Identification of ADP-Ribose Acceptor Sites on In Vitro Modified Proteins by Liquid Chromatography–Tandem Mass Spectrometry

  • Mario Leutert
  • Vera Bilan
  • Peter Gehrig
  • Michael O. Hottiger
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1608)

Abstract

Protein ADP-ribosylation is a covalent, reversible posttranslational modification (PTM) catalyzed by ADP-ribosyltransferases (ARTs). Proteins can be either mono- or poly-ADP-ribosylated under a variety of physiological and pathological conditions. To understand the functional contribution of protein ADP-ribosylation to normal and disease/stress states, modified protein and corresponding ADP-ribose acceptor site identification is crucial. Since ADP-ribosylation is a transient and relatively low abundant PTM, systematic and accurate identification of ADP-ribose acceptor sites has only recently become feasible. This is due to the development of specific ADP-ribosylated protein/peptide enrichment methodologies, as well as technical advances in high-accuracy liquid chromatography–tandem mass spectrometry (LC-MS/MS). The standardized protocol described here allows the identification of ADP-ribose acceptor sites in in vitro ADP-ribosylated proteins and will, thus, contribute to the functional characterization of this important PTM.

Key words

ADP-ribosylation ADP-ribosylome ARTD PARP PARG Mass spectrometry Ti4+-IMAC enrichment Phosphoenrichment 

1 Introduction

Protein ADP-ribosylation is a covalent posttranslational modification (PTM) catalyzed by different ADP-ribosyltransferases (ARTs). These enzymes use nicotinamide adenine dinucleotide (NAD+) as a substrate to transfer the ADP-ribose (ADPr) moiety onto specific amino acid side chains, a process termed protein mono-ADP-ribosylation (MARylation). ARTs can also mediate poly-ADP-ribosylation (PARylation) by transferring the ADPr moiety onto an existing protein-bound ADP-ribose unit. Currently, 22 cellular human ARTs are known. They are subdivided into ARTCs (C for C2/C3 toxin like) and ARTDs (D for diphtheria toxin like, also called PARPs). While ARTCs are membrane-associated or secreted ARTs, human ARTDs form a family of 18 intracellular enzymes with confirmed or putative mono- or poly-ADP-ribosyltransferase activity [1, 2, 3].

Several enzymes that remove ADP-ribose from mono- and poly-ADP-ribosylated substrates have also been identified, rendering ADP-ribosylation a fully reversible PTM. Several mammalian ADP-ribosylhydrolases have been characterized so far, including poly-ADP-ribose glycohydrolase (PARG), ADP-ribosylhydrolase 3 (ARH3), both of which are able to hydrolyze poly-ADP-ribose, and the mono-ADP-ribosylarginine hydrolase 1 (ARH1). In addition, the macrodomain-containing proteins MacroD1, MacroD2, and C6orf130 have recently also been shown to exhibit mono-ADP-ribosylhydrolase activity [4, 5, 6].

To elucidate the functional role of protein ADP-ribosylation, systematical analysis of all ADP-ribosylated proteins and identification of their ADP-ribose acceptor sites are necessary. Comparable to many other PTMs, the fraction of ADP-ribosylated cellular proteins is very low. Thus, studying this group of modified proteins requires specific ADP-ribosylated protein/peptide enrichment methodologies. Mass spectrometry (MS)-based proteomics is probably the most powerful tool for the analysis of PTMs. However, the analysis of this PTM has proven to be very challenging for several reasons, including the highly transient nature and the low abundance of ADP-ribosylated proteins, the special physicochemical properties of the PTM (bulky, highly charged, heterogeneous structure, labile), and the number of different amino acids that were reported to be modified (acidic and basic amino acids with a primary amino group on the side chain) [7]. Characterization of ADP-ribose acceptor sites by MS has significantly improved following the development of high-resolution mass spectrometers and novel fragmentation techniques.

