cell-type-specific metabolic labeling, detection and ... · digest elution enrichment steps 21–25...

Cell-type-specific metabolic labeling, detectionand identification of nascent proteomes in vivoBeatriz Alvarez-Castelao1*, Christoph T. Schanzenbächer1,2, Julian D. Langer1,2 andErin M. Schuman 1*

A big challenge in proteomics is the identification of cell-type-specific proteomes in vivo. This protocol describes how tolabel, purify and identify cell-type-specific proteomes in living mice. To make this possible, we created a Cre-recombinase-inducible mouse line expressing a mutant methionyl-tRNA synthetase (L274G), which enables the labeling of nascentproteins with the non-canonical amino acid azidonorleucine (ANL). This amino acid can be conjugated to different affinitytags by click chemistry. After affinity purification (AP), the labeled proteins can be identified by tandem massspectrometry (MS/MS). With this method, it is possible to identify cell-type-specific proteomes derived from livinganimals, which was not possible with any previously published method. The reduction in sample complexity achieved bythis protocol allows for the detection of subtle changes in cell-type-specific protein content in response to environmentalchanges. This protocol can be completed in ~10 d (plus the time needed to generate the mouse lines, the desired labelingperiod and MS analysis).

Introduction

Identification of cell-type-specific proteomes and how these proteomes react to specific stimuli is afundamental question for understanding function and dysfunction of living organisms. Identificationof proteins by MS has become a key technique for the study of cellular biology, complementing RNAand DNA sequencing. Unlike DNA or RNA sequencing, protein identification lacks the possibility ofamplifying the target molecules, i.e., the cellular protein content. For this reason, the detection ofprotein species using MS is more challenging than detection of RNA molecules using RNA-seq. Thislimits the identification of some protein species, especially low-abundant proteins in complex proteinmixtures. To overcome this constraint and increase the dynamic range for protein detection, thesensitivity of the MS instruments has greatly improved, as have the bioinformatics tools for proteinidentification and analysis. From the point of sample preparation, different strategies for decreasingsample complexity have been developed1. None of these strategies, however, preserves cellularintegrity. Consequently, increasing sensitivity is often accompanied by a loss of information. This isespecially notable in some morphologically complex cell types, such as neurons, of which the longaxons and dendrites are lost during procedures aimed at sorting cells for analysis. One way toovercome this limitation is the use of bio-orthogonal strategies. These strategies are based on the useof chemical reporters carrying alkynes, which can be used for protein purification. This approach hasbeen widely used for the labeling of proteins, glycans and lipids2, taking advantage of a certain degreeof promiscuity of the relevant endogenous cellular machinery to incorporate non-canonical mole-cules. Furthermore, the use of the cellular machinery for the incorporation of chemical reportersmakes this strategy amenable to genetic manipulations3–5. For example, the expression of amethionyl-tRNA synthetase (MetRS) bearing a point mutation that alters the amino acid-binding site(MetRS L274G or MetRS*) allows for the incorporation of ANL6–9 into proteins instead ofmethionine (Fig. 1a). ANL has a slightly different structure from methionine and it is not recognizedby the cell’s endogenous MetRS. Therefore, ANL cannot be incorporated into proteins in cells that donot express mutated MetRS. Thus, the expression of MetRS L274G under cell-type-specific promoterslimits ANL incorporation into proteins synthesized in the respective cell type, thereby allowing formetabolic labeling of cell-type-specific proteomes. ANL carries a reactive azide group, which allowsany alkyne to be covalently attached to ANL using copper-catalyzed azide-alkyne ligation (BONCAT4

1Max Planck Institute for Brain Research, Frankfurt, Germany. 2Max Planck Institute of Biophysics, Frankfurt, Germany.*e-mail: [email protected]; [email protected]

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PROTOCOLhttps://doi.org/10.1038/s41596-018-0106-6

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mailto:[email protected]

mailto:[email protected]

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https://doi.org/10.1038/s41596-018-0106-6

(biorthogonal non-canonical amino acid tagging)). Different molecules, such as fluorophores orantigenic tags, can be derivatized with alkynes. Here, we used biotin alkynes to covalently attachbiotin to ANL in order to detect and affinity-purify cell-type-specific proteins. We engineered a Cre-lox mouse line in which the expression of GFP-2A-MetRS L274G is controlled by Cre-recombinaseexpression (Fig. 1b). Consequently, when MetRS L274G mice are crossed with animals expressing Creunder the control of a cell-type-specific promoter, metabolic labeling of proteins with ANL will occuronly in the cell type expressing Cre. The mutant MetRS is fused to GFP, and these proteins areseparated by a P2A peptide that will be cleaved after protein expression10; thus GFP allows for theverification of cell-type-specific expression of MetRS L274G using fluorescence microscopy or (anti-GFP) western blot. This approach enables metabolic labeling of proteins in as many cell types asspecific Cre-driver lines exist. This method not only allows for the isolation of cell-type-specificproteins; the enrichment of newly synthesized proteins decreases the complexity of the sample,increasing the sensitivity and enabling the detection of small changes in proteomes in response tochanges in the extracellular or intracellular environment. ANL, like natural amino acids, can be addedto the animal’s diet or introduced by intraperitoneal (i.p.) injection. We administered ANL in thedrinking water of mice to label newly synthesized proteins. Although ANL contains an azide group,this compound is very stable and non-toxic, as has been shown for similar compounds11.

Overview of the procedureThe procedure consists of four stages: (i) protein labeling, (ii) tissue collection and click chemistry,(iii) purification of labeled proteins, and (iv) protein identification by MS and analysis. In the firststage (Steps 1 and 2), ANL is administered to the mice and the proteins from the cell type of interestare labeled. In the second stage (Steps 3–20), an alkyne is bound to the ANL-containing proteins byclick chemistry. In the third stage (Steps 21–28), the cell-type-specific proteins are isolated using thealkyne as bait for AP of the labeled proteins. In the last stage (Steps 29–57), purified proteins areidentified by MS followed by bioinformatics analysis (Fig. 2).

Applications of the methodThe most compelling application of this technique is the possibility of labeling and identifyingproteins that were synthesized in vivo, in the natural environment of the cell type of interest. One

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Fig. 1 | ANL incorporation into the mutant MetRS and mouse design. a, Scheme showing the structure of the analogof methionine ANL; the tail of the artificial amino acid is larger than methionine. A point mutation in the methionyl-tRNA synthetase amino acid-binding pocket (MetRS L247G or MetRS*) allows for the loading of ANL onto themethionyl-tRNA. Therefore, ANL can be incorporated into nascent proteins. b, Graphic representing wild-type (WT)and knock-in (KI) alleles in the R26-MetRS-L264G mouse line. In R26-MetRS-L264G animals, the expressioncassette is located in the Rosa26 locus. Cre-dependent expression of GFP-2A-MetRS* is determined by a floxedpolyadenylation signal. Adapted with permission from Alvarez-Castelao et al.12, Springer Nature.

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major advantage of the method is that the researcher can choose when to label the proteins to beidentified, so it is possible to know when and where (in which cell type) the proteins were synthesized.Both timing and cell-type specificity make protein detection more sensitive to subtle changes incellular protein content. Using this approach, we purified and identified 3,476 proteins from twodifferent neuronal types in mice labeled with ANL for 21 d in vivo12. To explore the sensitivity of ourmethod, we examined how the proteome of the excitatory neurons of the hippocampus changed afterexposing the mice to an enriched environment (EE), which has been shown to modify brain circuitsand change the density of synapses13. We identified 225 proteins exhibiting differences in expressionlevels between mice housed in standard cages and mice housed in EE cages. This proteome plasticitymay underlie the changes in synaptic structures and neural circuits associated with the exposure ofrodents to EE12. In addition, it is possible to combine this method with other quantitative tags, suchas isobaric or isotope tags for the comparison of several samples in one MS experiment. This methodcan also be used in vitro in primary cultures or in tissue slices12, simply by transfecting MetRS L274Gcontrolled by the desired cell-type-specific promoter. The labeled and purified proteins can beanalyzed by western blot for the study of specific candidate proteins, with the possibility of studyingseveral proteins of interest with one AP step. Click chemistry can be performed in fixed tissue,allowing the visualization of newly synthesized proteins in situ. The use of an alkyne congugated witha fluorophore for the click chemistry, followed by imaging, provides a general readout of globalprotein synthesis in tissues or cell cultures with spatial resolution (also known as fluorescence non-canonical amino acid tagging (FUNCAT)14). When click chemistry is combined with the in situproximity ligation assay (PLA)15, it is even possible to visualize specific candidate proteins that weresynthesized (in vivo or in vitro) during a defined period of time in a specific cell type. The methoddescribed here can be also used for protein-transfer experiments, which require knowledge about thesource of proteins, for example, in parabiosis studies16,17. This approach is very clean because ANLcannot be recycled in the transference organism, provided it does not express MetRS L274G.

