Which methods are available to visualize and analyze newly synthesized proteins? A classical approach is the use of radioactively labeled methionine35. Typically, a culture (e.g., of a cancer sample and a normal specimen) is incubated for a defined period of 2238 | VOL.9 NO.9 | 2014 | nature protocols

time in the presence of 35S-labeled methionine, followed by the isolation of conditioned medium, separation by 2D gel electrophoresis and detection (for example, by staining with the colloidal dye Coomassie Brilliant Blue and then detecting the de novo– synthesized proteins by autoradiography) 36. An alternative method is SILAC, which uses stable nonreactive heavy isotopes to label specific amino acids. For example, SILAC can be used to compare samples incubated with amino acids with a natural isotopic composition with those incubated with labeled ones (e.g., arginine and lysine labeled with the stable, nonreactive heavy isotopes 15N rather than 14N). SILAC has typically been used for tissue culture cells. Leucine, lysine and methionine are essential amino acids that have been used in SILAC, a method that seeks to completely replace the labeled amino acid within the proteome, which is achieved after five cell doublings. However, when an excess of arginine is provided to the cells in the growth medium, isotope-labeled arginine can be converted to amino acids such as proline; this process can negatively affect quantification accuracy37. In C. elegans, this problem has been solved using a genetic ‘trick’, which consists of downregulating an enzyme that is required for the conversion of arginine to proline 32. Another successful quantitative proteomics study in C. elegans solved the conversion problem by only using labeled lysine 31, which does not get converted to other amino acids. In contrast to SILAC methods, the protocol described herein exposes samples to the noncanonical amino acid azidohomoalanine (AHA), which is incorporated in proteins in place of the natural amino acid methionine. Noncanonical amino acids contain a unique bio-orthogonal chemical motif, such as an azide or alkyne group, which is absent in the cells of organisms ranging from C. elegans to vertebrates38. These highly reactive groups (an azide group in the case of AHA) can be made to react with a fluorescent tag or with biotin that is fused to a linker molecule, provided that these molecules are coupled to a reactive alkyne group (Fig. 1). By adding AHA at any particular point of time to any experimental system, proteins synthesized after the addition of AHA can be visualized and identified as de novo–synthesized proteins. In a sequential labeling approach, an investigator can even add a second time point in protein synthesis by adding yet another reactive methionine analog, this time alkyne-labeled (e.g., homopropargylglycine), which is made to react with an azide-labeled fluorescent tag or biotin-linker molecule39. These reactions are examples of ‘click chemistry’ reactions and are copper-catalyzed, although catalysis by this metal is not a general pre requisite for this type of reaction. The concept of click chemistry was developed by Sharpless and colleagues40 to describe modular reactions of chemical building blocks that are wide in scope, proceed in very high yield and that generate only by-products that are easily removed. Click chemistry reactions typically proceed rapidly to completion owing to a high thermodynamic driving force, and they are highly selective, producing only a single product. Dieterich et al.41 showed in HEK 293 cells that AHA is metabolically incorporated into proteins. Importantly, the newly synthesized proteins are not subject to increased degradation41. AHA crosses the cell membrane and is incorporated into the nascent polypeptide chain by the endogenous methionyl-tRNA synthetase (MetRS) with a charging rate of 1/390 relative to methionine in Escherichia coli42, although rates in C. elegans may differ43. Upon reacting with an alkyne-labeled marker, these clicked proteins can

protocol

© 2014 Nature America, Inc. All rights reserved.

Figure 1 | Bio-orthogonal labeling workflow. (a) Azidohomoalanine (AHA) is attached in vivo to methionyl-tRNA synthetase in place of methionine, which follows AHA incorporation into the nascent polypeptide. (b) Three possible approaches to identify via AHA incorporation de novo–synthesized proteins are used in this protocol: click chemistry followed by either western blotting or fluorescence microscopy, and iTRAQ quantitative mass spectrometry. Scheme adapted from Liang et al.22.

be visualized and quantified by immunoblotting or fluorescence microscopy, and they can be identified by mass spectrometry. Similar approaches have been applied to sugars44, lipids45, virus particles46 and nucleic acids47. It was only in 2012, however, that de novo–synthesized proteins were identified with click chemistry in vivo48. AHA was metabolically incorporated into newly synthesized proteins in larval zebrafish, in the absence of obvious toxic effects or an alteration of the simple behaviors that were analyzed in the study, such as spontaneous swimming and escape responses48. Before the publication of the study by Hinz et al.48, Laughlin and colleagues had identified cell surface glycans with click chemistry in larval zebrafish44,49 and in C. elegans50.

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Protocol overview and experimental design In the PROCEDURE, detailed instructions are provided on how to achieve metabolic incorporation of AHA into newly synthesized proteins. We also describe the synthesis of the compounds needed for click chemistry, and we provide an experimental outline to visualize, quantify and identify de novo–synthesized proteins by immunoblotting and fluorescence microscopy (using click chemistry) and by iTRAQ quantitative mass spectrometry (Fig. 2). Worm growth and labeling. Starting with a plate culture, worms are transferred into a flask to establish a liquid culture. The culture is then synchronized by bleaching gravid adult worms. The eggs surviving the bleaching process are made to hatch in the absence of food, which arrests the development of the newly hatched worms at the first larval stage (L1). Development is re-initiated by feeding the larvae. The day after the larvae have been fed, bioorthogonal labeling is initiated by concentrating the worm culture and placing it in a medium containing AHA. Upon completion of the labeling, worms are collected by sedimentation and washing. Time is allowed for digestion of any remaining bacteria in the gastrointestinal tract, to remove potential contamination owing to AHA incorporation into bacteria. Worms are then processed either for immediate use or stored at −80 °C. Three types of analyses. Depending on the research question being addressed, labeling can be followed by three alternative

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De novo protein synthesis analyzed in C. elegans We sought to extend these methods to enable the visualization and identification of newly synthesized proteins in vivo in C. elegans. We therefore developed two click-chemistry protocols based on two previously established in vitro methods, which were termed bio-orthogonal noncanonical amino acid tagging (BONCAT)41,51 and fluorescent noncanonical amino acid tagging (FUNCAT)52, incorporating our own work22. Specifically, we present a protocol detailing three complementary, mutually alternative types of in vivo analysis of de novo protein synthesis: ‘click chemistry’ followed either by western blotting or fluorescence microscopy, as well as iTRAQ quantitative mass spectrometry for the identification of de novo–synthesized proteins (Figs. 1 and 2).

