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Sep 2020
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Thermal Proteome Profiling to Identify Protein-ligand Interactions in the Apicomplexan Parasite Toxoplasma gondii
弓形虫热蛋白质组分析以识别顶复体寄生虫中的蛋白质-配体相互作用   

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Abstract

Toxoplasma gondii is a single-celled eukaryotic parasite that chronically infects a quarter of the global population. In recent years, phenotypic screens have identified compounds that block parasite replication. Unraveling the pathways and molecular mechanisms perturbed by such compounds requires target deconvolution. In parasites, such deconvolution has been achieved via chemogenomic approaches—for example, directed evolution followed by whole-genome sequencing or genome-wide knockout screens. As a proteomic alternative that directly probes the physical interaction between compound and protein, thermal proteome profiling (TPP), also known as the cellular thermal shift assay (CETSA), recently emerged as a method to identify small molecule–target interactions in living cells and cell extracts in a variety of organisms, including unicellular eukaryotic pathogens. Ligand binding induces a thermal stability shift—stabilizing or destabilizing proteins that change conformationally in response to the ligand—that can be measured by mass spectrometry (MS). Cells are incubated with different concentrations of ligand and heated, causing thermal denaturation of proteins. The soluble protein is extracted and quantified with multiplexed, quantitative MS, resulting in thousands of thermal denaturation profiles. Proteins engaging the ligand can be identified by their compound-dependent thermal shift. The protocol provided here can be used to identify ligand-target interactions and assess the impact of environmental or genetic perturbations on the thermal stability of the proteome in T. gondii and other eukaryotic pathogens.


Graphic abstract:



Thermal proteome profiling for target identification in the apicomplexan parasite T. gondii.


Keywords: Thermal proteome profiling (热蛋白组分析), CETSA (细胞热迁移实验), Toxoplasma (弓形虫), Parasite (寄生虫 ), Proteomics (蛋白质组学)

Background

Target deconvolution is a major challenge for the wealth of compounds identified through phenotypic screening. Chemogenomic approaches, such as directed evolution or drug screens, have been the favored tools for target identification in eukaryotic parasites (Paquet et al., 2017; Cowell et al., 2018; Luth et al., 2018; Rosenberg et al., 2019; Harding et al., 2020). Such approaches require culturing parasites and host cells under compound treatment for extended periods and often identify pathways indirectly affected by a small molecule rather than the target itself. In contrast, several proteomic methods developed in the past decade directly identify interactions between compounds and protein targets (McClure and Williams, 2018; Conway et al., 2021). For example, enrichment of interacting proteins can be performed with derivatized compounds for affinity-purification followed by mass spectrometry (MS). However, these approaches require specialized chemistry and introduce a linker and other chemical groups to the compound of interest, which may affect its behavior.


Thermal proteome profiling (TPP), also known as the cellular thermal shift assay (CETSA), offers a label-free approach that can be performed in a variety of formats that preserve cellular physiology, including in situ (Dai et al., 2019; Mateus et al., 2020b). Interactions with a target are identified by a compound-dependent shift in the protein’s thermal profile. Cells or cell extracts are treated with the compound and heated to induce thermal denaturation. Aggregated proteins are removed, and soluble proteins are quantified by MS to generate melting curves for each protein. TPP has recently identified the targets of antiparasitic compounds in the apicomplexan parasites Plasmodium falciparum (Dziekan et al., 2019; Lu et al., 2020) and Toxoplasma gondii (Herneisen et al., 2020), as well as in the trypanosome Leishmania donovani (Corpas-Lopez et al., 2019).


The application of TPP extends beyond target deconvolution (Becher et al., 2018; Dai et al., 2018 and 2019; Sridharan et al., 2019; Mateus et al., 2020b). Alterations to protein state and stability may arise from conformational changes, post-translational modifications, altered localization, and interactions with other proteins and biomolecules such as metabolites and nucleic acids. For example, we performed TPP on parasites lacking mitochondrial DegP2 to identify proteins with altered stability based on the loss of this protease (Harding et al., 2020). Genetic perturbations in conjunction with functional proteome profiling are in the early stages (Mateus et al., 2020a) and may be especially well-suited to map the unannotated parts of parasite proteomes.


While TPP has been performed predominantly in mammalian systems, it is expanding to other organisms (Corpas-Lopez et al., 2019; Dziekan et al., 2019; Volkening et al., 2019; Lu et al., 2020; Herneisen et al., 2020; Harding et al., 2020; Jarzab et al., 2020). We believe this approach pairs particularly well with the study of eukaryotic parasites, whose evolutionary divergence complicates identifying molecular pathways by genomic annotation or bioinformatic analysis. For that reason, we provide a detailed protocol describing our thermal profiling pipeline developed for the organism T. gondii. Below, we identify key considerations for selecting a TPP workflow appropriate for the researcher’s biological question. Step-by-step guidelines follow.


Types of experiment

In this protocol, we stratify steps by choice of material and treatment. TPP can be performed on live parasites or parasite lysates and by melting samples over a range of 10 temperatures (“temperature range”) or over a range of 10 compound concentrations melted at a single temperature (“concentration range”). These variations give rise to four permutations described in the Procedure as (B) Lysate Temperature Range Experiment, (C) Parasite Temperature Range Experiment, (D) Lysate Concentration Range Experiment, and (E) Parasite Concentration Range Experiment. Each experiment has advantages and disadvantages (Franken et al., 2015; Dai et al., 2019; Mateus et al., 2020b). For example, experiments using live cells are more physiological but combine direct and indirect effects. Lysate experiments may more directly identify ligand-protein interactions, but loss of cellular compartmentalization can also lead to non-physiological interactions. Concentration range experiments yield more information-rich thermal profiles, but real interactions may be missed if the thermal challenge temperatures are suboptimal (too low or too high) and the overall coverage of the proteome is reduced due to global denaturation.


Treatment conditions

We have performed thermal profiling experiments on extracellular parasites to avoid the added complexity of the host proteome and confounding effects from compound permeability and host metabolism. Compound treatments are often performed on intracellular parasites; however, the appropriate concentration of compound for the thermal profiling experiment should be determined by assays using extracellular parasites. The thermal profiling experiment should mimic assay conditions as closely as possible. Considerations include the amount of time needed for the compound to arrive at diffusion and binding equilibria, the buffer in which the equilibration takes place, and equilibration temperature (e.g., room temperature vs. 37°C). Mammalian studies have often performed the incubation in PBS (Reinhard et al., 2015; Savitski et al., 2014; Franken et al., 2015). We have used a buffer composition resembling the ionic makeup of the host cytosol (Herneisen et al., 2020; Harding et al., 2020). Buffers should lack serum, which would overwhelm parasite signals that can be quantified by MS.


We aim to process 25 µg of protein per reference sample, which in our experience corresponds to the material from 1 × 107 extracellular parasites of the type I RH strain. We subject concentration-range samples to at least two different thermal challenge temperatures; we have found 54°C and 58°C to work well while still providing sufficient coverage of the proteome. The thermal challenge temperatures may need to be optimized for each experiment; further commentary is provided in the Data analysis section.