Comparison with other methodsClassically, metabolic protein turnover has been measured using SILAC (stable isotope labeling byamino acids in cell culture). The main advantage of SILAC is that it is quantitative. It is, however, notcell-type specific, and the labeled proteins cannot be purified. Therefore, the resulting samples displaya high protein complexity, which limits MS sensitivity. In vivo studies using SILAC are possible18,19,but they are limited by the high cost of the isotopically labeled amino acids. A newer technique,CTAP20 (cell-type-specific labeling using amino acid precursors), is also based on isotopic labeling,

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Fig. 2 |Workflow for cell-type-specific protein labeling and purification.Workflow for protein labeling in mice by adding ANL to the drinking water orby intraperitoneal injection. The tissue under study is collected and the proteins containing ANL are clicked (BONCAT); then the labeled proteins areisolated by affinity purification and eluted by cleaving the alkyne. Purified proteins are identified by MS. The corresponding steps from the protocol areindicated. Strept, streptavidin. Adapted with permission from Alvarez-Castelao et al.12, Springer Nature.

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but in this case, the isotopic labeling relies on amino acid precursors. For the application ofCTAP, the necessary enzymes for the biosynthesis of essential amino acids must be exogenouslyexpressed, enabling cell-type-specific labeling. Nevertheless, to date, this technique has not beenimplemented in vivo, and, similar to SILAC, labeled proteins cannot be isolated from the lysates usingthis method. Labeling proteins with radioactive isotopes is another classic approach to studyingprotein turnover. Radioactive labeling is very sensitive and can be used for the study of generalprotein turnover or, in combination with immunoprecipitation, for candidate-based studies21.However, radioactively labeled proteins cannot be identified by MS, and the labeling is not cell-typespecific. In the past few years, puromycylation has become a widely used technique for the labeling ofnewly synthesized proteins22. The antibiotic puromycin is incorporated into peptide chains at the Asite of the ribosome and labeled proteins can later be detected by puromycin-specific antibodies. Theadvantage of puromycylation being easy to perform is counterbalanced by the fact that this techniquegenerates many truncated or aberrant proteins23–25. It is therefore not suitable for studies involvingfunctional proteins. In vivo labeling with puromycin is limited to very short periods of time (min-utes), owing to its toxicity. Some attempts have been made to label in a cell-type-specific manner withpuromycin26,27, but so far this technique has not been implemented in vivo. Finally, if we compareANL labeling with labeling with AHA (L-azidohomoalanine), the other widely used artificial aminoacid, the main advantage of ANL is that it can be used for cell-type-specific labeling because of itsrequirement for exogenous expression of MetRS L274G14,28,29, whereas AHA is incorporated intoevery cell type by the endogenous MetRS. Table 1 provides a concise comparison of methods formetabolic labeling.

The main limitation to the approach described here is potentially, limited ability to identifyproteins in cell types that have very low abundance in a large volume of tissues. If the cell type ofinterest is present in low numbers and is sparsely distributed in a large tissue volume, this methodmay not be suitable for a proteomics study. However, the method offers a good opportunity to studythe proteome of a low-abundance cell type if it is clustered in a specific part of the tissue that issuitable for microdissection, increasing the signal-to-noise ratio. But it is also important to take intoaccount the rate of synthesis and that the accessibility of ANL might vary between cell types30.

Expertise needed to implement the protocolFor the implementation of the protocol, it is necessary to have experience in the handling andmanagement of mouse colonies. Basic knowledge of biochemistry is strongly recommended. MSexpertise is required for the processing and analysis of the samples.

Experimental designThe experimental design depends on the scientific question and the cell type of interest. For in vivoexperiments, ANL can be administered by adding it to the drinking water or by i.p. injections. Thetwo administration systems work equally well, but, depending on the duration of the labeling, it mightbe better to use one system or the other. For short periods of time (up to 1 week), i.p. injection issuitable, injecting the mice once or twice per day. It is important to take into account that a certain

Table 1 | Comparison of metabolic protein labeling methods

Method Suitable formicroscopy

Microscopy forspecific proteins ofinterest

Proteinpurificationpossible

Cell-type-specificlabeling

In vivolabeling

In vitrolabeling

Radioactivity No No Yesa No Yesb Yes

SILAC No No No No Yes Yes

CTAP No No No Yes ? Yes

Puromycin Yes Yesc Yesc Yesc Yesc Yesc

AHA Yes Yes Yes No Yes Yes

MetRS*/ANL Yes Yes Yes Yes Yes Yesd

aOnly candidate-based by immunoprecipitation of the protein of interest. bRadioactivity is potentially harmful and highly toxic. cGenerated proteins areoften truncated; complicating the study of protein transport/half-life measurements, only short labeling times can be used due to toxicity; puromycincan be incorporated at every amino acid position in the protein, so the labeled non-truncated proteins can be substantially different from the naturalones. dMetRS* can be exogenously expressed in the cell type of interest.




expertise is needed for i.p. injections, in order to avoid loss of the injected substance. For longerperiods of labeling, we advise adding ANL to the drinking water, thus avoiding excessive physicalmanipulation of the mice. The number of replicates required per experiment varies depending on thescientific question; based on our studies, we estimate that three to six biological replicates per MScondition will be necessary (replicates add accuracy to the results; this is a general premise for anyMS-based study). Animals that do not express the MetRS* gene are always needed as a negativecontrol for the background, e.g., due to non-specific binding of proteins in the enrichment steps. Inour study, the minimum number of neurons used per proteome was 130,000–200,000 for the Pur-kinje proteome12,31, and the entire cerebellum was used as starting material. With this startingmaterial, we obtained ~30–150 ng of purified protein (estimated by MS and using as standard amixture of tryptic peptides from HeLa). As a rule of thumb, for neuronal samples, if GFP expressionfrom the cell-type-specific X-Cre::MetRS* line is not detectable by western immunoblot when 5–10 μgof the tissue lysate is loaded, it is very likely that the technique will not be sensitive enough for theidentification of the specific cell-type proteins by MS. General considerations for the experimentaldesign are given below.

ANL intakeWhen ANL is administered via the drinking water, we recommend an average daily ANLintake of 0.9 ± 0.1 (mean ± s.d.) mg/day/g of body weight for 21 d when aiming to label andidentify neuronal populations of the brain. Lower dosages or shorter labeling times might beused for the labeling of other cell types. When ANL is administered by i.p. injection, we recommendtesting different ANL doses in the range of 4–400 mM. A 400 mM dosage administered by i.p. (i.p.volume: 10 ml/kg) once per day for 1 week is recommended for labeling some neuronalpopulations (such as excitatory neurons of the hippocampus). Lower dosages (e.g., 4 mM) canpotentially be used for labeling other cell types or when longer labeling periods are desired. The idealconcentration of ANL should be determined on a case-by-case basis, depending on the proteinsynthesis rate of the cell type to be labeled, the desired duration of the labeling and the specificexperimental question.

Mouse dietLow-methionine chow (0.1% (wt/wt); see recipe in the Supplementary Methods) can be administeredto the mice. This step is optional, but we recommend it for the labeling of neuronal proteins. Notethat the methionine content of non-defined mice chow is variable. Raising the mice with a defineddiet will provide more reproducible results and avoid potential methionine content variation betweendifferent batches of mouse chow.

Sample sizeAnimal numbers per proteome will largely depend on the abundance of the cell type under study. Inthe case of specific neuronal types, when labeling neurons with an approximate population of130,000–200,000 neurons per brain or more, one brain is enough for protein purification. For theinitial experiments to establish the administration route, and the amount and the duration of ANLadministration, the use of small groups of animals is possible, because the results of regular BONCAT(global detection of labeled proteins using western blot (Steps 1–17); e.g., using a biotin alkyne tagand then an anti-biotin antibody) are generally consistent among duplicates (see Fig. 3 for an exampleof two different ANL administration protocols).

Replicates per proteomeTaking into account the intrinsic variability of in vivo experiments, we recommend that users obtainat least three to four replicates per proteome or condition; it is a general rule in MS that morereplicates lead to enhanced reliability and robustness of the protein identification. If the aim is to findsubtle differences between conditions, we consider a minimum of six replicates to be necessary.Owing to many critical steps in the protocol, we recommend starting with two to three times thenumber of animals as calculated for the desired number of biological replicates.

ControlsWe recommend the following controls: a positive control for click chemistry, for example, any type ofcell labeled with AHA (for a basic in vitro labeling protocol, see ref. 4); and a negative control forbackground, obtained by labeling with ANL a mouse line not expressing MetRS*, using exactly the




same labeling conditions that are used for the X-Cre::MetRS* mouse line under study. We recom-mend using as control line the MetRS* without Cre; the use of wild-type (WT) mice from the samestrain is also possible. We did not observe any differences in the background levels between the afore-mentioned mouse lines. At the end of the protocol, the obtained peptides in the two samples will becompared and will be used as a background proteome.