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types of in vivo analyses of de novo protein synthesis, either alone or in combination: western blotting, fluorescence microscopy and iTRAQ quantitative mass spectrometry. Western blotting is recommended to provide a global measure of de novo protein synthesis in response to internal or external stimuli; fluorescence microscopy will define which cell types in the worm have undergone de novo protein synthesis in response to different stimuli, and iTRAQ labeling followed by mass spectrometry is the method of choice to determine, in a quantitative manner, which proteins are de novo– synthesized and to which functional categories they belong. Western blotting. Worms are sonicated in a buffer containing SDS and Triton X-100 until complete cell lysis is achieved, which is followed by denaturation of the worms’ genomic DNA and degradation of DNA and RNA. After centrifugation and concentration steps, the lysate undergoes a click chemistry reaction by the addition of the triazole ligand, the biotin-alkyne tag and a copper bromide suspension for catalysis. Incubation under constant agitation for a prolonged period of time will yield a turquoise pellet, which can be analyzed by western blotting, using an antibiotin antibody. Immunofluorescence. The cuticle of the worms is permeabilized by repeated freeze-thaw cycles. The worms are fixed and subjected nature protocols | VOL.9 NO.9 | 2014 | 2239

AHA labeling

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outperforms SILAC and sample fractionation with combined fractional diagonal ­chromatography (COFRADIC) not only in the number of unique proteins identified but also in analysis time53.

Limitations A general limitation inherent to the pro­ cess of detecting proteins synthesized Click chemistry L1 Adults L1 L4/YA Age Adults and subsequent analysis de novo in response to a specific stimulus Western blotting is the background due to protein turno3d 1,300 1,300 220,000 1,000,000 Population (1st gen) (1st gen) (2nd gen) (3rd gen) ver. A challenge specific to the present protocol is the difficulty in detecting lowFluorescence micr. 6d Plate Liquid culture Medium abundance proteins, which stems from the 3 mentioned low charging rate to MetRS of iTRAQ quant. proteomics weeks AHA relative to methionine. Notably, if a methionine analog with a higher charging | Figure 2 Protocol workflow. rate than AHA were used, this modification might result in pronounced toxicity to a series of incubation and washing steps in borate buffer and associated with the protocol, which would limit its applicability. other buffers. A click chemistry reaction mix is prepared with One should also take into consideration that the incorporation of AHA into de novo–synthesized proteins via MetRS can be measthe triazole ligand, the alkyne-coupled fluorochrome Chromeo 546-Alkyne, tris(2-carboxyethyl)phosphine (TCEP) and copper ured only semiquantitatively. Furthermore, owing to N-terminal sulfate. Overnight incubation is followed by extensive washing, post-translational processing and acetylation, AHA can only be reliably detected in proteins that contain at least one nonterminal as well as by incubation with primary and secondary antibodies (such as to visualize distinct neuronal subtypes), if desired. DAPI methionine. A related consideration is that proteins will show difcan be added as a final step. After several washing steps, the worms ferent degrees of AHA incorporation depending on the number of methionine residues that they contain, and hence this protoare mounted on coverslips. col may introduce an inherent bias in protein detection. Finally, Mass spectrometry. In the present analytical approach, if the we have found that increasing AHA concentration and prolongpurpose of the experiment is specifically to isolate de novo– ing incubation times together leads to motor impairments in ­synthesized proteins, a purification step of biotinylated proteins C. elegans. As reported below, the thrashing assay reveals that a is required41. If the purpose is to determine both changes in the 3-h incubation with 4 mM AHA does not cause reduced numbers total and the de novo proteome with age and in response to heat of thrashes, whereas a 24-h incubation does. By contrast, incubashock, as we have done22, such a step is not performed. Worms are tion with only 2 mM AHA for 24 h does not cause impairments. If sonicated in a cocktail that contains protease inhibitors, and then AHA incubation conditions beyond those described in the current the protein concentration is determined. The sample is reduced protocol are to be investigated, toxicity assays such as the trashing with TCEP, alkylated with S-methyl methanethiosulfonate assay of motor function or a fluorescent reporter–based assay of (MMTS) and digested with trypsin. The sample is processed fur- the unfolded protein response (UPR) should be used. ther and labeled with iTRAQ reagents. Loading onto a strong cation exchange (SCX) column yields several fractions, which are Potential applications of the protocol analyzed by (nanoLC-ESI-MS/MS) mass spectrometry. The mass Despite the caveats detailed above, the techniques that we spectrometry data are analyzed to determine the relative protein describe herein for orthogonal labeling and detection of de novo– levels in the global and AHA-labeled pools. ­synthesized proteins in C. elegans represent useful additions to the repertoire of experimental tools that can be applied to studies Advantages of bio-orthogonal labeling compared with other using this popular model organism. Before analyzing and clicking methods the samples, subcellular fractionations may be performed. Click A major advantage of the approach outlined in this protocol is the chemistry can then be used in combination with 2D differential gel versatility of the types of analyses that can be performed. Although electrophoresis to compare the proteomes of specific sub­cellular SILAC, for example, is a mass spectrometry method typically compartments, as previously suggested54. Our protocol can also used in tissue culture cells, which enables the analysis of usually be used in combination with or to complement SILAC31,32. two, in some cases three, samples, the approach outlined in our The capacity to detect de novo–synthesized proteins would protocol enables the experimenter to perform the identical biobe useful in numerous contexts, including in the investigation orthogonal labeling reaction (i) to visualize new protein synthesis; of immune responses to pathogens 55,56 and of the response to (ii) to perform western blots as a global measure of de novo pro- environmental perturbations 57, and in comparing transcriptein synthesis; and (iii) to perform quantitative proteo­mics to tional and translational outputs arising from alterations in gene compare up to eight different samples (if eight tags are avail- regulation58. Of particular relevance here, with a simple and able—this protocol covers the use of four tags) in a single well-characterized nervous system, C. elegans represents an ideal experiment using iTRAQ. A recent study found that iTRAQ experimental system in which to investigate de novo protein Time

© 2014 Nature America, Inc. All rights reserved.