In the past few years, several methods for the identification of ADP-ribose acceptor sites on in vitro and in vivoADP-ribosylated proteins have been published. Zhang et al. [8] established an enrichment protocol based on the isolation of ADP-ribosylated peptides by boronate affinity chromatography and subsequent modified peptide elution using hydroxylamine (NH2OH). This methodology leaves a characteristic mass signature of 15.01 Da at the ADP-ribose acceptor site [8]. A major drawback of this protocol is that the chemical reaction employed here limits the detection of ADP-ribosylated amino acids to glutamates and aspartates only. Other groups have also used boronate affinity enrichment, but in combination with acidic elution, which leaves the ADP-ribose moiety intact and leads to a release of all bound peptides. This, unfortunately, resulted in high background of unmodified peptides. Additionally, hydroxylamine treatment has also been used as a stand-alone procedure without any enrichment, but this method only seems useful for strongly ADP-ribosylated targets [9, 10].

Phosphoproteomic approaches were also found to co-enrich ADP-ribosylated peptides, and protocols have been optimized for the specific enrichment of ADP-ribosylated or phosphoribosylated peptides [11, 12, 13, 14]. Chapman et al. [13] and Daniels et al. [14] used phosphodiesterases to reduce the mono- and poly-ADP-ribosylation modification (MAR or PAR, respectively) to a protein-bound phosphoribose. The resulting phosphoribosylated peptides are subsequently enriched using either Fe(III)-immobilized metal ion affinity chromatography (IMAC) or TiO2 microspheres. The conversion of protein-bound MAR or PAR to phosphoribose leads to a detectable mass signature of 212.01 Da.

The most recent enrichment approach published by Martello et al. [15] makes use of PARG enzymatic treatment to convert in vivo PARylated peptides into MARylated peptides, which are subsequently enriched by the ADP-ribose binding protein Af1521 and described in the Chap. 11 of this book. This technique allows the accurate and reproducible identification of ADP-ribose acceptor sites in vivo. However, this enrichment strategy has so far not been tested or optimized for in vitro modified proteins.

Here, we thus describe an updated protocol using a Ti4+-IMAC enrichment based on the work done by Chapman et al. [13] and Daniels et al. [14] to map ADP-ribose acceptor sites on in vitroADP-ribosylated proteins. This protocol is more readily applicable to a variety of different samples than the boronate affinity chromatography-based protocol described by our group in the previous edition of this book for in vitro modified proteins [9]. More importantly, this new methodology is not biased against specific ADP-ribose acceptor sites. The problem that phosphorylated peptides might co-enrich with phosphoribosylated/ADP-ribosylated peptides and interfere with the sensitivity of the detection is not an obstacle due to the low complexity of in vitro modified samples.

2 Materials

All solutions prepared with type 1 analytical grade water.

2.1 ADP-Ribosylation Assay and PARG Treatment

  1. 1.

    hARTD1 is expressed and purified from insect cells as carboxyl-terminal His-tagged protein and stored in liquid nitrogen.

     
  2. 2.

    hPARG is expressed and purified from insect cells as carboxyl-terminal His-tagged protein and stored in liquid nitrogen (seeNote1).

     
  3. 3.

    10 mM β-Nicotinamide adenine dinucleotide (NAD+) (>99% Sigma–Aldrich as hydrate) stored at −80 °C.

     
  4. 4.

    10 mM PJ-34 (≥98% Sigma-Aldrich as hydrochloride hydrate) stored at −20 °C.

     
  5. 5.

    ADP-ribosylation buffer (always prepare freshly): 50 mM Tris–HCl pH 8.0, 4 mM MgCl2, 250 μM DTT, and cOmplete™ EDTA-free Protease Inhibitor Cocktail.

     

2.2 FASPTrypsin Digestion

  1. 1.

    5× disulfide bond reduction buffer: 250 mM DTT, 250 mM Tris–HCl pH 8.2, and 5 M urea.

     
  2. 2.

    0.5 ml Microcon 30 kDa centrifugal filter units with Ultracel-30 membranes (Millipore, MRCF0R030).

     
  3. 3.

    Urea buffer: 8 M urea in 50 mM Tris–HCl pH 8.2.

     
  4. 4.

    Iodoacetamide solution: 0.05 M iodoacetamide in urea buffer (kept protected from light).

     
  5. 5.

    0.5 M NaCl.

     
  6. 6.

    0.05 M ammonium bicarbonate (prepare freshly).

     
  7. 7.

    Sequencing grade modified trypsin (Promega).

     

2.3 ADP-Ribosylated Peptide Enrichment

  1. 1.

    MagReSyn® Ti4+-IMAC from ReSyn Biosciences.

     
  2. 2.