Materials

Biological materials● X-Cre::MetRS* mouse line (Cre-recombinase-inducible mouse line; Jackson Laboratory, stock no.028071): we recommend the use of homozygote animals for MetRS* expression, if possible; Cre can beexpressed homo- or heterozygotically. ! CAUTION All experiments with animals should be performedin accordance with relevant local guidelines and regulations, especially regarding animal welfare andgenetic engineering. The protocol used for this paper was approved by the local government office (RPDarmstadt; protocols: V54-19c20/15-F122/14 and V54-19c20/15-F126/1012) and was compliant withthe Max Planck Society rules.

ReagentsProtein labeling and click chemistry● Low-methionine chow (ssniff custom, see in the Supplementary Methods)● Water (molecular biology grade, Sigma, cat. no. W4502)● ANL, synthesized as described previously for AHA32, with the following modification: use Boc-L-Lys-OH (N-alpha-tert-butyloxycarbonyl-L-lysine; Iris Biotech, cat. no. BAA1107.0005, CAS no. 13734-28-6) as starting material instead of Boc-Dab (Boc-protected diaminobutyric acid; Iris Biotech, cat. no.1087.0005, CAS no. 25691-37-6) in the same molar amounts. Alternatively, it can be obtained from IrisBiotech (cat. no. HAA1625). Other sources of ANL have not been tested. c CRITICAL ANL purity isvery important for its administration to the mice.

● Maltose (Sigma, cat. no. M9171)● SDS (Sigma, cat. no. 05030) ! CAUTION SDS is toxic upon inhalation; use a lab coat, gloves anda fume hood.

● Triton X-100 (Sigma, cat. no. T9284)● Triazole ligand (Sigma, cat. no. 678937)● Biotin alkyne (Thermo, cat. no. B10185)

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Fig. 3 | Dosage of ANL in drinking water. a, BONCAT signal from the hippocampi of CaMK2a-Cre::R26-MetRS*(MetRS*) mice. Mice were provided with ANL in the drinking water at/for the indicated dose and duration. A strongBONCAT signal was obtained from the mice expressing the mutant MetRS* but not in WT or MetRS* animalsprovided with ANL or water, respectively. b, Quantification of the western blots shown in a. All experiments withanimals were performed with permission from the local government office (RP Darmstadt; protocols: V54-19c20/15-F122/14 and V54-19c20/15-F126/1012) and were compliant with the Max Planck Society rules. Adapted withpermission from Alvarez-Castelao et al.12, Springer Nature.




● Disulfide tag (DST)-alkyne (synthesized as reported in ref. 33, in which it is referred to as probe 20)

c CRITICAL Do not use strong reducing agents when using this alkyne.● Copper (I) bromide (99.999% (wt/wt); Sigma, cat. no. 254185) c CRITICAL Purity is very important;other brands can be used if they are of the same purity. Store desiccated at room temperature (RT:19–21 °C) for up to 1 year.

● DMSO (Sigma, cat. no. 276855)● Complete EDTA-free protease inhibitor (PI; Roche, cat. no. 04693132001) ! CAUTION PI is toxic; use alab coat and gloves. c CRITICAL PI must be EDTA-free.

● Benzonase (Sigma, cat. no. E1014)● PD SpinTrap G-25 columns (GE Healthcare) c CRITICAL Other columns were tested, and aconsiderable amount of protein was lost; other brands can be used, but researchers should double-check for protein loss.

● NeutrAvidin beads (Pierce, cat. no. 29200)● β-Mercaptoethanol (Sigma, cat. no. M6250) c CRITICAL β-Mercaptoethanol is toxic; use a lab coat,gloves and a fume hood.

● SYPRO Ruby stain (Sigma, cat. no. S4942)● Pierce BCA Protein Assay Kit (Thermo, cat. no. 23225) ! CAUTION Materials in the Pierce BCAProtein Assay Kit are toxic to aquatic life.

● N-ethylmaleimide (NEM; Sigma, cat. no. 04259) ! CAUTION NEM is toxic; use a lab coat, gloves and afume hood.

● HCl (VWR, cat. no. 30024.290) ! CAUTION HCl is toxic; use a lab coat, gloves and a fume hood.● NaOH (Fluka, cat. no. 35256) ! CAUTION NaOH is corrosive; use a lab coat, gloves and a fume hood.● Ammonium bicarbonate (Sigma, cat. no. 09830) ! CAUTION Ammonium bicarbonate is toxic; use alab coat, gloves and a fume hood.

● NuPAGE 4–12% Bis-Tris protein gels (Thermo Fisher Scientific, cat. no. NP0322BOX)● NuPAGE MOPS SDS running buffer (Thermo Fisher Scientific, cat. no. NP0001)● NuPAGE LDS sample buffer (Thermo Fisher Scientific, cat. no. NP0007)● NuPAGE sample reducing agent (Thermo Fisher Scientific, cat. no. NP0009)● Novex Tris-glycine transfer buffer (Thermo Fisher Scientific, cat. no. LC3675)● Immobilon-P membrane (PVDF, 0.45 µm; Millipore, cat. no. IPVH00010)● Chicken antibody anti-GFP (Aves, cat. no. 1020)● Polyclonal rabbit anti-biotin antibody (Cell Signaling, cat. no. 5567)● Tris-buffered saline (TBS; Sigma, cat. no. T5912)● Odyssey blocking buffer (in TBS; LI-COR)● Tween 20 (Sigma, cat. no. P1379)

Secondary antibodies for western blot● IRDye 680RD Goat anti-Rabbit IgG (LI-COR)● IRDye 800RD Donkey anti-Chicken IgG (LI-COR)

Mass spectrometry● Dulbecco’s PBS (Thermo, cat. no. 14190-094)● EDTA-free protease inhibitor cocktail (Roche, cat. no. 04693132001) ! CAUTION EDTA-free proteaseinhibitor cocktail is toxic; use a lab coat and gloves.

● Microcon 10-kDa centrifugal filter unit with Ultracel-10 membrane (Merck, cat. no. MRCPRT010)● Urea powder (Sigma, cat. no. U5378)● Water (HiPerSolv Chromanorm; VWR Chemicals, cat. no. 83645.290)● Acetonitrile (ACN; LC–MS grade; Carl Roth, cat. no. AE70.1) ! CAUTION Acetonitrile is toxic; use alab coat, gloves and a fume hood.

● Tris (hydroxymethyl)-aminomethane (CellPure, purity ≥99.9% (wt/wt); Carl Roth, cat. no. 3170)! CAUTION Tris (hydroxymethyl)-aminomethane is toxic; use a lab coat, gloves and a fume hood.! CAUTION Ammonium bicarbonate is toxic; use a lab coat, gloves and a fume hood.

● NaCl (VWR Chemicals, cat. no. 27810.295)● Formic acid (Optima LC–MS grade; Fisher Chemical, cat. no. A117-50) ! CAUTION Formic acid istoxic; use a lab coat, gloves and a fume hood.

● Tris(2-carboxyethyl)phosphine hydrochloride (Thermo Scientific, cat. no. 20490)● Iodoacetamide (IAA; Thermo, cat. no. A39271) ! CAUTION IAA is toxic; use a lab coat and gloves.

c CRITICAL Store IAA in the dark for up to 1 year.




● Endoproteinase rLys-C (Promega, cat. no. V1671)● Trypsin (premium grade, MS approved, from porcine pancreas; Serva, cat. no. 37284.01)● Trifluoroacetic acid (TFA; purity ≥99.9% (vol/vol), for peptide synthesis; Carl Roth, cat. no. P088.1)! CAUTION TFA is toxic; use a lab coat, gloves and a fume hood.

● Acetic acid (glacial, purity 100% (wt/wt); Merck Millipore, cat. no. 1.00063.1000) ! CAUTION Aceticacid toxic; use a lab coat, gloves and a fume hood.

● ZipTip C18 (Merck, cat. no. ZTC18S960)● Dimethylsulfoxide (LC–MS grade; Thermo Scientific, cat. no. 85190)

EquipmentProtein labeling and click chemistry● Rotisserie rotator● Lyophilizer● Heating block/thermal mixer● Manual vortex● Centrifuges● Infrared imaging system (Odyssey; LI-COR)

Protein mass spectrometry● SpeedVac● HPLC system (Dionex, model no. U3000 RSCLnano) coupled to a high-resolution tandem massspectrometer (e.g., Thermo Orbitrap Elite, Q Exactive Plus/HF, FusionLumos or Bruker Impact-II).See ref. 34 for a review

● Reversed-phase column: particle size, 3 µm; C18; length, 20 mm (Acclaim PepMap 100; Thermo FisherScientific, cat. no. 164535)

● Analytical column: particle size, 2 µm; C18; length, 50 cm (Acclaim PepMap RSLC; Thermo FisherScientific, cat. no. 164540)

● Electrospray ionization (ESI) source (nanoFlex; Thermo Fisher Scientific, cat. no. ES071)● Mass spectrometer (Q Exactive Plus; Thermo Fisher Scientific, cat. no. IQLAAEGAAPFALGMBDK)

Software for analyzing large-scale mass-spectrometric data sets● For database search: MaxQuant (http://www.maxquant.org/) or MS Amanda/Sequest as part of theProteome Discoverer package (Thermo Fisher Scientific)

● For data evaluation: Perseus (https://maxquant.net/perseus/) or R project (http://www.r-project.org)

Reagent setup1× PBS1× PBS is prepared with 137 mM sodium chloride, 2.7 mM potassium chloride, 4.3 mM disodiumhydrogen phosphate and 1.4 mM potassium dihydrogen phosphate. Adjust the pH to 7.4 or 7.8 withHCl or NaOH. Filter the solution and store at 4 °C to avoid changes in concentration, for up to3 months. Check and re-adjust the pH before each experiment. c CRITICAL Following this specificrecipe when preparing 1× PBS is recommended; slightly less reactivity during click chemistry can occurwith alternative compositions.