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protocol synthesis in a range of neurobiological contexts, such as during aging or associative learning59,60. We also suggest that our protocol could be adapted for rodents. In these more complex systems, the inducible and tissue-specific expression of mutant tRNA transferases to enable the spatially and temporally controlled incorporation of the bio-orthogonal

label into nascent polypeptide chains could extend the utility of our methods, as suggested previously61. This notion is supported by the finding that AHA and other noncanonical amino acids have been successfully incorporated into proteins, sugars, lipids and nucleic acids using a range of model systems derived from several experimental species22,41,44,48–52.

© 2014 Nature America, Inc. All rights reserved.

MATERIALS REAGENTS  CRITICAL When you are working with C. elegans, aseptic technique must be used. All glass vials, flasks, media and reagents need to be sterilized before use. Worm growth, liquid culture and synchronization • C. elegans strains: wild-type, N2 - Variety Bristol; CB6081 bus-17(e2800), obtained from the Caenorhabditis Genetics Center (CGC) at the University of Minnesota • E. coli strains: HB101 and OP50 obtained from the CGC, and methionineauxotrophic CAG 18491 obtained from The Coli Genetic Stock Center CGSC (Yale University) • Milli-Q water  CRITICAL Milli-Q water is used for the preparation of all aqueous solutions and other relevant applications, except for bioorthogonal labeling and preparation of click chemistry reagents, for which molecular biology–grade water is necessary (see below). • Difco LB broth Lennox (BD, cat. no. 240230) • KH2PO4 (Chem-Supply, cat. no. PA009) • K2HPO4 (Sigma-Aldrich, cat. no. P3786) • Bacto agar (BD, cat. no. 214030) • NaCl (Ajax Finechem, cat. no. 465) • Bacteriological peptone (BD, cat. no. 211677) • CaCl2 (Ajax Finechem, cat. no. 127) • Cholesterol (Sigma-Aldrich, cat. no. C8667) • MgSO4 (Sigma-Aldrich, cat. no. 230391) • EDTA (Sigma-Aldrich, cat. no. E5134) • FeSO4 (Sigma-Aldrich, cat. no. 215422) • MnCl2 (Sigma-Aldrich, cat. no. 203734) • ZnSO4 (Sigma-Aldrich, cat. no. 204986) • CuSO4 (Sigma-Aldrich, cat. no. 203165) • Citric acid monohydrate (Sigma-Aldrich, cat. no. C1909) • Potassium citrate tribasic monohydrate (Sigma-Aldrich, cat. no. P1722) • Streptomycin sulfate salt (Sigma-Aldrich, cat. no. S6501) • Nystatin (10,000 U/ml; Sigma-Aldrich, cat. no. N1638) • Sodium hypochlorite 12.5% (wt/vol) (Ajax Finechem, cat. no. 485) • NaOH pellets (Ajax Finechem, cat. no. 482) Bio-orthogonal labeling • Na2HPO4 (Sigma-Aldrich, cat. no. 71643) • Molecular biology–grade water (Sigma-Aldrich, cat. no. W4502)  CRITICAL Molecular biology–grade is only required for bio-orthogonal labeling and preparation of click chemistry reagents. For all other applications that require the use of water, use Milli-Q water. • Cycloheximide (200 mg/ml in ethanol; Sigma-Aldrich, cat. no. C7698) • 4-Azido-l-homoalanine (AHA; ABCR, cat. no. AB259208)  CRITICAL Keep the powder desiccated at room temperature (RT, 20–24 °C). • Diethyl ether (Ajax Finechem, cat. no. 1725) • NaH2PO4 (Sigma-Aldrich, cat. no. 71505) Click chemistry for western blot detection • MgCl2 (Sigma-Aldrich, cat. no. M2670) • Triton X-100 (Sigma-Aldrich, cat. no. T8787) • SDS (Sigma-Aldrich, cat. no. L4390) • Complete EDTA-free protease inhibitor tablets (Roche, cat. no. 11873580001) • Liquid nitrogen (provided by companies such as BOC) • Benzonase nuclease (Sigma-Aldrich, cat. no. E1014) • DMSO (Sigma-Aldrich, cat. no. D2650) • Tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (triazole ligand; Sigma-Aldrich, cat. no. 678937)  CRITICAL Store the desiccated powder at RT. • Biotin-PEG3-propargylamide (biotin-alkyne tag, synthesized as described in Box 1). Please note that, as an alternative to biotin-PEG3-propargylamide, biotin alkyne (without PEG linker) can be used (Lumiprobe, cat. no. C37B0)