Lysis conditions

The final lysis buffer composition should contain 0.5-1% IGEPAL CA-630 (also known as NP-40), which provides a balance between solubilizing membrane proteins without re-solubilizing aggregated proteins (Reinhard et al., 2015). For most experiments, the lysis buffer should contain protease inhibitors (and optionally phosphatase inhibitors, depending on the focus of the experiment) and benzonase for digestion of nucleic acids prior to the SP3 cleanup. If the compound of interest is thought to affect proteases, phosphatases, or nucleic acid binding activity, then these supplements should be omitted until after the Separation of Soluble and Aggregated Protein. Our lysis buffers have had an ionic composition similar to PBS (Herneisen et al., 2020) and an intracellular-like buffer (Harding et al., 2020), depending on the application. The ionic composition of the buffer (e.g., presence of ATP and metabolites) can substantially influence the melting behavior of proteins (Lim et al., 2018; Sridharan et al., 2019). The concentration of parasite lysate also influences melting behavior; therefore, it is crucial to count the number of parasites prior to lysis and use a consistent lysis buffer volume for the number of parasites. Following harvest, parasites should be resuspended at least once in a wash buffer that is similar in composition to the lysis buffer (but lacking detergents) to dilute cell culture contaminants, such as serum proteins.

Materials and Reagents

  1. T12.5 flask (e.g., Corning Falcon Tissue Culture Flasks, catalog number: 29185-298)

  2. T175 flask (e.g., CELLSTAR® Filter Cap Cell Culture Flasks, catalog number: 82050-872)

  3. 15-cm dish (e.g., Corning Falcon® Tissue Culture Dishes, catalog number: 25383-103)

  4. Corning® 150 ml Bottle Top Vacuum Filter, 0.22 µm Pore 13.6 cm2 CA Membrane (Corning, catalog number: 430624)

  5. 50 ml conical tube (Corning, catalog number: 430829)

  6. Human foreskin fibroblast (HFF) cells (ATCC, catalog number: SCRC-1041)

  7. T. gondii cell lines (RH, e.g., ATCC 50838 or PRA-319)

  8. T. gondii filter (Whatman Pop-Top and Swin-Lok Plastic Filter Holders for 47 mm membrane filter size, e.g., VWR catalog number: 28163-089, with GE Healthcare Whatman Nuclepore Hydrophilic Membrane 3 or 5 µm circles, catalog number: 111112 or 111113)

  9. Cell scraper (Corning® Small Cell Scraper, catalog number: 3010)

  10. Protein low-bind tube (e.g., EppendorfTM LoBind Microcentrifuge Tubes, 1.5 ml Thermo Fisher Scientific, catalog number: 13698794)

  11. 8-strip PCR tubes (e.g., Genesee Scientific, catalog number: 27.125 U)

  12. Thickwall polycarbonate open-top ultracentrifuge tubes (0.2 ml, 7 × 20 mm; Beckman Coulter, catalog number: 343775)

  13. Protein low-bind 96-well plate (Eppendorf, catalog number: 951032905)

  14. Syringes 20 ml (BD Biosciences, catalog number: 302830)

  15. Hydrophobic Sera-Mag Speed Beads (GE Healthcare, catalog number: 65152105050250, ~50 mg/ml, keep at 4°C until use)

  16. Hydrophilic Sera-Mag Speed Beads (GE Healthcare, catalog number: 45152105050250, ~50 mg/ml, keep at 4°C until use)

  17. DMEM (Thermo Fisher Scientific, catalog number: 11965118, keep at 4°C until use)

  18. Newborn Calf Serum USA origin, heat Inactivated, sterile-filtered, suitable for cell culture (Sigma-Aldrich, catalog number: N4762-500ML, keep at -80°C until use)

  19. 10 mg/ml gentamicin (Life Technologies, catalog number: 15710072, room temperature)

  20. 200 mM L-glutamine (Life Technologies, catalog number: 25030081, keep at -20°C until use)

  21. 250 U/µl benzonase (Sigma-Aldrich, catalog number: E1014-25KU, store at -20°C)

  22. 100× Halt Protease Inhibitor Cocktail (Life Technologies, catalog number: 87786)

  23. IGEPAL® CA-630 viscous liquid (Sigma-Aldrich, catalog number: I3021-50ML)

  24. 10× PBS suitable for tissue culture (e.g., VWR, catalog number: 45001-130)

  25. DC Protein Assay (Bio-Rad, catalog number: 5000116)

  26. Tris(2-carboxyethyl)phosphine (TCEP; Pierce, catalog number: 20490; keep at -20°C until use)

  27. Methyl methanethiosulfonate (MMTS; Thermo Fisher Scientific, catalog number: 23011, keep at 4°C)

  28. Ethyl alcohol, Pure 200 proof, HPLC/spectrophotometric grade (Sigma-Aldrich, catalog number: 459828-1L)

  29. Sequencing-grade trypsin (e.g., Promega, catalog number: V5113, keep at -80°C until use)

  30. Triethylammonium bicarbonate buffer 1.0 M, pH 8.5 (Sigma-Aldrich, catalog number: T7408-100ML, keep at 4°C)

  31. Pierce Quantitative Fluorometric Peptide Assay (Thermo Fisher Scientific, catalog number: 23290, keep at 4°C until use)

  32. TMT10plex Isobaric Label Reagent Set (Thermo Fisher Scientific, catalog number: 90110, keep at -20°C until use)

  33. 50% hydroxylamine (Thermo Fisher Scientific, catalog number: 90115)

  34. Pierce high pH fractionation kit (Thermo Fisher Scientific, catalog number: 84868, keep at 4°C until use)

  35. Ultra-high-performance liquid chromatography (UPLC)-MS acetonitrile (Thermo Fisher Scientific, catalog number: A9561)

  36. UHPLC-MS water (Thermo Fisher Scientific, catalog number: W81)

  37. Pierce Formic Acid, LC-MS Grade (Thermo Fisher Scientific, catalog number: 28905)

  38. DMEM + 3% CFS (see Recipes)

  39. PBS (see Recipes)

  40. 10% IGEPAL CA-630 (also known as NP-40) (see Recipes)

  41. 10× CETSA buffer (see Recipes)

  42. CETSA wash buffer (see Recipes)

  43. CETSA lysis buffer (see Recipes)

  44. 1 M TCEP stock solution (see Recipes)

  45. 200 mM MMTS stock solution (see Recipes)

  46. Buffer A (see Recipes)

  47. Buffer B (see Recipes)

Equipment

  1. CO2 incubator (Thermo Fisher Scientific Forma Steri-Cycle 370, catalog number: 370)

  2. Clinical benchtop centrifuge (Eppendorf, model: Centrifuge 5810R, catalog number: 022625101)

  3. Microcentrifuge (Eppendorf, model: Centrifuge 5424R [discontinued], alternatives include Centrifuge 5425/5425 R)

  4. Minicentrifuge (VWR Galaxy Mini Centrifuge, catalog number: 37000-700)

  5. Hemocytometer (VWR Counting Chamber, catalog number: 1517O-173)

  6. Thermal cyclers (Bio-Rad C1000 TouchTM Thermal Cycler with Dual 48/48 Fast Reaction Module, catalog number: 1851148 and Bio-Rad C1000 TouchTM Thermal Cycler with 96-Deep Well Reaction Module, catalog number: 1851197)