ANL administration by drinking waterPrepare 1% (wt/vol) ANL + 0.7% (wt/vol) maltose dissolved in the usual drinking water of the miceand sterilize by filtration. This solution is stable for several weeks at 4 °C. c CRITICAL Water con-taining ANL should be the only source of drinking water in the cage. Use sterilized water bottles formice. The bottles should be replaced every 2–3 d, including the ANL solution. Mouse body weight andwater intake should be evaluated daily and ANL-containing bottles should be carefully scrutinized forcontamination or clogging due to maltose precipitation.

ANL administration by i.p. injectionsANL can be dissolved up to 400 mM in water, PBS pH 7.4 or NaCl, depending of the concentration ofANL used, to achieve an isotonic solution (osmolarity ≈ 300 and pH 6.5/8). This solution is stable for5–6 weeks at 4 °C.



http://www.maxquant.org/

https://maxquant.net/perseus/

http://www.r-project.org


PBS + PIPBS + PI is 1× PBS supplemented with one tablet of complete EDTA-free PI per 50 ml of PBS.

20% (wt/vol) SDSSDS is diluted to 20% (wt/vol) in molecular biology–grade water. This solution is stable for severalmonths at RT.

Biotin Aalkyne preparationDissolve the biotin alkyne in DMSO up to 5 mM.

20% (vol/vol) Triton X-100Triton X-100 is diluted to 20% (vol/vol) in molecular biology–grade water. This solution is stable forseveral months at RT.

Lysis bufferLysis buffer is 1% (wt/vol) SDS, 1% (vol/vol) Triton X-100, PI (diluted 1:4,000 (vol/vol)), and benzonase(diluted 1:1,000 (vol/vol)) in PBS, pH 7.4, + PI. This solution is stable for several months at RT.

TTBS bufferDilute TBS in water following the manufacturer’s instructions and add Tween 20 up to 0.4% (vol/vol) final.

c CRITICAL Add PI and benzonase immediately before use, according to the manufacturer’s instructions.

Triazole ligand solutionTriazole ligand solution is 200 mM stock solution in DMSO. c CRITICAL Avoid exposure of the solutionto air and water, and use a fresh aliquot of DMSO to prepare the stock solution. Divide the triazole ligandsolution into aliquots of 20 µl and store at −20 °C. This solution is stable for 2–3 months; after melting,always double-check that there is no precipitation. Triazole reactivity is variable among batches.

PD SpinTrap G-25 column exchange bufferPD SpinTrap G-25 column exchange buffer is 0.04% (wt/vol) SDS and 0.06% (vol/vol) Triton X-100in 1× PBS, pH 7.8, + PI. This solution is stable for several months at RT. c CRITICAL Add PIimmediately before use, according to the manufacturer’s instructions.

Ammonium bicarbonate bufferAmmonium bicarbonate is dissolved in molecular biology–grade water to a 50 mM concentration.This solution is stable for 5–6 weeks at RT.

NeutrAvidin-binding bufferNeutrAvidin-binding buffer is 1% (vol/vol) Triton X-100, 0.15% (wt/vol) SDS in 1× PBS, pH 7.4, +PI. This solution is stable for several months at RT. c CRITICAL Add PI immediately before use,according to the manufacturer’s instructions.

NeutrAvidin wash buffer 1NeutrAvidin wash buffer 1 is 1% (vol/vol) Triton X-100, 0.2% (wt/vol) SDS in 1× PBS, pH 7.4, + PI.This solution is stable for several months at RT. c CRITICAL Add PI immediately before use,according to the manufacturer’s instructions.

NeutrAvidin wash buffer 2NeutrAvidin wash buffer 2 is 50 mM ammonium bicarbonate + PI. This solution is stable for5–6 weeks at RT. c CRITICAL Add PI immediately before use, according to the manufacturer’sinstructions.

NeutrAvidin elution bufferNeutrAvidin elution buffer is 5% (vol/vol) β-mercaptoethanol and 0.03% (wt/vol) SDS. Prepare freshbefore use.

IodoacetamideDissolve IAA in molecular biology–grade water up to 0.5 M, immediately before adding it to the samples.




0.1 M Tris–HClTris–HCl, pH 8.5, is diluted to 0.1 M in LC–MS-grade water. This solution is stable for 5–6 weeks atRT. Double-check the pH value before use.

Urea A (UA) bufferUA buffer is 8 M urea dissolved in 0.1 M Tris–HCl, pH 8.5. c CRITICAL Prepare UA buffer freshbefore use.

Urea B (UB) bufferUB buffer is 8 M urea dissolved in 0.1 M Tris–HCl, pH 8.0. c CRITICAL Prepare UB buffer freshbefore use.

TCEP solution for MSDissolve TCEP up to 10 mM in UA buffer. c CRITICAL Prepare TCEP solution fresh before use.

0.5 M NaClNaCl is diluted to 0.5 M in LC–MS grade water. This solution is stable for 5–6 weeks at RT.

Wetting bufferWetting buffer is 100% (vol/vol) ACN. This solution is stable for 5–6 weeks at RT.

Equilibration bufferEquilibration buffer is 0.1% (vol/vol) TFA in LC–MS-grade water. This solution is stable for2–3 weeks at RT.

Elution bufferElution buffer is 90% (vol/vol) ACN and 0.1% (vol/vol) TFA in LC–MS-grade water. This solution isstable for 2–3 weeks at RT.

Loading buffer for MSLoading buffer for MS is 5% (vol/vol) ACN with 0.1% (vol/vol) formic acid in LC–MS-grade water.This solution is stable for 2–3 weeks at RT.

Buffer ABuffer A is 5% (vol/vol) dimethylsulfoxide and 0.1% (vol/vol) formic acid in LC–MS-grade water.This solution is stable for 2–3 weeks at RT.

Buffer BBuffer B is 5% (vol/vol) dimethylsulfoxide, 15% (vol/vol) water, 80% (vol/vol) ACN and 0.08% (vol/vol) formic acid. This solution is stable for 2–3 weeks at RT.

rLys-C solutionReconstitute the powder in the resuspension buffer provided with the kit (aliquots can be stored at−20 °C for up to 6 months).

Trypsin solutionReconstitute the powder in 50 mM acetic acid up to a concentration of 1 µg/µl (aliquots can be storedat −20 °C for up to 6 months).

0.1% (vol/vol) TFA0.1% (vol/vol) TFA is diluted in LC–MS-grade water. Prepare TFA fresh before use.

Procedure

In vivo protein labeling ● Timing days to weeks, depending on the experimental design! CAUTION In many countries, the application of this procedure in live mice will require specialpermission from the responsible animal welfare authorities. Mice might lose some weight during the




first weeks of ANL administration, especially if >1% ANL solution is used, or if the ANL is not pureenough; we recommend that the user request permission for a maximum weight loss of 20% of bodyweight during the experiment. Other than that, we did not observe any major impact of ANLadministration on mouse health.1 (Optional) Protein labeling (Steps 1 and 2). Start feeding the mice with the low-methionine diet

1 week before starting the administration of ANL. This step is optional, but we recommend it forthe labeling of neuronal proteins.? TROUBLESHOOTING

2 Supply X-Cre::MetRS* mice and control mice not expressing the mutant MetRS with drinking watercontaining up to 1% ANL for the desired time period (e.g., 21 d) or by i.p. injection (up to 400 mMANL solution) once or twice per day (morning and night).

c CRITICAL STEP Take into account that both room humidity and chow type will influencethe water intake of the mice. It is important to keep all these parameters constant to obtainreproducible experiments or replicates. Check mouse health and water intake daily accordingto animal welfare regulations. Depending on the source and purity of the ANL, the animalsmay be reluctant to drink ANL–maltose water. ANL purity has less impact when administeredby i.p. injection.? TROUBLESHOOTING

Protein purification ● Timing 8 h3 Tissue collection (Steps 3 and 4). Prepare tubes for the dissected tissue, weigh each of them and place

them on dry ice.4 Sacrifice the mice by an approved method according to regional animal welfare laws (e.g., by CO2 or

cervical dislocation), dissect the tissue pieces of interest, collect them and freeze them in thecorresponding tubes. When necessary, pieces of tissue from several animals can be collected andstored in the same tube. Weigh the tubes again after tissue collection to determine the wet weightof the tissue.

j PAUSE POINT Tissue pieces can be stored at −80 °C for several months.5 Preparation of samples for click chemistry (Steps 5–8). Homogenize the tissue in 12–15× lysis buffer

per milligram of wet tissue, with the help of a manual homogenizer, until the tissue is completelylysed. For example, a piece of tissue with a weight of 20 mg will be resuspended in 240 µl of lysisbuffer (20 × 12 = 240). Add more lysis buffer if the lysate does not appear clear afterhomogenization.

c CRITICAL STEP The suggested tissue/buffer ratio is recommended for brain tissue. Other tissuesmight need different buffer/tissue ratios.