    70% ethanol.

     
  3. 3.

    Loading Buffer: 1 M glycolic acid in 80% acetonitrile.

     
  4. 4.

    Wash buffer: 80% acetonitrile and 0.1% acetic acid in H2O.

     
  5. 5.

    Elution buffer: 50 mM Tris–HCl pH 8, 10 mM diammonium hydrogen phosphate, and 5% acetonitrile.

     

2.4 Stage Tip Desalting

  1. 1.

    C18 Empore high-performance extraction disks (3 M).

     
  2. 2.

    100% methanol.

     
  3. 3.

    Stage Tip Solution A: 0.5% acetic acid in H2O.

     
  4. 4.

    Stage Tip Solution B: 80% acetonitrile and 0.5% acetic acid in H2O.

     
  5. 5.

    Stage Tip Elution Solution: 60% acetonitrile and 0.5% acetic acid in H2O.

     

2.5 Mass Spectrometry

  1. 1.

    HPLC solvent A: H2O containing 0.1% formic acid.

     
  2. 2.

    HPLC solvent B: Acetonitrile containing 0.1% formic acid.

     
  3. 3.

    Frit column (inner diameter 75 μm, length 15 cm) packed with reversed phase material (C18-AQ, particle size 1.9 μm, pore size 120 Å, Dr. Maisch GmbH, Germany).

     
  4. 4.

    Instrumentation: Orbitrap Fusion Tribrid mass spectrometer (Thermo Scientific, San Jose, CA), connected to an Easy-nLC 1000 HPLC system (Thermo Scientific) (seeNote2).

     

3 Methods

3.1 Overview of the Protocol

In vitro auto- and especially trans-ADP-ribosylation reactions are often not efficient and lead to a low abundance of ADP-ribosylated proteins. We, therefore, recommend ADP-ribosylated peptide enrichment following a previously described phosphoenrichment technique that is based on immobilized titanium ion affinity chromatography [16]. This protocol facilitates modified peptide enrichment and increases the possibilities of a successful analysis, including ADP-ribosylation site determination. In vitro reactions should be carried out according to the optimized protocols for the different ADP-ribosyltransferases. The original protocol used phosphodiesterases to reduce mono- and poly-ADP-ribosylation modifications (MAR or PAR, respectively) to a protein-bound phosphoribose and to enrich subsequently phosphoribosylated peptides. We have, however, found that treatment of ADP-ribosylated proteins with poly(ADP-ribose) glycohydrolase (PARG), which reduces the complexity of PAR to protein-bound mono-ADP-ribose, works very reliable and allows efficient ADP-ribose enrichment as well. PARG-treated proteins are further digested with trypsin using filter-aided sample preparation (FASP) protocol [17]. The ADP-ribosylated peptides are finally enriched with magnetic microspheres with chelated Ti4+ ions. This protocol is optimized to use only very mild buffers for the binding, washing, and peptide elution steps in order to preserve the ADP-ribose and its linkage to the modified amino acid residue. Samples are desalted using a C18 Stage Tip protocol [18] and analyzed on an Orbitrap Fusion Tribrid mass spectrometer. HCD fragmentation has previously been shown to lead to reproducible identification of ADP-ribosylated peptides, and this method allows the accurate identification of the modified amino acid [19]. Mascot searches are performed to identify ADP-ribose acceptor sites by setting ADP-ribosylation as a variable modification for lysine, arginine, glutamate, and aspartate. A representative, annotated spectrum of an identified ADP-ribosylated ARTD1 peptide after the Ti4+ IMAC enrichment and the HCD ADP-ribose fragmentation pattern are shown in Fig. 1.
Fig. 1

HCD Fragmentation of an ADP-ribosylated peptide. (a) Representative annotated spectrum for the ARTD1 peptide VGHSIRHPDVEVDGFSELR that was found to be ADP-ribosylated on E76. ADP-ribose fragmentation ions are shown in red. (b) Nomenclature of ADP-ribose fragments as described by Hengel et al. [20]. The ADP-ribose fragment ions with strong signals in the HCD MS/MS spectra are shown

3.2 ADP-Ribosylation Assay and PARG Treatment

  1. 1.