• Propargylamine (Sigma-Aldrich, cat. no. 81825) • 24-[(3aS,4S,6aR)-hexahydro-2-oxo-1H-thieno[3,4-d]imidazol-4-yl]-4,20dioxo-9,12,15-Trioxa-5,19-diazatetracosanoic acid 2,3,5,6-tetrafluorophenyl ester (TFP-PEG3-biotin; Pierce, cat. no. 21219) • Copper (I) bromide (CuBr), 99.999% (Sigma-Aldrich, cat. no. 254185) • Bradford assay reagents (Quick Start Bradford protein assay 500, Bio-Rad, or equivalent) Western blotting • Glycerol (Sigma-Aldrich, cat. no. G7893) • 2-Mercaptoethanol, 99% (Sigma-Aldrich, cat. no. M3148) • Trizma-HCl (Sigma-Aldrich, cat. no. T5941) • Bromophenol blue solution (Sigma-Aldrich, cat. no. 18046) • 30% (wt/vol) Acrylamide/bisacrylamide (37.5:1) aqueous solution (Sigma-Aldrich, cat. no. A3699); store it at 4 °C in the dark until the expiry date indicated on the bottle ! CAUTION This toxic substance should be handled in a fume cupboard. • Ammonium persulfate (APS; Sigma-Aldrich, cat. no. A3678) • TEMED (N,N,N ′,N ′-tetramethylethylenediamine; Sigma-Aldrich, cat. no. T9281) • Glycine (Sigma-Aldrich, cat. no. G8898) • Trizma base, minimum 99.9% (Sigma-Aldrich, cat. no. T1503) • Concentrated HCl (32%; Merck Millipore, cat. no. 100319) • Nitrocellulose membrane (0.2 µm; Bio-Rad, cat. no. 162-0112) • Methanol (494437) • Tween 20 (Sigma-Aldrich, cat. no. P1379) • BSA (Sigma-Aldrich, cat. no. A9647) • Alkaline phosphatase–conjugated mouse anti-biotin antibody (Sigma-Aldrich, cat. no. A6561) or horseradish peroxidase (HRP)-conjugated goat anti-biotin antibody (Cell Signaling Technology, cat. no. 7075S) • HRP-conjugated goat anti-mouse antibody (Santa Cruz, cat. no. sc-2005) • Immobilon HRP substrate and peroxide solution (Merck Millipore, cat. no. WBKLS0100) Preparation of worms for fluorescence microscopy • Spermidine hydrochloride (Abcam, cat. no. ab120057) • PIPES, minimum 99% (Sigma-Aldrich, cat. no. P6757) • Formaldehyde solution, 37% (vol/vol; Sigma-Aldrich, cat. no. F1635) • KCl (Sigma-Aldrich, cat. no. P9333) • H3BO3 (Sigma-Aldrich, cat. no. B6768) • 2-Mercaptoethanol, minimum 98% (Sigma-Aldrich, cat. no. M3148) • DTT (Sigma-Aldrich, cat. no. D9779) • Hydrogen peroxide solution, 30% (vol/vol) in water (Sigma-Aldrich, cat. no. H1009) Click chemistry for fluorescence microscopy • Chromeo 546-Alkyne (BCAL-008; BaseClick) • 0.5 M Tris(2-carboxyethyl)phosphine hydrochloride solution (TCEP; Sigma-Aldrich, cat. no. 646547) • Copper sulfate (CuSO4; Sigma-Aldrich, cat. no. C1297) • Sodium azide (NaN3; Sigma-Aldrich, cat. no. 609374) ! CAUTION This highly toxic substance should be handled in a fume cupboard, while wearing appropriate protective clothing, including a dust mask. • Goat serum (Sigma-Aldrich, cat. no. G9023) • Primary antibodies: e.g., mouse anti-acetylated tubulin (1:100; Sigma-Aldrich, cat. no. T7451) • Secondary antibodies: e.g., goat anti-mouse Alexa Fluor 568 (1:500; Life Technologies, cat. no. A-11019) • DAPI (Sigma-Aldrich, cat. no. D9542) • Fluoromount-G (SouthernBiotech, cat. no. 0100-01) Mass spectrometry • Triethylammonium bicarbonate buffer (TEAB; Sigma-Aldrich, cat. no. T7408)

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protocol Box 1 | Synthesis of biotin-PEG3-propargylamide, the biotin-alkyne tag ● TIMING 40 min

© 2014 Nature America, Inc. All rights reserved.

Synthesis of N1-[19-[(3aS,4S,6aR)-hexahydro-2-oxo-1H-thieno[3,4-d]imidazol-4-yl]-15-oxo-4,7,10-Trioxa-14-azanonadec-1yl]-N4-2-propyn-1-yl-butanediamide (biotin-PEG3-propargylamide, see structure) 1. Add 50 mg (72 µmol) of TFP-PEG3-biotin slowly to excess neat propargylamine while stirring until complete dissolution is achieved. 2. After 10 min, add the solution dropwise to ~2 ml of anhydrous diethyl ether, whereupon a white precipitate forms. 3. Collect the precipitate by centrifugation at 13,400g at RT for 5 min, decant the supernatant and rinse the pellet with several drops of anhydrous diethyl ether. 4. Dry the product in vacuo to generate the final product as a fine white powder (38 mg, 65% yield). NMR spectroscopy analytical data In agreement with published data68, the following 1H NMR (400 MHz, CD3OD) spectrum features are assigned to the desired product: δ 4.52 (1H, dd, J = 7.8, 4.5 Hz, NHCHCH), 4.33 (1H, dd, J = 7.8, 5.0 Hz, NHCHCH2), 3.97 (2H, d, J = 2.5 Hz, NHCH2CCH), 3.68-3.49 (12H, m), 3.30-3.22 (5H, m), 2.96 (2H, dd, J = 12.7, 5.0 Hz, CHCH2S), 2.60 (1H, t, J = 2.5 Hz, NHCH2CCH), 2.50 (1H, t, J = 3.5 Hz, CH2CONH), 2.23 (4H, t, J = 7.4 Hz, OCH2CH2O), 1.88-1.58 (8H, m), 1.50-1.43 (2H, m).

• Protease inhibitor cocktail for fungal and yeast cells (Sigma-Aldrich, cat. no. P-8215) • Phosphoric acid (85%; Sigma-Aldrich, cat. no. W290017) • Tris(2-carboxyethyl)phosphine hydrochloride (TCEP; Sigma-Aldrich, cat. no. C4706) • S-Methyl methanethiosulfonate (MMTS; Fluka, cat. no. 64306) • Trypsin (Promega, cat. no. V5111) • iTRAQ 4-plex peptide labeling reagents (AB Sciex, cat. no. 4352135) • Ethanol (AB Sciex, cat. no. 4352156) • KH2PO4 (Sigma-Aldrich, cat. no. P0662) • Acetonitrile (Merck Millipore, cat. no. 1.00030.4,000) • KCl (Sigma-Aldrich, cat. no. P9541) • Formic acid (Fluka, cat. no. 94318) Toxicity assays • zcIs13[Phsp-6::gfp] transgenic C. elegans (strain SJ4100) received from the CGC (University of Minnesota), which is funded by the US National Institutes of Health Office of Research Infrastructure Programs (P40 OD010440) • zcIs4[Phsp-4::gfp] transgenic C. elegans (strain SJ4005) received from the CGC (University of Minnesota) • Tetramisole hydrochloride (Sigma-Aldrich, cat. no. T1512) EQUIPMENT • Petri dishes (Falcon, cat. no. 351029) • Marienfeld graduated glass pipettes or low-binding pipette tips (Sorenson, cat. no. 10230T) • Dissecting microscope (MVX10 Macro Zoom fluorescence microscope, Olympus) • Cooled digital color camera (DP70, Olympus) • Confocal microscope (e.g., Zeiss LSM 710) • Spectrophotometer to measure bacterial density (BioPhotometer, Eppendorf) • Sonicator (VCX 130, Sonics VibraCell) • DNA LoBind tubes, 1.5 ml (Eppendorf, cat. no. EPP0030108.051) • Amicon Ultra 0.5-ml centrifugal filter units supplied with microcentrifuge tubes, one to collect the filtrate and the other to recover the concentrated sample (Merck Millipore, cat. no. UFC500396) • Rotisserie rotator • Temperature-controlled shaker for microcentrifuge tubes (Thermomixer, Eppendorf) • Western blotting equipment: For SDS PAGE, Mini-Protean 3 Dodeca cell (Bio-Rad, cat. no. 165-8000); for wet transfer, Mini Trans-Blot cell (Bio-Rad, cat. no. 170-3930); for imaging of blots, VersaDoc model 4000 (Bio-Rad) 2242 | VOL.9 NO.9 | 2014 | nature protocols