6 Heat the homogenate to 75 °C for 15 min.7 Centrifuge at 10 °C for 15 min at 16,000g and transfer the supernatant to a new tube.8 Measure the protein amount of each sample using a BCA protein assay kit and adjust all samples

to the same protein concentration with lysis buffer. Protein concentration should range between 2and 4 μg/μl.

j PAUSE POINT Tissue lysates can be stored at −80 °C for several months.9 Alkylation (Step 9). Dilute the sample by the addition of 3 volumes of PBS, pH 7.4, + PI. Add 0.5 M

IAA to a final concentration of 20 mM to alkylate the samples. Incubate the samples at RT in thedark for 2 h. Add another 20 mM of IAA and incubate in the dark for 2 additional hours.

c CRITICAL STEP Depending on the tissue of interest, reduction with TCEP before the alkylationstep might be needed; a detailed explanation of how to do this can be found in ref. 35.

c CRITICAL STEP Dissolve the IAA just before adding it to the samples.10 Buffer exchange (Step 10). Equilibrate the PD SpinTrap G-25 columns with PD SpinTrap G-25

column exchange buffer and proceed with the buffer exchange of the samples, accordingly to themanufacturer’s recommendations. Prepare 80-µl aliquots of the eluted samples and freeze them at−80 °C.

c CRITICAL STEP If you use columns different from those described above, make sure that theproteins are not lost during the buffer exchange process. This can be done by performing anotherBCA assay and comparing the results to the values obtained in Step 8. Using the PD SpinTrap G-25columns, we usually recover 80–90% of the protein sample.

j PAUSE POINT Lysates can be stored at −80 °C for several months.




Click chemistry test reaction ● Timing 36 h (two overnight steps)

c CRITICAL We recommend testing a small fraction of the sample to evaluate the labeling success in thespecific tissue and experiment. The aim of Steps 11–17 of the Procedure is to evaluate the labelingefficiency for proteins with the Cre-line of choice and to identify and discard samples from animals withpoor labeling.11 Take 40 μl of the samples from Step 10 and add 80 μl of PBS, pH 7.8, + PI.

c CRITICAL STEP The ratio of lysate/PBS used here needs to be constant for all the samples.12 Thaw aliquots of biotin alkyne and triazole ligand solution. Set up reactions in sets of approximately

six samples. For each set of samples with a reaction volume of, e.g., 120 μl, add in the followingorder and vortex for 20 s after adding each of the reagents: 1.5 μl of triazole ligand solution (stock40 mM), 1.5 μl of biotin alkyne (stock 5 mM) and 1 μl of Cu(I) bromide (stock 10 mg/ml).

c CRITICAL STEP Fresh copper bromide in DMSO suspension must be prepared immediatelybefore adding it to the reaction by pipetting up and down three or four times (not by vortexing).The setup of the reactions has to be done quickly (<2 min per set of six samples).

13 Incubate the samples overnight at 4 °C with constant rotation on a sample mixer witha 360° rotator.

14 Spin down at 16,000g for 5 min at 4 °C. A small pellet should be visible.15 Transfer the supernatant to a new tube and keep at 4 °C.

j PAUSE POINT Samples can be stored at −20 °C until further analysis.16 Western blot analysis (Steps 16–18). Mix 40 μl of the sample from Step 15 with 10 μl of SDS–PAGE

loading buffer according to the manufacturer’s instructions, and boil the samples at 90 °C for10 min.

17 Run SDS–PAGE gels and transfer them to a PVDF membrane, using a traditional wet transfersystem; block the membrane in Odyssey blocking buffer for 30 min at RT and incubate overnight at4 °C with the primary anti-GFP and anti-biotin antibodies (both diluted 1:1,000 (vol/vol) inOdyssey blocking buffer). Wash the blots three times with TTBS buffer and incubate with thesecondary antibodies (diluted 1:5,000 (vol/vol) in Odyssey blocking buffer) for 1 h at RT. Wash theblots three times with TTBS buffer and scan the blots using an infrared imaging system.? TROUBLESHOOTING

18 Select samples from animals with similar labeling efficiency to be prepared for proteomic analysisby MS. For mouse lines with very high ANL labeling (~20× more signal in the X-Cre::MetRS*compared with the control), it is possible to perform the AP with NeutrAvidin beads (Steps 21–24),using a non-cleavable biotin alkyne followed by on-bead digestion before MS. Also direct click, asshown in refs. 36,37, could be tested. For samples with lower labeling, the use of a cleavable biotinalkyne (DST-alkyne) or an alkyne with a specific elution method is mandatory; this step favors thespecific elution of labeled proteins versus proteins non-specifically bound to the beads.

Alkyne dosage optimization ● Timing 2 d, including two overnight steps19 Thaw the necessary aliquots of the selected samples from Step 10 to test several dosages (from 5 to

50 μM) of the alkyne (DST or any other). See an example of alkyne dosage in Fig. 4. For purificationof neuronal proteins, we recommend using ~20 μM of the DST alkyne. Perform Steps 12–17 todetermine the best signal-to-noise ratio by comparing the X-Cre::MetRS* samples to the negativecontrol.

c CRITICAL STEP Do not use strong reducing agents (such as DTT or β-mercaptoethanol) to runthe gels clicked with the DST-alkyne. We recommend loading the samples with 5 mM NEM.

20 Click the remaining samples from Step 10 with the amount of alkyne that yields the best signal-to-noise ratio. Scale up the click reactions (Steps 12–17).

c CRITICAL STEP Small changes in alkyne concentration can greatly change the result of the clickchemistry. Be sure that identical volumes are pipetted after scaling up the reactions.

ANL-labeled protein purification using DST alkyne ● Timing 23 h (one overnight step)21 ANL-labeled protein purification (Steps 21–25). Take the clicked samples and perform a buffer

exchange using the PD SpinTrap G-25 columns with NeutrAvidin-binding buffer (Step 10). Freezea 40-µl aliquot from each sample and keep the rest of the sample on ice.

22 Wash the NeutrAvidin high-capacity beads three times with NeutrAvidin-binding buffer. For thewashes, use 10 volumes of buffer per volume of dry beads. After each wash, resuspend the beadswith a light vortex, and sediment the beads by centrifugation at 3,000g for 5 min at RT.




23 After the last wash, calculate the volume of the dry beads, add the same volume of NeutrAvidin-binding buffer (1:1 (vol/vol)), and carefully resuspend the beads.

24 Mix 100 μg of clicked sample (in an approximate volume of 200 μl) with 4 μl of the beads obtainedin the previous step. Incubate the mixture in a rotator overnight at 4 °C.

c CRITICAL STEP This basic reaction setup might have to be up- or downscaled: the amount ofbeads used per microgram of total protein strongly depends on the amount of labeled proteins permicrogram of total protein.

25 Sediment the beads by centrifugation at 3,000g for 5 min at 4 °C. Transfer the supernatant to a newtube, freeze a 40-µl aliquot from each sample and keep the rest of the sample at −80 °C.

26 Protein elution (Step 26). Wash the beads three times quickly and then three times for 10 min eachin a rotator; perform all the washes first with binding buffer, then with NeutrAvidin Wash buffer 1,next with PBS (pH 7.4) + PI, and finally with NeutrAvidin wash buffer 2 (washes are performed asin Step 22). Elute the clicked proteins from the beads two times, incubating for 30 min at RT in arotator with one bead volume of NeutrAvidin elution buffer each time. Sediment the beads bycentrifugation at 3,000g for 5 min at RT, and combine the two elutions.

27 Sample evaluation (Step 27). Take 1/3 (by volume) of the combined eluate from the NeutrAvidinbeads obtained in the Step 26, run an SDS–PAGE gel and stain it with silver staining or SYPRORuby by following the manufacturer’s instructions. Freeze the rest of the sample. See an example ofeluted samples in Fig. 5a and Supplementary Fig. 1.? TROUBLESHOOTING

j PAUSE POINT Eluted samples can be stored at −80 °C for up to 1 year.28 Sample lyophilization (Step 28). Lyophilize the remaining samples to remove the β-mercaptoethanol,

then resuspend each sample in 40 µl of UA buffer. The amount of time needed for this step willdepend on the volume to be lyophilized.