  7. Benchtop ultracentrifuge (Beckman Ultra MAX [discontinued], alternatives include the Optima MAX-XP and Optima MAX-TL)

  8. Thermo mixer (Eppendorf, model: ThermoMixer C, catalog number: 5382000023 with 1.5 ml SmartBlock, catalog number: 5360000038)

  9. Magnetic stand (Invitrogen Dynamag 2, catalog number: 12321D)

  10. Vacuum centrifuge (SavantTM Universal SpeedVacTM Vacuum System, catalog number: SPD111V and, catalog number: UV5450)

  11. Lyophilizer (Labconco FreeZone Triad Freeze Dryer, catalog number: 794001030)

  12. Orbitrap mass spectrometer (Thermo Fisher Scientific Q Exactive HFX [discontinued] or Exploris 480, catalog number: BRE725533) with optional FAIMS Pro Interface (Thermo Fisher Scientific, catalog number: FMS02-10001)

  13. MS-coupled LC system (Thermo Fisher Scientific EASY-nLC 1200, catalog number: LC140) with Acclaim PepMap 100 75 µm × 2 µm nanoViper trapping column (Thermo Fisher Scientific, catalog number: 164946) and PepMap RSLC C18 3 µm, 100A, 75 µm × 15 cm analytical column (Thermo Fisher Scientific, catalog number: ES900)

  14. Pierce formic acid, LC-MS grade (Life Technologies, catalog number: 28905)

  15. UPLC-MS acetonitrile (Thermo Fisher Scientific, catalog number: A9561)

  16. UPLC-MS water (Thermo Fisher Scientific, catalog number: W81)

Software

  1. Proteome Discoverer, version 2.4 (Thermo Fisher Scientific)

  2. R, version 4.0 or later: https://cran.r-project.org/

  3. Tidyverse package, version 1.3: https://cran.r-project.org/web/packages/tidyverse/index.html

  4. TPP package, release 3.12: https://bioconductor.org/packages/TPP/

Procedure

This protocol assumes readers are familiar with T. gondii parasite and host cell propagation. For standard reviews, see Roos et al. (1994) and Jacot et al. (2020).


  1. Parasite harvest

    1. Infect T175 flasks or 15-cm dishes with confluent HFFs with 2 × 107-5 × 107 RH tachyzoites each, which is equivalent to parasites from one fully lysed T12.5 flask, 40-48 h before the assay. Enough T175’s should be infected to harvest 4 × 108 parasites for the assay. The yield may vary depending on host cell age, parasite strain, and treatment; in our experience, 3-4 15-cm dishes are usually sufficient to achieve this number of parasites.

    2. When the parasites have fully lysed from the monolayer, scrape the flask and collect the media containing extracellular parasites. A fully lysed monolayer contains an abundance of extracellular parasites and few remaining attached host cells. Remove host cell debris by passing the media through a 3 µm filter into one 50 ml conical vial per flask or dish.

    3. Concentrate the parasite solution by centrifuging the conicals at 1,000 × g for 10 min at room temperature in a centrifuge with swinging bucket rotors. Discard the supernatant. Resuspend the parasite pellet in 1 ml of wash buffer (lysis buffer without detergents, inhibitors, or enzymes) and transfer the parasite suspension to a 1.5 ml protein low-bind tube.

    4. Create a 1:500 dilution of the parasite suspension and count using a hemocytometer.

    5. Centrifuge the parasites at 1,000 × g for 10 min at room temperature. Discard the supernatant.

    6. Depending on the desired treatment, proceed to section (B) Lysate Temperature Range Experiment, (C) Parasite Temperature Range Experiment, (D) Lysate Concentration Range Experiment, or (E) Parasite Concentration Range Experiment.


  2. Lysate temperature range experiment

    1. Parasite lysis

      1. Resuspend the parasite suspension in 100 µl lysis buffer per 2 × 107 parasites (see lysis considerations in the Background). Sufficient parasites (4 × 108) should be harvested for at least 1.1 ml of lysate, with a small amount of excess to account for pipetting error in the steps below.

      2. Allow lysis to proceed on ice for 15 min with occasional mixing by pipetting.

    2. Compound treatment

      1. Prepare a compound dilution in the lysis buffer at 2× the desired final concentration and a vehicle solution with an equivalent amount of DMSO (or appropriate vehicle). Aliquot 550 µl of each solution into a 1.5 ml protein low-bind tube.

      2. Combine 550 µl of parasite lysate with 550 µl of the 2× compound or vehicle solution and gently pipette to mix. The compound is now at the desired final concentration.

      3. Aliquot 100 µl of the parasite suspension with vehicle or compound into ten labeled PCR tubes corresponding to the anticipated melting temperatures (see below).

      4. Allow the solution to equilibrate at room temperature or at 37°C for at least 5 min (see treatment considerations in the Background).

    3. Thermal challenge

      1. Briefly collect the liquid in the bottom of the tubes using a mini-centrifuge with PCR tube adapter. Centrifuge for approximately 3 s.

      2. Place the PCR tubes in the appropriate orientation on the thermal cycler, such that the tubes with lysate match the desired temperature.

        Note: The thermal cycler program should be started in advance so that the wells are at temperature when the tubes are added; the precise sequence depends on the temperature gradient that can be achieved by the user’s thermal cycler. We have used melting temperatures of 37°C, 41°C, 43°C, 47°C, 50°C, 53°C, 56°C, 59°C, 63°C, and 67°C split across two PCR strip tubes in 48-well thermal cyclers.

      3. Allow denaturation to occur for 3 min.

      4. Quickly remove the tubes from the thermal cycler and place on ice for 5 min.

      5. Briefly collect evaporated liquid in the bottom of the tubes by using a mini-centrifuge with PCR tube adapter. Centrifuge for approximately 3 s.

      6. Proceed to the step described below, F. Separation of Soluble and Aggregated Protein.


  3. Parasite temperature range experiment

    1. Compound treatment

      1. Prepare a compound solution at 2× the desired final concentration and a vehicle solution with an equivalent amount of DMSO (or appropriate vehicle). Aliquot 550 µl of each solution into a 1.5 ml protein low-bind tube.

      2. Combine 550 µl of parasite suspension with 550 µl of the 2× compound or vehicle solution and gently but thoroughly pipette to mix. The compound is now at the desired final concentration.

      3. Aliquot 100 µl of the parasite suspension with vehicle or compound into ten labeled PCR tubes corresponding to the anticipated melting temperatures (see below).

      4. Allow the compound to equilibrate with the parasites at room temperature or at 37°C for at least 5 min (see treatment considerations in the Background).

    2. Thermal challenge

      1. Briefly collect evaporated liquid in the bottom of the tubes by using a mini-centrifuge with PCR tube adapter. Centrifuge for approximately 3 s.

      2. Place the PCR tubes in the appropriate orientation on the thermal cycler, such that the tubes with lysate match the desired temperature.