• Rocking plate for antibody incubations (PROMAX 1020, Heidolph Instruments) • Mass spectrometer: TripleTOF 5600 (AB Sciex) or equivalent highresolution, high-mass-accuracy and fast-scanning mass spectrometer • NanoLC system: NanoLC-Ultra or NanoLC-400 system (Eksigent) • Analytical column: ProteCol C18 (150 µm × 10 cm; 3 µm particle size; 300 Å pore size; SGE) • SCX HPLC: Agilent 1100 quaternary HPLC system with polysulphoethyl A column (2.1 mm × 10 cm, 5 µm, 200 Å) • SuperFrost Plus glass slides (Thermo Scientific, cat. no. J1800AMNZ) • Coverslips, glass, 22 × 22 mm (HD Scientific Supplies) • Bruker Avance DRX400 (400.1 MHz) NMR spectrometer or equivalent, or access to such a piece of equipment Software • ImageJ 64-bit software (National Institutes of Mental Health) • Prism5 (GraphPad Software) • Olympus DP controller (Olympus, version 2.1.1.183) • Movie player (such as VLC for .avi files; thrashing assay; http://www.videolan.org/) • ProteinPilotV4.2 (AB Sciex) • UniProtKB/SwissProt release 2012 (http://www.uniprot.org/) REAGENT SETUP NaOH, 10 M  Dissolve 400g of NaOH pellets in water to a final volume of 1 liter. This solution can be stored at RT for at least 1 year. Potassium phosphate buffer  Prepare a 1-liter solution containing 0.8 M KH2PO4 and 0.2 M K2HPO4; adjust the pH to 6.0 with 10 M NaOH and autoclave the buffer. This buffer can be stored at RT for at least 1 year. Cholesterol stock solution  Prepare a 2.6 mM solution of cholesterol in 100% ethanol. This solution can be stored at −20 °C for several months. Nematode growth medium (NGM) agar plates  Prepare 1.5 liters of a medium containing 1.7% (wt/vol) Bacto agar, 0.3% (wt/vol) NaCl and 0.25% (wt/vol) bacteriological peptone. Autoclave, wait for the medium to cool down to 55 °C, and then add CaCl2 to a final concentration of 1 mM, cholesterol stock solution to a final concentration of 2.6 µM, potassium phosphate buffer to a final concentration of 25 mM and MgSO4 to a final concentration of 1 mM. Transfer the medium thus prepared to 10-cm Petri dishes. The plates can be stored at 4 °C for 1 month and at RT for several days before drying out. Trace metals solution  Prepare a solution containing 5 mM EDTA, 4.5 mM FeSO4, 1 mM MnCl2, 1 mM ZnSO4 and 0.2 mM CuSO4. Store it in the dark before use. S basal  Prepare a mixture containing 100 mM NaCl, 6 mM K2HPO4 and 44 mM KH2PO4. Autoclave the mixture and then wait for it to cool down to

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protocol 55 °C before adding cholesterol stock solution to a final concentration of 2.6 µM. This medium can be stored at RT for at least 1 year. Potassium citrate solution, pH 6.0  Prepare a solution containing 0.095 M citric acid monohydrate and 0.9 M potassium citrate tribasic monohydrate; adjust the pH to 6 with 10 M NaOH. Autoclave the solution. This solution can be stored at RT for at least 1 year. S medium  To 1 liter of S basal, add MgSO4 to a final concentration of 3 mM, CaCl2 to a final concentration of 3 mM, potassium citrate solution (pH 6.0) to a final concentration of 10 mM and 10 ml of trace metals solution. Prepare this medium immediately before use. AHA stock solution  Prepare a 20 mM stock solution of 4-azido-lhomoalanine in molecular biology–grade water. This solution can be stored for up to 1 month at 4 °C. AHA–S medium  Immediately before incubating the worms, add AHA stock solution to S medium to obtain the desired AHA concentration. Streptomycin solution  Prepare a 0.4% (vol/vol) solution of streptomycin sulfate salt in Milli-Q water. This solution can be stored at −20 °C for several months. Liquid growth medium (LGM) with or without HB101  To prepare 50 ml of medium, add 36 ml of S medium, 1.5 ml of nystatin and 200 µl of strepto­ mycin. If needed, add 12.5 ml of concentrated HB101 to the medium (see their preparation below). This medium should be prepared immediately before use. Bleaching solution  Prepare a solution containing 7.5% (wt/vol) sodium hypochlorite and 1.6 M NaOH. Freshly prepare this solution before use. Seeding NGM agar plates with OP50 bacteria  Establish bacterial cultures by inoculating 20 ml of LB broth with a single OP50 colony and incubate it on a shaker at 200 r.p.m. overnight at 37 °C. The next day, seethe 10-cm NGM agar plates by adding a 0.2-ml drop of OP50 liquid culture. Use a sterilized, bent Pasteur pipette to spread the drop to create an even OP50 lawn in the center of the plate. Leave the plates upright overnight at RT to allow the OP50 lawn to grow. Store the seeded plates in a sealed container at 4 °C for a maximum of 4 weeks. Concentrated HB101 bacteria  Establish a bacterial starter culture by ­inoculating 5 ml of LB broth containing 0.4% (vol/vol) streptomycin with a single HB101 colony and incubate on a shaker at 200 r.p.m. for 6 h at 37 °C. Add the starter culture to 1 liter of LB broth containing 0.4% (vol/vol) streptomycin in a flat-bottom conical flask and incubate it on a shaker at 200 r.p.m. overnight at 37 °C. Next, centrifuge the culture at 5,200g for 15 min at RT and resuspend in 20 ml of S medium. The concentrated bacteria are divided into 15- to 20-ml aliquots, which can be stored at −20 °C for several months. M9 buffer, 1×  Prepare a solution containing 86 mM NaCl, 42 mM Na2HPO4 and 22 mM KH2PO4. Autoclave the solution, wait for it to cool to 55 °C, and then add MgSO4 to a final concentration of 1 mM. This buffer can be stored at RT for at least 1 year. PBS, 10×  Prepare a solution containing 20 mM NaH2PO4, 80 mM Na2HPO4 and 1.5 M NaCl. This solution can be stored at RT for at least 1 year. PBS-MC  Prepare 1× PBS supplemented with 1 mM MgCl2 and 0.1 mM CaCl2. Chill it on ice. Prepare the solution immediately before use. PBS-MC-PI  Supplement 1× PBS-MC with EDTA-free protease inhibitor cocktail.  CRITICAL Prepare the solution freshly before use according to the EDTA-free protease inhibitor cocktail manufacturer’s instructions, and chill it on ice. Triton X-100, 20% (vol/vol)  Prepare this solution in molecular biology– grade water. This solution can be stored at RT for at least 1 year. SDS solution, 20% (wt/vol)  Prepare this solution in molecular biology– grade water. This solution can be stored at RT for at least 1 year. Complete EDTA-free protease inhibitor solution  Dissolve one tablet of complete EDTA-free protease inhibitor in 1 ml of water to obtain a 50× stock solution. The solution can be stored at –20 °C for up to 6 months. Worm lysis buffer  Prepare a solution containing 0.5% (wt/vol) SDS, 2% (vol/vol) 50× complete EDTA-free protease inhibitor solution and 1% (vol/vol) Triton X-100 in 1× PBS. This buffer can be stored at 4 °C for 1–2 weeks. Triazole ligand stock solution  Prepare a 200 mM solution of triazole ligand in DMSO and store it in 10-µl aliquots at −20 °C for up to 6 months. Avoid exposure to air or water.