Sample preparation for mass spectrometry ● Timing 3 d

c CRITICAL Steps 29–35 must be performed at RT (unless otherwise specified):29 Protein alkylation (Steps 29–35). Load the samples onto the filter (Microcon-10) unit and centrifuge

at 18,000g for 15 min at RT, and discard the flow-through from the collection tube.30 Add 100 µl of UA buffer to the filter unit and centrifuge at 18,000g for 15 min at RT and discard the

flow-through from the collection tube.31 Add 100 µl of 10 mM TCEP working solution and incubate at RT for 20 min.32 Centrifuge the samples at 18,000g for 15 min at RT and discard the flow-through from the

collection tube.33 Add 100 µl of 0.05 M IAA solution and mix at 600 r.p.m. in the thermal mixer for 1 min, followed

by incubation for 20 min in the dark without mixing.

KDa

250

50

37

150

100

75

25

Met

RS* W

T

Alkyne (μM)

Anti-biotin

6080 40 30

Met

RS* W

T

Met

RS* W

T

Met

RS* W

T

Fig. 4 | Alkyne dosage testing. Western immunoblot showing that the click chemistry efficiency depends on theamount of alkyne used. WT and Gad2-Cre-MetRS* (MetRS*) mice were provided with 1% (wt/vol) ANLadministered in the drinking water for 21 d, and then the cortex was dissected and clicked with different amounts ofthe biotin alkyne. Labeled proteins were detected by western blot with anti-biotin antibody. All experiments withanimals were performed with permission from the local government office (RP Darmstadt; protocols: V54-19c20/15-F122/14 and V54-19c20/15-F126/1012) and were compliant with the Max Planck Society rules.




34 Centrifuge the filter units at 18,000g for 15 min at RT. Discard the flow-through from the collection tube.35 Add 100 µl of UB buffer to the filter unit and centrifuge at 18,000g for 15 min at RT. Repeat this

step twice. Discard the flow-through from the collection tube. The proteins are now immobilized onthe membrane. The amounts are typically invisible to the eye.

36 Protein digestion (Steps 36–39). Add 40 µl of UB buffer with Lys-C (enzyme/protein ratio = 1:50(wt/wt)) and mix at 600 r.p.m. in the thermal mixer for 1 min, followed by incubation of the filterunits at 37 °C for 20 h without further agitation.

37 Transfer the filter units to new collection tubes, and add 120 µl of 50 mM ammonium bicarbonatebuffer with trypsin solution (enzyme/protein ratio = 1:100 (wt/wt)). Mix at 600 r.p.m. in thethermal mixer for 1 min, followed by incubation of the filter units at 37 °C for 20 h without furtheragitation.

38 Centrifuge the filter units at 18,000g for 15 min at RT; then add 50 µl of 0.5 M NaCl and centrifugethe filter units at 18,000g for 10 min at RT.

39 Add 150 µl of 0.5 M NaCl to the filter and centrifuge at 18,000g for 20 min at RT. Combine theeluate from this step with the eluate from Step 38.

40 Peptide desalting with ZipTips (Steps 40–45). Equilibrate C18 ZipTips by aspirating and dispensing10 µl of wetting buffer up and down three times slowly (3 s per pipetting step, i.e., 3 s up and 3 sdown), followed by pipetting 3 × 10 µl of wetting buffer into waste.

41 Pipette 3 × 10 µl of equilibration buffer into waste (slowly, 3 s per pipetting step).42 Acidify the sample by adding TFA up to a final concentration of 0.1% (vol/vol). Take 10 µl of the

sample and pipette up and down ten times (slowly, 3 s per pipetting step).43 Pipette 3 × 10 µl of 0.1% (vol/vol) TFA into waste (slowly, 2 s per pipetting step).44 Elute the desalted samples with 10 µl of elution buffer by pipetting up and down 10× (slowly, 3 s per

pipetting step) and transfer the eluate to a new tube.45 Evaporate the eluates in a SpeedVac (at RT).

j PAUSE POINT Samples can be stored at −20 °C for several weeks.

LC–MS/MS analysis ● Timing 3.5 h per sample46 Reconstitute the sample in 20 µl of loading buffer before MS measurement.47 Load the reconstituted samples on reversed-phase columns (trapping column: particle size = 3 µm,

C18, length = 20 mm; analytical column: particle size = 2 µm, C18, length = 50 cm) using a nano-HPLC system.

36

BO

NC

ATG

FP

KDa

25

250

50

37

150

100

75

25

20

15

WT

Met

RS* W

T

Met

RS*

Purifiedproteins

12 16 20 24 28 3212

16

20

24

28

32

36

Pro

tein

inte

nsiti

es in

Met

RS

*(lo

g 2 s

cale

)

Protein intensities in WT(log2 scale)

Purkinje neuron proteome(‘unique’ + enriched)

WT1,687 6

GAD2::MetRS*

4

a b c

Enriched proteins

Fig. 5 | Cell-type-specific protein purification. a, Western blot showing the BONCAT reactions of WT and GAD2-Cre::R26-MetRS* from mice supplied with drinking water containing 1% (wt/vol) ANL for 21 d (left panel) and SYPRORuby staining (right panel) of the purified/eluted proteins constituting the Purkinje proteome (MetRS* lane). b, Plotshowing the higher abundance in GAD2-Cre::R26-MetRS* mouse samples compared with unlabeled samples (peptideintensities) of proteins detected in both groups. c, The Purkinje neuron proteome was obtained by the union ofproteins unique to or enriched (more than threefold the levels of WT) in at least two sample replicates from GAD2-Cre::R26-MetRS* mice. All experiments with animals were performed with permission from the local governmentoffice (RP Darmstadt; protocols: V54-19c20/15-F122/14 and V54-19c20/15-F126/1012) and were compliant with theMax Planck Society rules. Adapted with permission from Alvarez-Castelao et al.12, Springer Nature.




MS analysis ● Timing 2 d or more, depending of the number of samples48 Load the samples into the mass spectrometer and elute the peptides in gradients of water-based

buffer A and ACN-based buffer B. Ramp gradients from 4 to 48% buffer B in 178 min at a flow rateof 300 nl/min.

49 Ionize the peptides eluting from the column using an ESI source and analyze them using a ThermoScientific Q Exactive Plus mass spectrometer (or an equivalent high-resolution tandem massspectrometer used for high-throughput proteomics). For LC–MS/MS analysis, run the massspectrometer in a data-dependent mode, using the following parameters:

Parameter Value

Polarity Positive

Full MS

Microscans 1

Resolution 70,000

AGC target 1 × 106 ion counts

Maximum ion time 120 ms

Scan range 350–1,400 m/z

dd-MS2

Microscans 1

Resolution 35,000

AGC target 5 × 105 ion counts

Maximum ion time 120 ms

Loop count 12

Isolation window 4.0 m/z

Isolation offset 0.0 m/z

Scan range 200–2,000 m/z

Fixed first mass None

Normalized collision energy 30

dd Settings

Minimum AGC target 1.2 × 104 ion counts

Charge exclusion Unassigned, 1, ≥6Peptide match Preferred

Exclude isotopes On

Dynamic exclusion 30 s

AGC, automatic gain control.

Bottom-up proteomics analysis ● Timing 2 d or more, depending on the number ofsamples to be measured50 Analyze raw MS datasets using MaxQuant or other computational platforms suitable for processing

high-resolution MS spectra (e.g., Proteome Discoverer). See the ‘Experimental design’ section foranalysis details.

51 Calculate peptide identifications with a false-discovery rate (FDR) ≤0.01 for multiple comparisons,and proteins with one or more unique peptide per protein included for subsequent analyses.

52 Quantify proteins by measuring peptide ion intensity in ≥4 consecutive full scans, using a label-freeapproach (LFQ).

53 Analyze identified proteins and log2 protein abundances using a bioinformatics platform (e.g.,Perseus, R project).

54 Compare proteins purified in the X-Cre::MetRS* samples to the corresponding WT (or othercontrol) samples, and calculate the fold enrichment of each protein by subtracting the X-Cre::MetRS* intensity from the intensity measured in the paired WT (or other control) sample. See anexample of this in Fig. 5b.

55 Consider only proteins detected exclusively in the ANL-treated X-Cre::MetRS* samples orfound to be ≥3 times enriched in the ANL-treated X-Cre::MetRS* sample for further analysis(or significantly enriched using, e.g., a paired t test, P ≤ 0.05). Proteins with no enrichment butquantified in both X-Cre::MetRS* and WT are counted as overlapping proteins. See an example ofthis in Fig. 5c.




56 Analyze differential protein abundance between conditions by a two-sided t test (permutation-based FDR) for two sample groups or by ANOVA (followed by post hoc tests, e.g., Fisher’s leastsignificant difference, P ≤ 0.05) for three or more sample groups. If the aim is to compare twoproteomes or two conditions, the relative intensities and abundance changes between the twoconditions should be analyzed after all data are normalized (mean centering).

57 Visualize data using GO enrichment analyses (e.g., GOrilla, http://cbl-gorilla.cs.technion.ac.il/),segmentation analyses, principal component analyses, heat maps and volcano plots, using, e.g., thePerseus software package and protein network analyses using String (e.g., http://www.string-db.org).