        Note: The thermal cycler program should be started in advance so that the wells are at temperature when the tubes are added; the precise sequence depends on the temperature gradient that can be achieved by the thermal cycler. We have used melting temperatures of 37°C, 41°C, 43°C, 47°C, 50°C, 53°C, 56°C, 59°C, 63°C, and 67°C split across two PCR strip tubes and 48-well thermal cyclers.

      3. Allow denaturation to occur for 3 min.

      4. Quickly remove the tubes from the thermal cycler and place on ice for 5 min.

    3. Parasite lysis

      1. Briefly collect evaporated liquid in the tubes by using a mini-centrifuge with PCR tube adapter. Centrifuge for approximately 3 s.

      2. Add 20 µl of 6× lysis buffer to each tube and gently pipette to mix (see treatment considerations in the Background). Allow the parasites to lyse on ice for at least 15 min.

      3. Proceed to the step described below, F. Separation of Soluble and Aggregated Protein.


  4. Lysate concentration range experiment

    1. Parasite lysis

      1. Resuspend the parasites suspension in 100 µl lysis buffer per 2 × 107 parasites (see lysis considerations in the Background). Sufficient parasites (4 × 108) should be harvested for at least 1.1 ml of lysate, with a small amount of excess to account for pipetting error in the steps below.

      2. Allow lysis to proceed on ice for 15 min with occasional mixing by pipetting.

    2. Compound treatment

      1. Prepare a dilution series of ten concentrations of the compound, including vehicle alone, at 2× the desired final concentration in lysis buffer. Aliquot 110 µl of the 2× compound solution into a PCR tube.

        Note: We advise ensuring that the same concentration of vehicle is maintained across all samples by preparing the dilution series into a lysis buffer containing a vehicle concentration equal to that of the highest compound concentration.

      2. Aliquot 110 µl of the parasite lysate into the PCR tubes containing 2× compound solution. The compound is now at the final desired concentration, and the volume in each tube is 220 µl.

      3. Use a multichannel pipette to gently mix the lysate and transfer half the volume (110 µl) to another set of PCR tubes. There are now two sets of 10 tubes with 110 µl per tube.

      4. Allow the solution to equilibrate at room temperature or at 37°C for at least 5 min (see treatment considerations in the Background).

    3. Thermal challenge

      1. Briefly collect the liquid in the bottom of the tubes by using a mini-centrifuge with PCR tube adapter. Centrifuge for approximately 3 s.

      2. To induce thermal denaturation, place one set of tubes on a thermal cycler pre-warmed to 54°C and the other set of tubes in a deep-well thermal cycler pre-warmed to 58°C (see considerations in the Background).

        Note: The thermal cycler program should be started in advance so that the wells are at temperature when the tubes are added.

      3. Allow denaturation to occur for 3 min.

      4. Quickly remove the tubes from the thermal cycler and place on ice for 5 min.

      5. Proceed to the step described below, F. Separation of Soluble and Aggregated Protein.


  5. Parasite concentration range experiment

    1. Compound treatment

      1. Prepare a concentration range of ten compound solutions, including vehicle, at 2× the desired final concentration in lysis buffer. Aliquot 110 µl of the 2× compound solution into a PCR tube.

        Note: We advise ensuring that the same concentration of vehicle is maintained across all samples by preparing the dilution series into a lysis buffer containing a vehicle concentration equal to that of the highest compound concentration.

      2. Aliquot 110 µl of the parasite suspension into the PCR tubes containing 2× compound solution. The compound is now at the final desired concentration, and the volume in each tube is 220 µl.

      3. Use a multichannel pipette to gently mix the parasite suspension and transfer half the volume (110 µl) to another set of PCR tubes. There are now two sets of 10 tubes with 110 µl of parasites in compound solution.

      4. Allow the compound to equilibrate with the parasites at room temperature or at 37°C for at least 5 min (see treatment considerations in the Background).

    2. Thermal challenge

      1. Briefly collect the liquid in the tubes by using a mini-centrifuge with PCR tube adapter. Centrifuge for approximately 3 s.

      2. To induce thermal denaturation, place one set of tubes on a thermal cycler pre-warmed to 54°C and the other set of tubes in a deep-well thermal cycler pre-warmed to 58°C (see considerations in the Background).

        Note: The thermal cycler program should be started in advance so that the wells are at temperature when the tubes are added.

      3. Allow denaturation to occur for 3 min.

      4. Quickly remove the tubes from the thermal cycler and place on ice for 5 min.

    3. Parasite lysis

      1. Briefly collect evaporated liquid in the tubes by using a mini-centrifuge with PCR tube adapter. Centrifuge for approximately 3 s.

      2. Add 20 µl of 6× lysis buffer to each tube and gently pipette to mix (see treatment considerations in the Background). Allow the parasites to lyse on ice for at least 15 min.

      3. Proceed to the step described below, F. Separation of Soluble and Aggregated Protein.


  6. Separation of soluble and aggregated proteins

    Below, we describe the two separation methods we have used for isolation of soluble proteins. For more information, see Note 1.

    1. Ultracentrifugation method

      1. Transfer the heat-challenged lysates (a volume of approximately 100 µl) to ultracentrifuge tubes pre-chilled on a bed of ice.

        Note: The minimum volume of these tubes is 100 µl. Using lower volumes risks unbalancing the centrifuge rotor.

      2. Load the tubes into a pre-chilled TLA-100 rotor in a benchtop ultracentrifuge (e.g., Beckman Ultra MAX) chilled to 4°C. The TLA-100 rotor can fit up to 20 tubes, which is enough for the two treatment conditions of a temperature-range experiment or two challenge temperatures of a concentration-range experiment. The tubes must be appropriately balanced to avoid damage to the rotor and ultracentrifuge.

      3. Centrifuge the samples at 100,000 × g for 20 min at 4°C using an ultracentrifuge. To calculate the appropriate rpm, use the rotor radius specifications and an online calculator such as https://www.beckman.com/centrifuges/rotors/calculator.

      4. Gently remove the rotor, taking care not to disturb the tubes, and immediately transfer the tubes to ice. If available, work in a cold room.

      5. Remove the top ~80% by volume of the supernatant and transfer to a pre-chilled protein low-bind tube. It is critical not to disrupt the pellet, which contains aggregated proteins and the membranous fraction.

      6. Proceed to the next section, F. Protein Cleanup and digestion with the SP3 protocol.

    2. Filter plate method

      1. Pre-wet the filter plate with 100 µl of 1× lysis buffer (with compound/treatment, if applicable). Place the filter plate on top of a 96-well plate. Centrifuge at 500 × g in a swinging-bucket centrifuge for 5 min, until the solution passes through the filter and into the 96-well plate. Discard the solution.

      2. Place the filter plate over a clean protein low-bind or polypropylene 96-well plate. Transfer the heat challenged lysates (~100 µl) to the equilibrated filter plate and centrifuge at 500 × g for 5 min at 4°C to separate the soluble protein from aggregates. Soluble proteins pass through the filter into the 96-well plate.