Biotin-alkyne tag solution  Prepare a 25 mM stock solution of the biotin-alkyne tag in 1 × PBS and store it in 50-µl aliquots at −20 °C in the dark for up to 6 months. Tris buffer, 1.5 M, pH 8.8  Dissolve 118.2 g of Trizma-HCl in water, adjust the pH to 8.8 with concentrated HCl and adjust the volume to 500 ml with water. This buffer can be stored at RT for at least 1 year. Tris buffer, 1.0 M, pH 6.8  Dissolve 78.8 g of Trizma-HCl in water, adjust the pH to 8.8 with concentrated HCl and adjust the volume to 500 ml with water. This buffer can be stored at RT for at least 1 year. APS solution, 10% (wt/vol)  Dissolve 1 g of APS in 10 ml of water and divide it into 1-ml volumes. The aliquots can be stored at −20 °C for several months. Separation gel, 10% (wt/vol)   To make a 10-ml gel solution sufficient to prepare two gels, combine the following reagents: water (8 ml), 30% (wt/vol) acrylamide/bisacrylamide solution (6.7 ml), 1.5 M Tris buffer (pH 8.8; 2.5 ml) and 20% (wt/vol) SDS (100 µl). Add 10% (wt/vol) APS (200 µl) and TEMED (8 µl). Prepare it freshly before pouring gels; once APS and TEMED are added, the gel will immediately begin to polymerize. Stacking gel  To make an 8-ml stacking gel solution that is sufficient to prepare two gels, combine the following reagents: water (5.6 ml), 30% (wt/vol) acrylamide/bisacrylamide solution (1.3 ml), 1.0 M Tris buffer (pH 6.8; 1 ml) and 20% (wt/vol) SDS (40 µl). Next, add 10% (wt/vol) APS (80 µl) and TEMED (8 µl). Prepare it freshly before pouring gels; once APS and TEMED are added, the gel will immediately begin to polymerize. Sample buffer, 4×  Prepare a buffer containing 9.2% (wt/vol) SDS, 20% (vol/vol) glycerol, 20% (vol/vol) 2-mercaptoethanol, 25% (vol/vol) 1 M Trizma-HCl and 0.2% (vol/vol) bromophenol blue solution. This buffer can be stored at RT for at least 1 year. SDS running buffer, 10×, pH 8.3  Prepare a solution containing 2 M glycine, 1% (wt/vol) SDS and 0.25 M Trizma base (the pH should be 8.3 without the need to adjust). This buffer can be stored at RT for at least 1 year. SDS-free running buffer, 10×  Prepare a solution containing 2 M glycine and 0.25 M Trizma base. This buffer can be stored at RT for at least 1 year. Wet transfer buffer, 1×  Directly before running the gel, prepare a solution containing 10% (vol/vol) 10× SDS-free running buffer and 20% (vol/vol) methanol. Prechill the buffer at 4 °C before use. Tris-buffered saline, 10×  Prepare a solution containing 1.5 M NaCl and 0.1 M Trizma base. Adjust the pH to 7.4 with concentrated HCl. This buffer can be stored at RT for at least 1 year. Tris-buffered saline-T  Prepare a solution containing 0.1% (vol/vol) Tween 20 in 1× Tris-buffered saline, pH 7.4. This buffer can be stored at RT for at least 1 year. Na-PIPES, 1 M  Add 324.3 g of PIPES to 1 liter of water, and then add 10 M NaOH (~13 ml) until PIPES is dissolved. This solution can be stored at RT for at least 1 year. Modified Ruvkun’s witches brew, 2×  Prepare a solution containing 160 mM KCl, 40 mM NaCl, 20 mM EDTA, 10 mM spermidine hydrochloride, 30 mM Na-PIPES, 50% (vol/vol) methanol and 4% (vol/vol) formaldehyde in Milli-Q water. This solution can be stored at 4 °C for 1–2 weeks. Tris-Triton buffer  Prepare a solution containing 0.1 M TRIZMA base (pH 7.4), 1% (vol/vol) Triton X-100 and 1 mM EDTA in water. This buffer can be stored at RT for at least 1 year. Borate stock buffer, 40×  Prepare a solution containing 1 M H3BO3 and 0.5 M NaOH in Milli-Q water; titrate it to pH ≥9.5 with 10 M NaOH. This buffer can be stored at RT for at least 1 year. Borate buffer, 1×  Prepare a solution containing 2.5% (vol/vol) 40× borate stock buffer and 0.01% (vol/vol) Triton X-100 in Milli-Q water, and adjust the pH to 9.5 with 10 M NaOH. This buffer can be stored at RT for at least 1 year. DTT in borate buffer  Add 100 µl of the 30% (vol/vol) hydrogen peroxide solution to 10 ml of 1× borate buffer. Mix it well and use the buffer immediately. 1% (vol/vol) solution of 2-mercaptoethanol in borate buffer  Add 10 µl of 2-mercaptoethanol to 10 ml of 1× borate buffer. Mix it well and use the buffer immediately. Chromeo 546-Alkyne stock solution  Prepare a 200 mM Chromeo 546-Alkyne solution in DMSO. Store it in 50-µl aliquots at −20 °C and protect it from light and moisture. The solution can be stored at –20 °C for up to 1 month. nature protocols | VOL.9 NO.9 | 2014 | 2243

protocol

© 2014 Nature America, Inc. All rights reserved.