    For in vitro auto-ADP-ribosylation of ARTD1, incubate 20 pmol hARTD1 in the presence of 10 pmol annealed double-stranded oligomer (5-GGAATTCC-3) and 100 nM NAD+ in ADP-ribosylation buffer (seeNote3). Reaction volume: 50 μl, reaction conditions: 15 min, 30 °C. Terminate the reactions by adding PJ-34 (ADP-ribosylation inhibitor) to a final concentration of 10 μM. To generate larger amounts of modified target protein, several reactions can be run in parallel (seeNote4).

     
  2. 2.

    To reduce the complexity of PAR and generate MARylated proteins, samples are incubated with 5 pmol hPARG. Adjust the MgCl2 and NaCl buffer concentrations to 10 mM and 50 mM, respectively, and incubate for 1 h at 37 °C.

     
  3. 3.

    For the identification of ADP-ribose acceptor sites in peptides, proceed immediately with the FASPtrypsin digestion, or, alternatively, freeze the proteins at −20 °C.

     

3.3 FASPTrypsin Digestion

  1. 1.

    Add 5× reduction buffer to the protein sample to achieve 1×, and incubate at 37 °C for 30 min to reduce the disulfide bonds.

     
  2. 2.

    Load up to 250 μl of reduced sample onto the Microcon-30 kDa centrifugal filter unit. Centrifuge at 14,000 × g for ~15–20 min at room temperature (RT). Repeat until the sample is completely loaded onto the filter.

     
  3. 3.

    Add 200 μl of urea buffer to the filter unit. Centrifuge at 14,000 × g for ~20 min at RT.

     
  4. 4.

    Add 100 μl iodoacetamide solution to the filter unit. Gently shake for 5 min and centrifuge at 14,000 × g for ~15–20 min at RT.

     
  5. 5.

    Add 100 μl of urea buffer to the filter unit. Centrifuge at 14,000 × g for ~ 15 min at RT. Repeat step twice.

     
  6. 6.

    Add 100 μl of 0.5 M NaCl to the filter unit. Centrifuge at 14,000 × g for ~15–20 min at RT. Repeat step once.

     
  7. 7.

    Add 100 μl of ammonium bicarbonate solution to the filter unit. Centrifuge at 14,000 × g for ~15–20 min at RT. Repeat step twice.

     
  8. 8.

    Transfer the filter units to new collection tubes.

     
  9. 9.

    Add 120 μl of trypsin (1:25 trypsin to protein), dissolved in ammonium bicarbonate solution, to the filter unit, and gently shake for 1 min.

     
  10. 10.

    Incubate the filter units at RT overnight in a humidity chamber.

     
  11. 11.

    The next day, centrifuge the filter units at 14,000 × g for ~15–20 min. The flow-through contains the digested proteins.

     
  12. 12.

    Re-elute the column with 80 μl of ammonium bicarbonate solution.

     
  13. 13.

    Dry the eluted peptides in a vacuum concentrator (seeNote5).

     

3.4 Enrichment of ADP-ribosylated Peptides

  1. 1.

    Thoroughly resuspend MagReSyn® Ti4+-IMAC microspheres to ensure homogeneous suspension.

     
  2. 2.

    Transfer 25 μl (0.5 mg) MagReSyn® Ti4+-IMAC to a 2 ml microcentrifuge tube.

     
  3. 3.

    Place the tube on a magnetic separator, allow 10 s for the microspheres to clear, and discard the storage solution.

     
  4. 4.

    Wash the microspheres, with gentle agitation, in 200 μl of 70% ethanol for 5 min.

     
  5. 5.

    Place the tube on the magnetic separator, and allow the microspheres to clear. Discard the ethanol solution.

     
  6. 6.

    Repeat steps 4 and 5.

     
  7. 7.

    Add 50 μl loading buffer to microspheres, and let stand for 60 s to equilibrate.

     
  8. 8.

    Place the tube on the magnetic separator and allow the microspheres to clear. Remove the loading buffer. Important: The microspheres equilibration step should be performed immediately before sample loading.

     
  9. 9.

    Repeat the equilibration process two additional times.

     
  10. 10.

    Mix dried protein digests with 100 μl of loading buffer, incubate for 5min at RT and add mixture to the equilibrated microsphere pellet (seeNote6).

     
  11. 11.

    Incubate at room temperature for 30 min with continuously shaking to ensure adequate sample and microsphere interaction.