      3. Transfer the soluble fraction from the 96-well plate to protein low-bind tubes. The volume of the soluble protein solution is reduced relative to the input volume and should be measured prior to the next step, G. Protein Cleanup and Digestion.


  7. Protein cleanup and digestion with the SP3 protocol

    1. Quantify protein abundance

      1. Determine the protein concentration in the reference sample (37°C for temperature-range experiments and the lowest compound concentration for concentration-range experiments) using a protein quantification assay, e.g., the DC Protein Assay (Bio-Rad), according to the manufacturer’s instructions. Diluent solutions should contain the compound of interest or vehicle, if applicable, as it may substantially alter absorbance readings. The amount of protein determined in this step will be used to calculate the amount of SP3 beads to use for sample cleanup and trypsin to add for digestion. We typically quantify 20-60 µg of soluble protein in the reference sample. The following steps assume a yield of 50 µg in the reference sample; adjust volumes accordingly for lower amounts of protein.

      2. Transfer a volume corresponding to 50 µg of protein in the reference sample to a new protein low-bind tube. Transfer the same volume of the remaining samples to protein low-bind tubes as well. Raise the volume to 100 µl with lysis buffer.

    2. Reduce cysteines

      1. Add 0.5 µl of a 1 M TCEP solution to each sample. The concentration of TCEP is now 5 mM.

      2. Incubate the samples at 55°C for 10 min, e.g., on a heat block or thermomixer.

    3. Alkylate cysteines

      Remove the tubes from 55°C and allow them to cool to room temperature. Add 7.54 µl of a 200 mM MMTS stock solution to bring the concentration to 15 mM. Allow the reaction to occur for 10 min at room temperature. Note: alternative protocols alkylate with iodoacetamide (IAA) in the dark. We prefer MMTS for in-solution digests due to its rapid reaction rate, stability, and lower non-specific alkylation (Müller and Winter, 2017), which can increase the number of peptide identifications following MS analysis. The choice of alkylating agent will determine search modification on cysteine, i.e., methylthio (+45.988 Da) for MMTS or carbamidomethyl (+57.021 Da) for IAA.

    4. Clean up samples using the SP3 protocol (Hughes et al., 2019). For more information, see Note 2.

      1. Prepare enough hydrophobic and hydrophilic Sera-Mag beads at 50 µg/µl for a 1:10 bead/protein (wt/wt) ratio relative to the reference sample. For example, to process 10 samples with a 50 µg reference sample, prepare 5 mg of beads.

        1. In a 1.5 ml tube, combine 50 µl of the 50 mg/ml hydrophobic beads with 50 µl of the 50 mg/ml hydrophilic beads.

        2. Place the beads on a magnetic rack and allow them to separate. Use a P200 pipette to remove and discard the supernatant.

        3. Wash the beads in 100 µl MS-grade water. Place the beads on a magnetic rack and again discard the supernatant.

        4. Resuspend the beads in 100 µl of MS-grade water for a final concentration of 50 µg/µl.

      2. Add 10 µl of 50 µg/µl beads to each sample. The bead/protein (wt/wt) ratio is now at least 10:1.

      3. Bind the proteins to the beads by adding a 4× volume of 100% HPLC-grade ethanol. For example, to the combined volume of 100 µl sample with 0.5 µl TCEP, 7.54 µl MMTS, and 10 µl Sera-Mag beads, add 472 µl 100% ethanol. Note: the protein solution is now 80% ethanol by volume. We found this proportion to be optimal for binding of T. gondii proteins to the Sera-Mag beads.

      4. Allow the proteins to aggregate with the beads by placing the tubes in a thermomixer and shaking at 1,000 rpm at 24°C for at least 10 min. The beads should “clump” upon binding protein.

      5. Place the tubes on a magnetic rack and allow the beads to separate, which takes approximately 30 s. Discard the supernatant into a waste stream that is appropriate for 80% ethanol.

      6. Wash the beads three times with 180 µl 80% ethanol, which can be prepared by diluting HPLC-grade ethanol with HPLC-grade water. Each time, allow the beads to magnetically separate from the solution for 30 s and dispose of the supernatant into an appropriate waste stream.

        Note: In the final rinse, remove as much of the ethanol wash solution as possible to minimize carryover during the enzymatic digestion step. We remove nearly all of the liquid by centrifuging the beads at 16,000 × g for 30 s and double-stacking a P200 and P10 tip to remove the supernatant.

    5. Digest proteins into peptides

      1. Prepare a trypsin digest solution in 50 mM TEAB at a 1:50 (wt/wt) protein:trypsin ratio. Prepare enough stock solution for the number of samples to be processed, e.g., 20 samples for a temperature range experiment with a control and treatment condition and with 10 melting temperatures each.

      2. Add 35 µl of digest solution to each tube. Gently move the beads into the liquid with the tip of a pipette, but avoid pipetting the beads, as they are sticky.

      3. Place the tubes in a thermo mixer warmed to 37°C and shake at 1,000 rpm overnight (16-18 h).

      4. Centrifuge the tubes at 16,000 × g for 1 min at room temperature to pellet the beads and collect evaporated liquid.

      5. Place the tubes on a magnetic rack and allow the beads to separate for 30 s. Transfer the aqueous supernatant, which contains the digested peptides, to a new protein low-bind tube.


    After the peptides have been eluted, samples can be snap-frozen in liquid nitrogen and dried in a lyophilizer with a condenser temperature of -80°C and chamber pressure of approximately 0 mbar. The peptides are typically lyophilized to a powder in four hours or fewer. The lyophilized peptides can be stored at -80°C for several months.


  8. Tandem mass tag labeling

    Sample multiplexing is performed with isobaric mass tags, which are commercially available in 10-plex and 16-plex format (Werner et al., 2014; Li et al., 2020). We keep working stocks of TMT 10-plex reagents at concentrations of 6.66 µg/µl (100 µg per 15 µl) in acetonitrile at -80°C for 3-6 months. We perform labeling at 2:1 (wt/wt) TMT:peptide (Zecha et al., 2019).


    A TMT labeling scheme should be selected in advance of labeling. Each temperature or concentration is labeled with one TMT channel. Two full 10-plex labeling reactions are performed per experiment: the 10 melting temperatures with vehicle and compound for temperature-range experiments or the 10 compound concentrations melted at two temperatures for concentration-range experiments. We have observed reporter ion interference when labeling sequentially (Brenes et al., 2019). Therefore, the labeling schemes shown in Figure 1 are recommended.



    Figure 1. Recommended TMT labeling strategy for temperature- and concentration-range experiments. t1/c1 refers to the lowest temperature or concentration in the experiment.


    1. Quantify peptide abundance with the Pierce fluorometric peptide assay

      Before starting, quantify the number of peptides in the reference sample (37°C for temperature range experiments and the lowest compound concentration for concentration range experiments) using the Pierce Fluorometric Peptide Assay according to manufacturer’s instructions. If peptides have been lyophilized, resuspend in 35 µl 50 mM TEAB pH 8.5. A 1:20 dilution is often sufficient to place the sample within the range of the standard curve, e.g., 0.5 µl of sample + 9.5 µl water. For a standard whole-proteome TMT reaction, use 25-50 µg of peptides in 35 µl of 50 mM TEAB, diluting the sample as necessary. Use equivalent volumes and dilutions of the non-reference samples. The steps below are written for samples containing 50 µg of peptides in 35 µl.