PBDTT (PBS-DMSO-Tween-Triton) buffer  Prepare a solution of 1× PBS supplemented with 1% (vol/vol) DMSO, 0.1% (vol/vol) Tween 20 and 0.5% (vol/vol) Triton X-100. This buffer can be stored at RT for at least 1 year. PBST-A  Prepare a solution in 1× PBS, which contains 1% (wt/vol) BSA, 0.5% (vol/vol) Triton X-100, 5 mM NaN3 and 1 mM EDTA. The solution can be stored at 4 °C for at least 6 months. PBST-B  Prepare a solution in 1× PBS, which contains 0.1% (wt/vol) BSA, 0.5% (vol/vol) Triton X-100, 5 mM NaN3 and 1 mM EDTA. The solution can be stored at 4 °C for at least 6 months. Blocking solution  Prepare a solution of 10% (vol/vol) goat serum and 5% (wt/vol) BSA in PBST-B. The solution can be stored at 4 °C for at least 6 months. Protein extraction buffer  Prepare a solution containing 0.25 M TEAB and 2% (wt/vol) SDS. This buffer can be stored at RT for at least 6 months. TCEP stock solution  Prepare a 100 mM solution of TCEP in water. The solution should be made before use.

MMTS stock solution  Prepare a 200 mM solution of MMTS. The solution can be stored at 4 °C for at least 6 months. SCX buffer A  Prepare a solution containing 5 mM KH2PO4 and 25% (vol/vol) acetonitrile. Adjust the pH to 2.7 with phosphoric acid. This buffer can be stored at RT for 1 month. SCX buffer B  Prepare a solution containing 5 mM KH2PO4, 350 mM KCl and 25% (vol/vol) acetonitrile. Adjust the pH to 2.7 with phosphoric acid. This buffer can be stored at RT for 1 month. nanoLC loading buffer  Prepare a solution containing 0.1% (vol/vol) formic acid and 2% (vol/vol) acetonitrile. This buffer can be stored at RT for 1 month. nanoLC ESI MS/MS buffer A  Prepare a solution containing 0.1% (vol/vol) formic acid. This buffer can be stored at RT for 1 month. nanoLC ESI MS/MS buffer B  Prepare a solution containing 0.1% (vol/vol) formic acid and 90% (vol/vol) acetonitrile. This buffer can be stored at RT for 1 month.

PROCEDURE Worm culture ● TIMING 10 d  CRITICAL An overview of the timing, culture conditions and number of worms needed for the labeling of ~25 samples with 15–20,000 L4 (larval stage 4) worms each is given in Figure 2, factoring in a 50% loss of worms during the extensive washing steps.  CRITICAL Please note that Box 2 provides information on the worm and bacterial strains that can be used in the protocol. 1| Use a sterilized scalpel to transfer a chunk of ~1 × 1 cm agar with L1 larvae to a large seeded NGM agar plate, and incubate it for 24 h at 20–25 °C. 2| Wash the worms (there should be ~1,300 of them) off the plate with S medium and transfer them into a 250-ml conical glass flask containing a 50-ml final volume of LGM. Cover the flask with aluminum foil.  CRITICAL STEP To avoid loss of worms, transfer them with sterilized glass pipettes or low-binding pipette tips rather than plastic pipettes. 3| Shake the conical glass flask at 150 r.p.m. at RT.  CRITICAL STEP Grow the culture up to a maximum of 1 million worms. We advise to regularly take small aliquots of the culture and to examine them using a dissecting microscope. Insufficient aeration or overcrowding may result in immobile dead worms or the formation of dauer-stage larvae, which are readily recognizable as they are thinner than regular larvae. 4| Monitor the culture daily under the microscope to observe developmental stage and numbers of worms, until ~220,000 adults are obtained, approximately after 7 d of incubation (Fig. 2). Please note that whenever the worm culture is no longer visibly cloudy, it means that the food supply is depleted and additional concentrated HB101 has to be added. ? TROUBLESHOOTING 5| Collect gravid adult worms in 50-ml tubes and centrifuge them with a low brake setting at 480g for 3 min at 15 °C. 6| Discard the supernatant and transfer at least 1 ml of compact worms to two or three 15-ml tubes. 7| Add bleaching solution in a 1:1 (vol/vol) ratio to the worms. Bleaching kills gravid adult worms and dissolves their carcasses, whereas eggs remain largely undamaged. Vortex the mixture vigorously for exactly 2 min, and top up the tube with 10 ml of Milli-Q water. Centrifuge the tube immediately with a low brake setting at 480g for 3 min at 15 °C. Discard the supernatant. 8| Repeat Step 7; this time, however, vortex for 90 s. 9| Confirm under the microscope that the C. elegans eggs are free of carcasses. If they are not, repeat Step 7 once more. 10| Wash the eggs in 10 ml of Milli-Q water and centrifuge the mixture thus obtained with a low brake setting at 480g for 3 min at 15 °C. Discard the supernatant. 2244 | VOL.9 NO.9 | 2014 | nature protocols

protocol Box 2 | Which strains of worms and bacteria to use for labeling?

© 2014 Nature America, Inc. All rights reserved.