     
  12. 12.

    Place the tube on the magnetic separator and allow the microspheres to clear. Discard the coupling supernatant.

     
  13. 13.

    Remove unbound sample by washing microspheres with 100 μl loading buffer for 30 s with gentle agitation.

     
  14. 14.

    Place the tube on a magnetic separator, and allow 10 s for the microspheres to clear. Remove the supernatant.

     
  15. 15.

    Remove nonspecifically bound peptides by resuspending the microspheres in 100 μl wash buffer for 2 min with gentle agitation.

     
  16. 16.

    Place the tube on a magnetic separator, and allow 10 s for the microspheres to clear and remove the supernatant.

     
  17. 17.

    Repeat steps 15 and 16 twice for a total of three washes.

     
  18. 18.

    Elute the bound peptides from the microspheres by adding 60 μl elution buffer and letting stand for 15 min. Ensure that the microspheres remain in suspension by gently agitating the tube.

     
  19. 19.

    Place the tube on the magnetic separator and allow the microspheres to clear. Remove the eluate and transfer it to a new tube.

     
  20. 20.

    Repeat the elution steps 18 and 19 twice for a final elution volume of 180 μl.

     

3.5 Stage Tip Desalting

  1. 1.

    Prepare Stage Tips by plugging two C18 disks in a 200 μl pipette tip (seeNote7).

     
  2. 2.

    Make a hole in the lid of a 1.5 ml Eppendorf tube and fit stage tip in. The tip should be tightly attached to the lid, and the tip should not touch the bottom of the tube.

     
  3. 3.

    Activate the Stage Tip by adding 200 μl 100% methanol to the stage tip, and centrifuge at 1000 × g for approx. 3 min.

     
  4. 4.

    Add 200 μl Stage Tip Solution B to the stage tip, and centrifuge at 1000 × g for approx. 3 min.

     
  5. 5.

    Add 200 μl Stage Tip Solution A to the stage tip, and centrifuge at 1000 × g for approx. 3 min.

     
  6. 6.

    Add 200 μl of your peptide sample, and centrifuge at 1000 × g for approx. 3 min. Repeat step until the whole sample is loaded.

     
  7. 7.

    Wash Stage Tip by adding 50 μl of Stage Tip Solution A, and centrifuge at 1000 × g for approx. 3 min until Stage Tip is completely dry.

     
  8. 8.

    Elute Stage Tip by adding 20 μl of Stage Tip Elution solution, and centrifuge at 1000 × g for approx. 1 min. Repeat elution step once more and combine elutions.

     
  9. 9.

    Partially dry the eluted samples in a vacuum concentrator (seeNote5). The samples can be stored at −20 °C or directly proceed by LC-MS/MS.

     

3.6 LC-MS/MS

All data are acquired on an Orbitrap Fusion Tribrid mass spectrometer connected to an Easy-nLC 1000 HPLC system (seeNote2). 4 μl of peptide sample in 0.1% formic acid are loaded and separated at a flow rate of 300 nl per min. The following LC gradient was applied: 0 min: 2% HPLC solvent B, 60 min: 30% B, 70 min: 97% B, and 80 min: 97% B.

Survey scans were recorded in the Orbitrap mass analyzer in the range of m/z 350–1800, with a resolution of 120,000 and a maximum injection time of 50 ms. Higher-energy collisional dissociation (HCD) spectra were acquired in the Orbitrap mass analyzer. A maximum injection time of 240 ms, an AGC target value of 5e5, and a resolution of 120,000 were used. The precursor ion isolation width was set to m/z 2.0, and the normalized collision energy was 35%. Charge state screening was enabled, and charge states 2–5 were included. The threshold for signal intensities was 5e4, and precursor masses already selected for MS/MS acquisition were excluded for further selection during 30 s.