    2. TMT labeling reaction

      1. Equilibrate the TMT reagents at room temperature for 3 min.

        Note: Record the reagent lot number and isotopic corrections for the batch. This information may be used to create a custom quantification method in Proteome Discoverer that corrects for isotopic impurities arising from natural carbon isotopes.

      2. Centrifuge the TMT reagents at 13,000 × g for 1 min and resuspend each vial in 120 µl of 100% MS-grade acetonitrile. Create 15 µl aliquots and store at -80°C for up to 6 months.

        Note: If resuspended TMT reagents will not be used for extended periods of time, lyophilize the reagents and store as a powder at -20°C.

      3. Add 15 µl of TMT reagent (100 µg) to the reference sample (50 µg protein in 35 µl 50 mM TEAB). If working with more or less peptide input, maintain the final vol/vol ratio of acetonitrile (TMT reagents) to aqueous buffer (TEAB buffer). For example, if labeling only 25 µg of peptides in 35 µl of TEAB, add 50 µg of TMT reagent in 7.5 µl and 7.5 µl of 100% acetonitrile to bring the final composition to 30% vol/vol acetonitrile.

      4. Centrifuge the tubes at 13,000 × g for 30 s to collect the liquid.

      5. Place the tubes in the thermomixer and shake at 400 rpm for 60 min at room temperature.

      6. Quench unreacted TMT reagent by adding 3.2 µl of 5% hydroxylamine per 50 µl reaction. Place the tubes in the ThermoMixer and shake at 400 rpm for 15 min at room temperature.

      7. Combine the samples in a 1.5 ml protein low-bind tube. Use the same pipette tip for all transfers to avoid losing peptides due to contact with new surfaces. The volume should now be approximately 530 µl.

      8. Flash-freeze the pooled sample in liquid nitrogen and lyophilize until dry. Note: sample volume may alternatively be reduced via vacuum centrifugation.

      9. Dry samples may be stored at -80°C for several months.

    3. Desalting and fractionation

      TMT-labeled samples should be fractionated prior to MS data acquisition to reduce isolation interference during MS analysis. We perform high pH reversed-phase peptide fractionation using HPLC (e.g., with Shimadzu LC-20AD; see Herneisen et al., 2020) or the Pierce High pH Reversed-Phase Peptide Fractionation Kit according to manufacturer’s instructions, which we have found provide equivalent coverage of the T. gondii proteome and also function as a desalting step. We pool samples into eight fractions for LC-MS. The fractions can be lyophilized and stored at -80°C indefinitely.


  9. MS data acquisition

    Data acquisition methods are highly dependent on facilities. At a minimum, TMT-labeled samples should be acquired using sufficient resolution to resolve the reporter ions and with a long gradient to separate the complex peptide mixtures and reduce co-isolation interference. Here, we describe the data acquisition protocol for our Exploris 480 orbitrap with FAIMS Pro interface coupled to an Easy-nLC 1200 system.

    1. Sample resuspension and injection

      1. Resuspend each lyophilized fraction in Buffer A to an estimated concentration of 0.5-1 µg peptides/µl. We typically resuspend each sample in 25 µl. Ensure that the lyophilizate is completely solubilized; it may help to thoroughly wash the sides of the tube and collect the liquid by centrifuging at 16,000 × g for 1 min.

      2. Transfer each resuspended fraction to an autosampler tube. Once the samples are resuspended, they should be kept at 4°C.

      3. Inject 0.5-1 µg of peptides for MS analysis (typically 1-2 µl). Samples belonging to the same TMT labeling experiment can be injected sequentially (i.e., the set of fractions). We perform a blank injection between different TMT labeling experiments to reduce carryover.

    2. LC gradient

      Our samples are separated over a 90-min gradient described in Table 1. The gradient includes an optional 12-minute seesaw for column maintenance. Our LC system includes a commercial trapping column (Acclaim PepMap 100 75 µm × 2 µm nanoViper) connected to a 15 cm commercial analytical column (PepMap RSLC C18 3 µm, 100A, 75 µm × 15 cm).


      Table 1. LC gradient used for TMT10-labeled T. gondii proteome

      Time Duration %B
      00:00 00:00 1
      01:00 01:00 6
      42:30 41:30 21
      63:15 20:45 36
      73:30 10:15 50
      74:00 00:30 100
      88:00 14:00 100
      91:00 03:00 2
      94:00 03:00 2
      97:00 03:00 98
      100:00 03:00 98


    3. MS acquisition settings

      Method parameters for the orbitrap Exploris 480 with FAIMS Pro interface are summarized in Table 2. In our experience, alternating between compensation voltages of -50 and -65 yielded best coverage of the T. gondii proteome. The ddMS2 resolution of 30,000 has been optimized for the TurboTMT scan option (Bekker-Jensen et al., 2020); users who elect not to use this setting should opt for a higher resolution.


      Table 2. MS acquisition settings for Orbitrap Exploris 480 with FAIMS Pro interface

      Parameter Setting
      Global
      Ion source
      Ion Source Type NSI
      Spray Voltage Static
      Positive Ion (V) 1800
      Gas Mode Static
      Ion Transfer Tube Temp (°C) 270
      FAIMS Mode Standard Resolution
      FAIMS Gas Time Dependent
      FAIMS Gas Table
      0 min 3 L/min gas
      1 min 0 L/min gas
      MS Global Settings
      Infusion Mode Liquid Chromatography
      Expected LC Peak Width (s) 30
      Advanced Peak Determination False
      Default Charge State 2
      Internal Mass Calibration Off
      EXP 1: TMT MS2 FAIMS – 50 CV
      Full Scan
      Orbitrap Resolution 120000
      Scan Range (m/z) 350-1200
      FAIMS Voltages On
      FAIMS CV (V) -50
      RF Lens (%) 40
      AGC Target Standard
      Maximum Injection Time Mode Auto
      Microscans 1
      Data Type Profile
      Polarity Positive
      Source Fragmentation Disabled
      Intensity
      Filter Type Intensity Threshold
      Intensity Threshold 5.0e3
      Charge State
      Include charge state(s) 2-5
      Include undetermined charge states: False
      Dynamic Exclusion
      Dynamic Exclusion Mode Custom
      Exclude after n times 1
      Exclusion duration (s) 30
      Mass tolerance 10 ppm
      Exclude isotopes True
      Perform dependent scan on single charge state per precursor only True
      Precursor Fit
      Fit threshold (%) 70
      Fit window (m/z) 0.7
      Data Dependent
      Data Dependent Mode Cycle Time
      Time between Master Scans (sec) 2
      ddMS2
      Multiplex Ions False
      Isolation Window (m/z) 0.7
      Isolation Offset Off
      Collision Energy Mode Fixed
      Collision Energy Type Normalized
      HCD Collision Energy (%) 36
      Orbitrap Resolution 30000
      TurboTMT TMT Reagents
      Scan Range Mode Define First Mass
      First Mass (m/z) 110
      AGC Target Standard
      Maximum Injection Time Mode Auto
      Microscans 1
      Data Type Centroid
      EXP 2: TMT MS2 FAIMS – 65 CV
      Full Scan
      Orbitrap Resolution 120000
      Scan Range (m/z) 350-1200
      FAIMS Voltages On
      FAIMS CV (V) -65
      RF Lens (%) 40
      AGC Target Standard
      Maximum Injection Time Mode Auto
      Microscans 1
      Data Type Profile
      Polarity Positive
      Source Fragmentation Disabled
      Intensity
      Filter Type Intensity Threshold
      Intensity Threshold 5.0e3
      Charge State
      Include charge state(s) 2-5
      Include undetermined charge states: False
      Dynamic Exclusion
      Dynamic Exclusion Mode Custom
      Exclude after n times 1
      Exclusion duration (s) 30
      Mass tolerance 10 ppm
      Exclude isotopes True
      Perform dependent scan on single charge state
      per precursor only
      True
      Precursor Fit
      Fit threshold (%) 70
      Fit window (m/z) 0.7
      Data Dependent
      Data Dependent Mode Cycle Time
      Time between Master Scans (sec) 2
      ddMS2
      Multiplex Ions False
      Isolation Window (m/z) 0.7
      Isolation Offset Off
      Collision Energy Mode Fixed
      Collision Energy Type Normalized
      HCD Collision Energy (%) 36
      Orbitrap Resolution 30000
      TurboTMT TMT Reagents
      Scan Range Mode Define First Mass
      First Mass (m/z) 110
      AGC Target Standard
      Maximum Injection Time Mode Auto
      Microscans 1
      Data Type Centroid