Troubleshooting

Troubleshooting advice can be found in Table 2.

Timing

Steps 1 and 2, protein labeling: days to weeks, experiment dependentSteps 3 and 4, tissue collection: 10 min per mouseSteps 5–8, preparation of samples for click chemistry: 1–2 h, depending on the number of samplesStep 9, alkylation: 4 hStep 10, buffer exchange: 30 min to 1 h, depending on the number of samplesSteps 11–15, click chemistry: 18 h (overnight step)Steps 16–18, western immunoblot: 18 h (overnight step)Steps 19 and 20, alkyne dosage optimization: 2 d (two overnight steps)Steps 21–26, ANL-labeled protein purification: 21 h (overnight step)Step 27, sample evaluation: 90 min

Table 2 | Troubleshooting table

Step Problem Possible reason Solution

1 and 2 Water intake of the mice istoo low (<1.5–2 ml per day)

Low-purity ANL stock Decrease the percentage of ANL in the drinking water

17 Failed click chemistry(no signal in the western blot)

-Too much detergent due toincomplete buffer exchange

-Perform an additional buffer-exchange step (Step 10)-Alternatively precipitate the proteins (using a standardprecipitation method in Wessel and Flugge38)

-Poor quality of the reagents -Prepare fresh Triazole ligand solution and double-check that thecopper (I) bromide used is 99.9% pure

Clicked proteins are not moreabundant in the X-Cre::MetRS*mouse than in the WT mouseanalyzed by western blot

-Too few cells for the labeling -If possible, dissect a region richer in your cell type of interest

-Inefficient click chemistry -See the solutions to the previous problem (‘Failed click chemistry’)

-Labeling levels too low -Revise the protocol of ANL administration to the mice

-Alkylation step was notefficient

-Use fresh dissolved IAA; alkylate for longer periods of time, up to3 h per step (Step 9)-Implement a reduction step with TCEP previous to alkylation(Step 9); follow the steps in Langraf et al.35

27 Purified proteins are not moreabundant in the X-Cre::MetRS*mouse than in the WT mouse

-Inefficient click chemistry -Run the collected aliquots from Steps 21 and 25 to verify properclick chemistry; if failed, see the solutions to the problem forStep 17-Use the dosage of alkyne with the best signal/noise labeling ratio(X-Cre::MetRS*/negative control, Steps 19 and 20) (Fig. 4)

-Excessive non-specific bindingto the beads

-Use the minimum amount of beads possible-Add a pre-clearing step before Step 10, by mixing the lysates withNeutrAvidin beads, incubate overnight at 4 °C, centrifuge thesamples, discard the beads and keep working with thesupernantant-Add a final quick wash with 1% (wt/vol) SDS

-Incubation time with the beadsis too long

-Reduce the incubation time of the samples with the beads

-Too much detergent was usedin the elution step

-Decrease the SDS concentration in the elution buffer

-Insufficient resuspension afterthe lyophilization step

-Gently resuspend the proteins with a pipette



http://cbl-gorilla.cs.technion.ac.il/

http://www.string-db.org


Step 28, sample lyophilization: 2–4 h, depending on the sample volumeSteps 29–35, protein alkylation: 3.5 hSteps 36–39, protein digestion: 2 d (two overnight steps)Steps 40–45, peptide desalting with ZipTips: 30 min per sampleSteps 46–48, LC–MS/MS analysis: 3.5 h per sampleSteps 49–57, MS analysis: 2 d or more, depending on the sample size

b

6

4

2

0

CaMK2-HC Hippocampus Gad2 Cerebellum Glia

× M

ethi

onin

e/10

0 A

As

a

0

5

10

15

20

Fre

quen

cy

25

30

35

1 34 67 100

133

166

199

232

265

298

331

364

397

430

463

496

529

562

595

628

661

694

727

760

793

826

859

892

925

958

991

1,02

41,

057

1,09

01,

123

1,15

61,

189

1,22

21,

255

1,28

81,

321

1,35

41,

387

1,42

01,

453

1,48

61,

519

1,55

21,

585

Hippocampus

Protein length

CaMKII-Cre::R26-MetRS*

Fig. 6 | Analysis of eluted proteins. a, Distribution of frequencies and protein lengths identified in either the wholehippocampal proteome or the CaMK2a-Cre::R26-MetRS* proteome (ANL-labeled and -purified proteins). Thehippocampus proteome is represented by red bars, and light blue bars represent the CaMK2a-Cre::R26-MetRS*proteome. The overlap between the two proteomes is represented by dark blue lines. b, Median methionine contentof purified ANL-labeled or -unlabeled proteomes. Note that the methionine content of the CaMK2a-Cre::R26-MetRS*(CaMK2-HC) and the GAD2-Cre::R26-MetRS* (Gad2) proteomes was not substantially different from those of theunlabeled proteomes from hippocampus, cerebellum or primary cultures of glia. The upper and lower bounds of theboxes indicate the 75 and 25 percentiles of the data, respectively, and the whiskers indicate the 95th (upper) and 5th(lower) percentiles. Camk2-HC: six biological replicates, hippocampus: three biological replicates, Gad2: threebiological replicates, cerebellum: four biological replicates and Glia: three biological replicates. All experiments withanimals were performed with permission from the local government office (RP Darmstadt; protocols: V54-19c20/15-F122/14 and V54-19c20/15-F126/1012) and were compliant with the Max Planck Society rules. Adapted withpermission from Alvarez-Castelao et al.12, Springer Nature.




Anticipated results

After delivering ANL in the drinking water of X-Cre::MetRS* mice for 21 d and using as startingmaterial the entire hippocampus or cerebellum, we identified a total of 533 proteins differentiallyexpressed between excitatory neurons of the hippocampus and inhibitory Purkinje neurons of thecerebellum (including unique proteins and common proteins expressed at different levels). We didnot find any bias in our study toward preferential identification of longer or shorter proteins, or forproteins containing more or less methionine (Fig. 6). It was not possible to consistently identify thepeptides containing ANL ± alkyne in the MS. This could be due to metabolic or chemical conversionof the ANL moiety leading to multiple products that are each only present in small quantities. But wedetected a significantly lower number of peptides containing methionine (P ≤ 0.001), consistent withthe idea that the purified proteins indeed contain ANL (Fig. 7).

One additional quality-control step involves the evaluation of each biological replicate on the basisof MS intensities. For example, the biochemical purification can suffer if the click chemistry is notoptimal, if there is too much non-specific binding or elution in the purification step, or if proteinrecovery after purification is poor (see ‘Troubleshooting’ section). All of these potential problems willresult in poor protein enrichment in the X-Cre::MetRS* sample, with similar MS intensities observedin the experimental and negative control samples (see Supplementary Fig. 2 and compare withFig. 5b,c). In this case, when the enrichment between X-Cre::MetRS* and control samples is notsignificant (using a t test, P > 0.05, or a static cutoff of more than threefold), the replicate can bediscarded. The exact fold-threshold of enrichment depends on the samples and data structure, butthis is a general, important quality-control step.

Reporting SummaryFurther information on research design is available in the Nature Research Reporting Summary.

Data availabilityThe datasets presented in this Protocol were originally generated in ref. 12. All data are available fromthe corresponding author on reasonable request.

Hippo-campus

Camk2-Cre::MetRS*

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Fig. 7 | Methionine content of the eluted peptides. Graph representing lower methionine content of the elutedpeptides in the ANL-labeled Camk2-Cre::R26-MetRS* (21 d) samples compared with peptides detected in no-ANL-labeled hippocampus extracts. These data suggest that most of the purified proteins had incorporated ANL (that isnot identified by MS). a, Percentage of methionine-free peptides identified in non-labeled crude hippocampusextracts and Camk2-Cre::R26-MetRS* proteome. b, Percentage of peptides with 1 and 2 methionines in non-labeledcrude hippocampus extracts and Camk2-Cre::R26-MetRS* proteome. In the graphs, the bars indicate the 99%confidence interval for biological variation. Hippocampus: three biological replicates; Camk2-Cre::MetRS*: sixbiological replicates. ***P ≤ 0.001. All experiments with animals were performed with permission from the localgovernment office (RP Darmstadt; protocols: V54-19c20/15-F122/14 and V54-19c20/15-F126/1012) and werecompliant with the Max Planck Society rules.




References

1. Feist, P. & Hummon, A. B. Proteomic challenges: sample preparation techniques for microgram-quantityprotein analysis from biological samples. Int. J. Mol. Sci. 16, 3537–3563 (2015).

2. McKay, C. S. & Finn, M. G. Click chemistry in complex mixtures: bioorthogonal bioconjugation. Chem. Biol.21, 1075–1101 (2014).

3. Laughlin, S. T., Baskin, J. M., Amacher, S. L. & Bertozzi, C. R. In vivo imaging of membrane-associatedglycans in developing zebrafish. Science 320, 664–667 (2008).