3.7 Database Analysis and Configuration of Mascot Modifications

MS data are analyzed as previously described [19]. MS and MS/MS spectra are converted into Mascot generic format (mgf) using Proteome Discoverer, v2.1 (Thermo Fisher Scientific, Bremen, Germany). All high-resolution HCD MS/MS spectra are deconvoluted using MS Spectrum Processor, v0.9 [21]. Searches were performed against the UniProtKB human database (taxonomy 9606, version 20140422), which includes 35′787 Swiss-Prot, 37′02 TrEMBL entries, 73′589 reversed sequences, and 260 common contaminants. Mascot 2.5.1 (Matrix Science) is used for peptide identification using the following search settings: singly charged b and y ion series, immonium ions, and water and ammonia loss ion series are searched. Enzyme specificity is set to trypsin, allowing up to four missed cleavages. The ADP-ribose variable modification is set to a mass shift of 541.0611, with scoring of the neutral losses equal to 347.0631 and 249.0862. The marker ions at m/z 428.0372, 348.0709, 250.0940, 136.0623 are ignored for scoring. Lysine, arginine, and glutamic and aspartic acid are set as variable ADP-ribose acceptor sites. Peptides are considered correctly identified when a Mascot score >20, and an expectation value <0.05 are obtained. To assess the location of the ADP-ribose acceptor sites, we use the site localization analysis provided by Mascot, which is based on the work by Savitski et al. [21] and was developed especially for phosphorylation. Due to the lack of a better estimate, we define correctness as having a confidence of ≧95% in the Mascot site localization analysis (seeNote8).

4 Notes

  1. 1.

    As an alternative to PARG treatment, enzymes converting ADP-ribose to phosphoribose (e.g., nudix hydrolases, snake venom phosphodiesterase I) can be used, but these require individually optimized reaction conditions and are expensive in the case of snake venom phosphodiesterase I [14, 22]. None of the available methods to date are capable of distinguishing between mono- and poly-ADP-ribose acceptor sites. We envision that a specific set of binding proteins with affinities for either PAR or MAR or conversion of PAR in to a specific moiety could solve this problem in the near future.

     
  2. 2.

    We measured all our samples on an Orbitrap Fusion Tribrid mass spectrometer, but it is also possible to conduct a similar analysis on other mass spectrometers with optimized machine settings.

     
  3. 3.

    Higher NAD+ concentrations can trigger the generation of very long ADP-ribose polymers that might interfere with trans-ADP-ribosylation or subsequent analysis.

     
  4. 4.

    We started our analysis with 50 μg ARTD1 and ended up with enough material for nine mass spectrometryinjections. The amount of initial starting protein and the peptide solution that is injected into the mass spectrometer need to be optimized depending on the efficiency of the ADP-ribosylation reaction, the HPLC, and the mass spectrometer used for the analysis.

     
  5. 5.

    Partial drying of the peptides (leave 1–2 μl) increases the overall yield.

     
  6. 6.

    To control for the enrichment and MS analysis, standard phosphopeptides can be added into the sample prior to sample preparation.

     
  7. 7.

    Video tutorial describing how to build and use the stage tips [18]: https://www.biochem.mpg.de/226863/Tutorials.

     
  8. 8.

    This method is not optimized for ADP-ribosyl modifications due to the lack of standard peptides with known modification sites. For this reason, even if Mascot states a correctness of 95% for the site localization, this value is arbitrary and cannot be validated experimentally. If required, other amino acid acceptor sites can be included as variable modification sites.

     

Notes

Acknowledgments

The authors would like to thank Paolo Nanni (member of the Functional Genomics Center Zurich, University of Zurich/ETH Zurich, Zurich, Switzerland) for advice and technical assistance. We also thank Felix R. Althaus (Institute of Pharmacology and Toxicology, University of Zurich-Vetsuisse) for providing hPARG-expressing baculovirus. Stephan Christen and Deena Leslie Pedrioli (both University of Zurich) provided editorial assistance and critical input during the writing. Work on ADP-ribosyltransferases in the laboratory of M.O.H is supported by Kanton of Zurich and the Swiss National Science Foundation (310030_157019).

Mario Leutert and Vera Bilan contributed equally to this chapter.

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Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Mario Leutert
    • 1
    • 2
  • Vera Bilan
    • 1
    • 2
  • Peter Gehrig
    • 3
  • Michael O. Hottiger
    • 1
  1. 1.Department of Molecular Mechanisms of DiseaseUniversity of ZurichZurichSwitzerland
  2. 2.Molecular Life Science PhD Program of the Life Science Zurich Graduate SchoolUniversity of Zurich/ETH ZurichZurichSwitzerland
  3. 3.Functional Genomics Center ZurichUniversity of Zurich/ETH ZurichZurichSwitzerland

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