Data analysis

  1. Protein quantification with Proteome Discoverer

    Following MS data acquisition, RAW files are processed using any of several analysis pipelines to obtain protein quantification from the MS/MS scans and reporter ion abundances. This protocol describes data processing using the Proteome Discoverer 2.4 software. Alternatives are documented elsewhere (Perez-Riverol et al., 2014; Franken et al., 2015).

    1. Load the data into Proteome Discoverer 2.4 by creating a new study and analysis.

      1. Select processing and consensus workflows that are appropriate for the instrument used for data acquisition and the reporter ion-based quantification method. We use the common templates provided by Thermo Fisher for the Q Exactive for our orbitrap platforms.

      2. Select TMT 10-plex as a quantification method. We create custom quantification methods with lot-specific corrections, but the default quantification method will suffice.

      3. Add the RAW spectrum files as fractions (if following the protocol here, add 8 RAW files per experiment). We analyze each set of fractions separately. For example, the vehicle treatment of a temperature range experiment would be analyzed separately from the compound treatment.

    2. Adjust settings in the Processing Workflow to conform to the experiment. We use default settings for the Minora Feature Detector, Spectrum Selector, and Percolator nodes (strict targeted FDR of 0.01 based on q-value with a relaxed FDR of 0.05). Major adjustments to the Sequest search engine node include

      1. Inputting the correct protein database (for T. gondii RH strains, the most recent release of the GT1 annotated proteins *.fasta, which can be found at https://toxodb.org/toxo/app/downloads/Current_Release/TgondiiGT1/fasta/data/).

      2. Selecting the desired dynamic modifications. We have used Oxidation (+15.995 Da) on M, Phosphorylation (+79.966 Da) on S/T/Y, and Acetylation (+42.011 Da) on the N terminus of the protein. Including additional dynamic modifications will increase the search space but may be common practice based on the conditions used in the protein workup steps.

      3. Selecting the appropriate static modifications: TMT 6-plex (+229.163 Da) on the peptide N terminus and K, and methylthio (+45.988 Da) on C. Note that use of other alkylating agents (e.g., IAA) will require an alternative modification on cysteine. For hyperperplexing with SILAC, see Note 3.

    3. Adjust the settings in the Consensus Workflow to enable downstream processing of melting curves:

      1. Use only unique peptides for quantification.

      2. Turn off scaling.

      3. For temperature range experiments, set Normalization Mode to none; it is important not to normalize abundances by channel, as protein abundance is globally decreasing at higher melting temperatures.

      4. For concentration range experiments, optionally set Normalization Mode to none. Data can be normalized in the TPP R package (see next section). We have also opted to normalize in Proteome Discoverer and forgo normalization in the TPP package.

      5. Optionally adjust the co-isolation threshold or Average Reporter S/N threshold. Lowering these thresholds may increase quantification but lower data quality.

    4. Upon completion of the analysis, export the protein-level quantification as a *.txt file.


  2. Curve Fitting

    Curve fitting is performed using the TPP R package, which has been extensively documented (Franken et al., 2015; Childs et al., 2019; Kurzawa et al., 2020). Recently, alternative thermal proteome profiling data analysis packages have been proposed (Dziekan et al., 2020), and users may develop their own custom normalization and curve fitting approaches. The output file from Proteome Discoverer must be modified to match the input format of the TPP package. Tables S1-S4 represent example output from Proteome Discoverer. Tables S5-S9 show the streamlined tables used as input to the TPP package, and Table S10 is representative output.

    Table S1. Temperature range, cells, replicate 1 output from the Proteome Discoverer 2.4 software.
    Table S2. Temperature range, cells, replicate 2 output from the Proteome Discoverer 2.4 software.
    Table S3. Temperature range, lysate, replicate 1 output from the Proteome Discoverer 2.4 software.
    Table S4. Temperature range, lysate, replicate 2 output from the Proteome Discoverer 2.4 software.
    Table S5. Temperature range, cells, replicate 1 trimmed input to the TPP R package.
    Table S6. Temperature range, cells, replicate 2 trimmed input to the TPP R package.
    Table S7. Temperature range, lysate, replicate 1 trimmed input to the TPP R package.
    Table S8. Temperature range, lysate, replicate 2 trimmed input to the TPP R package.
    Table S9. An example configuration table specifying the experiments, conditions, and replicates used for curve fitting in the TPP R package.
    Table S10. Example output from the TPP R package.


  3. Anticipated Results

    In a typical temperature-range experiment, we detect over 3,000 proteins, of which ~80% have quantification values sufficient for curve fitting. Our other proteomics experiments identify 4,600-4,800 proteins, indicating that the thermal challenge inherent to the thermal profiling approach reduces proteome coverage. We perform experiments in biological duplicate. Figure 2A reveals replicate variability in calculated protein melting temperatures. To generate a reference dataset, we performed thermal profiling on live parasites or lysates belonging to the T. gondii RH/TIR1 strain and hyperplexed the samples with SILAC (Harding et al., 2020; Herneisen et al., 2020); see Note 4. Aggregates were separated using the filter plate method described in section F of the Protocol. As observed for other organisms (Jarzab et al., 2020), proteome-wide thermal stability is greater in lysates than in cells (Figure 2B). To include thermostable proteins in our analysis, we calculated the numerical area under the curve (AUC) using the trapezoidal rule (Figure 2C). In contrast to the melting temperature, which requires at least 50% thermal denaturation, the AUC metric can be calculated for all proteins with complete thermal profiles. Figure 2D shows the relationship between melting temperature and AUC.