C. elegans strains The use of wild-type C. elegans (N2) is described in the main text of the present protocol; however, in principle, the mutant strain bus-17(e2800) (also sourced from CGC) could be used. The main features of the mutant C. elegans strain bus-17, in comparison with wild-type N2 and as relevant for the present protocol, are as follows: • bus-17 mutant worms69 have a more permeable cuticle than N2 worms. This characteristic facilitates AHA uptake and fluorescence staining. • Synchronization, however, is more effective for N2 worms. • N2 worms grow more quickly than bus-17 mutant worms. E. coli strains Both HB101 and OP50 can be used as bacterial food sources for C. elegans. There is a further option to use no or dead bacteria to reduce bacterial contamination in the samples analyzed by mass spectrometry, and to use a methionine-auxotrophic strain to increase AHA incorporation. • Live HB101 or OP50: food source to maintain C. elegans on plates; they can also be used for the AHA incubation step. • Dead OP50: Bacteria are heat-killed by completely submerging them in a 65 °C water bath for 3 h before AHA incubation. To determine that the bacteria are dead, streak a small amount on a fresh LB plate and incubate overnight at 37 °C, after which time the absence of bacterial colonies should be obvious. By feeding dead bacteria to worms during AHA labeling, the majority of the labeling comes from free AHA, i.e., not from that which has been passaged through the bacterial proteins. We have previously found that 150) in the survey scan sequentially subjected to MS/MS analysis. Acquire each MS/MS spectrum for 200 ms in the 100–1,500 m/z mass range with a total cycle time of ~2.3 s. (xiv) After peptide elution, clean the column with 95% (vol/vol) nanoLC-ESI-MS/MS buffer B for 15 min and then equilibrate with nanoLC-ESI-MS/MS buffer A for 25 min before the next sample is injected. (xv) For data processing, submit the experimental nanoLC-ESI-MS/MS data to ProteinPilot V4.2 using the combined fasta database of ‘Caenorhabditis elegans’ and ‘E. coli’ downloaded from SwissProt 2012 (http://web.expasy.org/docs/swissprot_guideline.html). Select iTRAQ 4-plex peptide labeled for ‘Sample Type’, MMTS for ‘Cysteine Alkylation’ and Met→ AHA in ‘Special Factors’. Select ‘Quantification’, ‘Bias Correction’, ‘Thorough ID’ and ‘Run False Discovery Rate Analysis’ in the ‘Specify Processing’ tab. A protein unused score better than 1.3 (better than 95% confidence) is used as the cutoff threshold for protein identification. Proteins with iTRAQ ratios larger than 1.2 or smaller than 0.82 and P values 90% confidence and the identified protein score is better than 95% confidence (ProteinPilot protein unused score >1.3). By using a less stringent threshold, more proteins can be identified. Figure 9 shows a representative spectrum of a Met→AHA modified peptide identified with iTRAQ4plex N-terminal labeling. Less than 0.1% of identified proteins are from E. coli, indicating virtually complete removal of the bacteria.

Acknowledgments This study was supported by the Estate of Dr. Clem Jones, AO, and by grants from the Australian Research Council (DP13300101932) and the National Health and Medical Research Council of Australia (APP1037746 and APP1003150) to J.G. Mass spectrometry was undertaken at The Australian Proteome Facility, with the infrastructure provided by the Australian Government through the National Collaborative Research Infrastructure Strategy. Some strains were provided by the CGC, which is funded by the US National Institutes of Health Office of Research Infrastructure Programs (P40 OD010440). AUTHOR CONTRIBUTIONS M.U., V.L., Y.L.C., S.B., X.S., T.Z., H.L. and S.B. performed the experiments; M.U., V.L., Y.L.C., S.B., X.S., T.Z., H.L., S.B., M.K., H.R.N. and J.G. analyzed the data; and M.U., V.L., Y.L.C., H.R.N. and J.G. wrote the manuscript with input from all authors. COMPETING FINANCIAL INTERESTS The authors declare no competing financial interests. Reprints and permissions information is available online at http://www.nature. com/reprints/index.html. 1. Taylor, R.C. & Dillin, A. Aging as an event of proteostasis collapse. Cold Spring Harb. Perspect. Biol. 3 (2011). 2. Kaeberlein, M. & Kennedy, B.K. Hot topics in aging research: protein translation and TOR signaling, 2010. Aging Cell 10, 185–190 (2011). 3. Giboureau, N., Som, I.M., Boucher-Arnold, A., Guilloteau, D. & Kassiou, M. PET radioligands for the vesicular acetylcholine transporter (VAChT). Curr. Top Med. Chem. 10, 1569–1583 (2010). 4. De Strooper, B. Proteases and proteolysis in Alzheimer disease: a multifactorial view on the disease process. Physiol. Rev. 90, 465–494 (2010). 5. Chen, C.C. et al. Visualizing long-term memory formation in two neurons of the Drosophila brain. Science 335, 678–685 (2012). 6. Götz, J., Chen, F., van Dorpe, J. & Nitsch, R.M. Formation of neurofibrillary tangles in P301L tau transgenic mice induced by Aβ42 fibrils. Science 293, 1491–1495 (2001). 7. Ittner, L.M. et al. Dendritic function of tau mediates amyloid-β toxicity in Alzheimer’s disease mouse models. Cell 142, 387–397 (2010). 8. Cajigas, I.J., Will, T. & Schuman, E.M. Protein homeostasis and synaptic plasticity. EMBO J. 29, 2746–2752 (2010). 9. Antonov, I., Kandel, E.R. & Hawkins, R.D. Presynaptic and postsynaptic mechanisms of synaptic plasticity and metaplasticity during intermediateterm memory formation in Aplysia. J. Neurosci. 30, 5781–5791 (2010). 10. Holt, C.E. & Schuman, E.M. The central dogma decentralized: new perspectives on RNA function and local translation in neurons. Neuron 80, 648–657 (2013). 11. David, D.C. et al. Proteomic and functional analysis reveal a mitochondrial dysfunction in P301L tau transgenic mice. J. Biol. Chem. 280, 23802–23814 (2005). 12. David, D.C. et al. β-Amyloid treatment of two complementary P301L tauexpressing Alzheimer’s disease models reveals similar deregulated cellular processes. Proteomics 6, 6566–6577 (2006). 13. Lim, Y.-A. et al. Aβ and human amylin share a common toxicity pathway via mitochondrial dysfunction. Proteomics 10, 1621–1633 (2010). 14. Rhein, V. et al. Amyloid-β and tau synergistically impair the oxidative phosphorylation system in triple transgenic Alzheimer’s disease mice. Proc. Natl. Acad. Sci. USA 106, 20057–20062 (2009).

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