4. Dieterich, D. C., Link, A. J., Graumann, J., Tirrell, D. A. & Schuman, E. M. Selective identification of newlysynthesized proteins in mammalian cells using bioorthogonal noncanonical amino acid tagging (BONCAT).Proc. Natl. Acad. Sci. USA 103, 9482–9487 (2006).

5. Elsasser, S. J., Ernst, R. J., Walker, O. S. & Chin, J. W. Genetic code expansion in stable cell lines enablesencoded chromatin modification. Nat. Methods 13, 158–164 (2016).

6. Ngo, J. T. et al. Cell-selective metabolic labeling of proteins. Nat. Chem. Biol. 5, 715–717 (2009).7. Mahdavi, A. et al. Engineered aminoacyl-tRNA synthetase for cell-selective analysis of mammalian protein

synthesis. J. Am. Chem. Soc. 138, 4278–4281 (2016).8. Yuet, K. P. et al. Cell-specific proteomic analysis in Caenorhabditis elegans. Proc. Natl. Acad. Sci. USA 112,

2705–2710 (2015).9. Link, A. J. et al. Discovery of aminoacyl-tRNA synthetase activity through cell-surface display of non-

canonical amino acids. Proc. Natl. Acad. Sci. USA 103, 10180–10185 (2006).10. de Felipe, P. et al. E unum pluribus: multiple proteins from a self-processing polyprotein. Trends Biotechnol.

24, 68–75 (2006).11. Griffin, R. J. The medicinal chemistry of the azido group. Prog. Med. Chem. 31, 121–232 (1994).12. Alvarez-Castelao, B. et al. Cell-type-specific metabolic labeling of nascent proteomes in vivo. Nat. Biotechnol.

35, 1196–1201 (2017).13. Bennett, E. L., Diamond, M. C., Krech, D. & Rosenzweig, M. R. Chemical and anatomical plasticity brain.

Science 146, 610–619 (1964).14. Dieterich, D. C. et al. In situ visualization and dynamics of newly synthesized proteins in rat hippocampal

neurons. Nat. Neurosci. 13, 897–905 (2010).15. tom Dieck, S. et al. Direct visualization of newly synthesized target proteins in situ. Nat. Methods 12,

411–414 (2015).16. Liu, Y. et al. Application of bio-orthogonal proteome labeling to cell transplantation and heterochronic

parabiosis. Nat. Commun. 8, 643 (2017).17. Liu, Y. et al. Addendum: application of bio-orthogonal proteome labeling to cell transplantation and het-

erochronic parabiosis. Nat. Commun. 9, 1052 (2018).18. Zanivan, S., Krueger, M. & Mann, M. In vivo quantitative proteomics: the SILAC mouse. Methods Mol. Biol.

757, 435–450 (2012).19. Fornasiero, E. F. et al. Precisely measured protein lifetimes in the mouse brain reveal differences across tissues

and subcellular fractions. Nat. Commun. 9, 4230 (2018).20. Gauthier, N. P. et al. Cell-selective labeling using amino acid precursors for proteomic studies of multicellular

environments. Nat. Methods 10, 768–773 (2013).21. Jansens, A. & Braakman, I. Pulse-chase labeling techniques for the analysis of protein maturation and

degradation. Methods Mol. Biol. 232, 133–145 (2003).22. Schmidt, E. K., Clavarino, G., Ceppi, M. & Pierre, P. SUnSET, a nonradioactive method to monitor protein

synthesis. Nat. Methods 6, 275–277 (2009).23. Goodman, C. A. & Hornberger, T. A. Measuring protein synthesis with SUnSET: a valid alternative to

traditional techniques? Exerc. Sport Sci. Rev. 41, 107–115 (2013).24. Starck, S. R., Green, H. M., Alberola-Ila, J. & Roberts, R. W. A general approach to detect protein expression

in vivo using fluorescent puromycin conjugates. Chem. Biol. 11, 999–1008 (2004).25. Marciano, R., Leprivier, G. & Rotblat, B. Puromycin labeling does not allow protein synthesis to be measured

in energy-starved cells. Cell Death Dis. 9, 39 (2018).26. Du, S. et al. Cell type-selective imaging and profiling of newly synthesized proteomes by using puromycin

analogues. Chem. Commun. 53, 8443–8446 (2017).27. Barrett, R. M., Liu, H. W., Jin, H., Goodman, R. H. & Cohen, M. S. Cell-specific profiling of

nascent proteomes using orthogonal enzyme-mediated puromycin incorporation. ACS Chem. Biol. 11,1532–1536 (2016).

28. McShane, E. et al. Kinetic analysis of protein stability reveals age-dependent degradation. Cell 167, 803–815.e21 (2016).

29. McClatchy, D. B. et al. Pulsed azidohomoalanine labeling in mammals (PALM) detects changes in liver-specific LKB1 knockout mice. J. Proteome Res. 14, 4815–4822 (2015).

30. Alvarez-Castelao, B. & Schuman, E. M. The regulation of synaptic protein turnover. J. Biol. Chem. 290,28623–28630 (2015).

31. Woodruff-Pak, D. S. Stereological estimation of Purkinje neuron number in C57BL/6 mice and its relation toassociative learning. Neuroscience 141, 233–243 (2006).

32. Link, A. J., Vink, M. K. & Tirrell, D. A. Preparation of the functionalizable methionine surrogate azidoho-moalanine via copper-catalyzed diazo transfer. Nat. Protoc. 2, 1879–1883 (2007).




33. Szychowski, J. et al. Cleavable biotin probes for labeling of biomolecules via azide-alkyne cycloaddition.J. Am. Chem. Soc. 132, 18351–18360 (2010).

34. Gillet, L. C., Leitner, A. & Aebersold, R. Mass spectrometry applied to bottom-up proteomics: entering thehigh-throughput era for hypothesis testing. Annu. Rev. Anal. Chem. 9, 449–472 (2016).

35. Landgraf, P., Antileo, E. R., Schuman, E. M. & Dieterich, D. C. BONCAT: metabolic labeling, click chemistry,and affinity purification of newly synthesized proteomes. Methods Mol. Biol. 1266, 199–215 (2015).

36. Schanzenbächer, C. T., Langer, J. D. & Schuman, E. M. Time- and polarity-dependent proteomic changesassociated with homeostatic scaling at central synapses. Elife 7, e33322 (2018).

37. Schanzenbächer, C. T., Sambandan, S., Langer, J. D. & Schuman, E. M. Nascent proteome remodelingfollowing homeostatic scaling at hippocampal synapses. Neuron 92, 358–371 (2016).

38. Wessel, D. & Flugge, U. I. A method for the quantitative recovery of protein in dilute solution in the presenceof detergents and lipids. Anal. Biochem. 138, 141–143 (1984).

AcknowledgementsWe thank C. Hanus, C. Glock, S. tom Dieck, A.R. Dörrbaum, I. Bartnik, B. Nassim-Assir, E. Ciirdaeva, A. Mueller, D.C. Dieterich andD.A. Tirrell for their contributions to the Nature Biotechnology paper12. We thank H. Geptin, D. Vogel., N. Fürst, I. Wüllenweber andF. Rupprecht for their excellent technical assistance. We thank E. Noll for the synthesis of ANL and P. Landgraf for the synthesis of theDST alkyne. We thank E. Northrup, S. Zeissler, S. Gil Mast and the animal facility of the MPI for Brain Research for their excellentsupport. Work in the laboratory of E.M.S. was supported by the Max Planck Society, the European Research Council, grants DFG CRC902 and 1080, and the DFG Cluster of Excellence for Macromolecular Complexes; this project received funding from the EuropeanResearch Council (ERC) under the European Union’s Horizon 2020 Research and Innovation Program (grant agreement no. 743216).B.A.-C. was supported by a Marie Curie IEF grant.

Author contributionsB.A.-C. and C.T.S. designed the experiments, and acquired, analyzed and interpreted the data. J.D.L. and E.M.S. designed the experiments,and analyzed and interpreted the data. E.M.S. and B.A.-C. wrote and revised the manuscript. All authors contributed to the writing andrevision of the article.

Competing interestsThe authors declare no competing interests.

Additional informationSupplementary information is available for this paper at https://doi.org/10.1038/s41596-018-0106-6.

Reprints and permissions information is available at www.nature.com/reprints.

Correspondence and requests for materials should be addressed to B.A-C. or E.M.S.

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Related linksKey references using this protocolAlvarez-Castelao, B. et al. Nat. Biotechnol. 35, 1196–1201 (2017): https://www.nature.com/articles/nbt.4016Yuet, K. P. et al. Proc. Natl. Acad. Sci. USA 112, 2705–2710 (2015): http://www.pnas.org/content/112/9/2705Ngo, J. T. et al. Nat. Chem. Biol. 5, 715–717 (2009): https://www.nature.com/articles/nchembio.200



https://doi.org/10.1038/s41596-018-0106-6

http://www.nature.com/reprints

https://www.nature.com/articles/nbt.4016

http://www.pnas.org/content/112/9/2705

https://www.nature.com/articles/nchembio.200


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