    Such a reference dataset can be used to select temperature ranges and thermal challenge temperatures for experiments involving compound treatment. Table 3 summarizes the distribution of melting temperatures from parasites and lysates from two different sets of experiments. The first experiment melted parasites or lysates over a temperature range of 37-67°C and separated soluble proteins from aggregates by ultracentrifugation (Herneisen et al., 2020). The second experiment, presented here, melted parasites or lysates over a temperature range of 41-73°C and separated aggregates with a filter plate. Researchers may reference the distribution most similar to their intended workflow. To detect compound-dependent thermal stabilization, concentration range experiments should be performed slightly above the melting temperature of the protein target under vehicle-treated conditions (Franken et al., 2015). In cases in which the protein target is not known, we have opted to perform the thermal challenges at two temperatures corresponding to the median and third quartile temperatures of the melting distribution. However, melting temperature often depends on the cellular environment. Figure 2E and F show melting temperatures and AUC values stratified by subcellular assignment by the MS-based LOPIT approach (Barylyuk et al., 2020). Some subcellular structures, such as the tubulin cytoskeleton and 20S subunit of the proteasome, prove particularly thermostable; detecting compound-dependent thermal shifts in proteins belonging to these substructures would require a high thermal challenge temperature. By contrast, proteins in the nucleus and nucleolus tend to be prone to precipitation, and using the median thermal challenge temperature would result in poor quantification and coverage of proteins in these substructures. Other organelles are particularly sensitive to cellular preparation; for example, components of the 60S ribosome co-melt in cells but exhibit disparate melting profiles in lysates. Therefore, researchers should leverage their observations and predictions about a compound’s mechanism of action to select the most appropriate thermal profiling parameters.



    Figure 2. Melting behavior of the T. gondii proteome. (A) Reproducibility of melting temperatures of proteins quantified in both replicates of an intact cell melting experiment. (B) Distribution of average melting temperatures of proteins quantified in both replicates of an intact cell and lysate temperature range experiment. (C) Depiction of how area under the curve (AUC) is calculated by numerical integration using the trapezoidal rule for the protein CDPK1. (D) Relationship between average melting temperature and AUC of proteins for which both values are available. Points with a lighter shade of gray were poorly fit to a sigmoidal melting curve (R2 < 0.8). (E) Distribution of average melting temperatures and AUC in cells or (F) lysates by LOPIT assignment from Barylyuk et al. (2020).


    Table 3. Distributions of melting temperatures from T. gondii lysates and intact cells from two different sets of experiments


    Ultracentrifugation (Herneisen et al., 2020) Filter plate (here)

    Lysate Cells Lysate Cells
    Min 44.8 43.5 47.6 43.9
    1st quartile 50.7 50.9 53.3 47.7
    Mean 53.4 53.8 56.7 52.5
    Median 52.7 53.4 55.5 52.3
    3rd quartile 55.4 56.4 59.1 56.3
    Max 65.6 66.9 72.0 72.1

Notes

  1. Following thermal challenge and global protein denaturation, soluble protein is separated from unfolded protein aggregates. The original CETSA protocol described centrifugation in a minifuge at 20,000 × g (Jafari et al., 2014), which was subsequently elevated to 100,000 × g in an ultracentrifuge to enhance the signal-to-noise ratio for MS analysis (Franken et al., 2015). Filter plates can be used as an alternative with the benefit of higher throughput (Mateus et al., 2018 and 2020a; Dziekan et al., 2020). After the soluble protein has been separated from the aggregates, samples can be snap-frozen in liquid nitrogen and stored at -80°C for several months.

  2. Solutions containing soluble proteins are cleaned up and processed using a modified SP3 protocol based on Hughes et al. (2019), which provides high capture and throughput that is well-suited for dilute and low-abundance TPP samples. Protein precipitation is not recommended as it can lead to uneven sample loss that degrades the quality of melting curves. The protocol has been optimized for T. gondii protein samples (Harding et al., 2020; Herneisen et al., 2020) and is compatible with TMT-labeling upon elution.

  3. To reduce MS time and run-to-run variability, we have hyperplexed TPP experiments using SILAC, as described elsewhere (Herneisen et al., 2020). This variation requires growing parasites in heavy and light SILAC media for 3 passages prior to the TPP experiment. Parasites grown in different media are treated as biological duplicates and are combined in equal weights prior to alkylation. Quantification values originating from the heavy samples are obtained by searching for peptides with heavy arginine (+10.008 Da) and the heavy Lysine-TMT6plex (+237.177 Da) modifications in Proteome Discoverer.

  4. The MS proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE (Perez-Riverol et al., 2019) partner repository with the dataset identifier PXD024912 and 10.6019/PXD024912.

Recipes

  1. DMEM + 3% CFS (used for routine parasite passaging) per 500 ml

    5 ml 200 mM glutamine

    500 µl 10 mg/ml gentamicin

    15 ml calf serum

    Filter the supplemented DMEM through a bottle top filter into a clean glass bottle that has not been washed with detergent.

  2. PBS

    100 ml 10× tissue culture-grade PBS

    900 ml deionized water

    Filter-sterilize and store at room temperature

  3. 10% IGEPAL CA-630 (also known as NP-40) (50 ml)

    5 ml IGEPAL CA-630

    45 ml deionized water

    Store at 4°C for 6 months

  4. 10× CETSA buffer (1 L)

    (50 mM NaCl, 1.42 M KCl, 10 mM MgCl2, 56 mM glucose, 250 mM HEPES pH 7.2)

    2.922 g NaCl

    105.86 g KCl

    2 g MgCl2

    10.1 g glucose

    59.575 g HEPES

    Add deionized water to 1 L and adjust the pH to 7.2 with KOH

    Sterile-filter the solution and store at 4°C

  5. CETSA wash buffer (1 ml)

    (5 mM NaCl, 142 mM KCl, 1 mM MgCl2, 5.6 mM glucose, 25 mM HEPES pH 7.2)

    100 µl 10× CETSA buffer

    900 µl deionized water

  6. CETSA lysis buffer (1 ml)

    100 µl 10× CETSA buffer

    80 µl 10% IGEPAL CA-360

    10 µl Halt protease inhibitors

    1 µl benzonase

    809 µl deionized water

  7. 1 M TCEP stock solution

    1 g TCEP HCl

    3.489 ml deionized water

    Store as 500 µl aliquots at -80°C and as 20 µl working aliquots at -20°C

  8. 200 mM MMTS stock solution

    200 mg MMTS

    7.924 ml isopropanol

    Store as 500 µl aliquots at 4°C

  9. Buffer A (100 ml)

    (0.1% formic acid in MS-grade water)

    100 ml MS-grade water

    100 µl >99% formic acid

    Sonicate for 10 min

  10. Buffer B (25 ml)

    (80% acetonitrile and 0.1% formic acid)

    20 ml MS-grade acetonitrile

    25 µl >99% formic acid

    5 ml MS-grade water

    Sonicate for 10 min

Acknowledgments

This research was supported by funds from a NIH grant (1R01AI144369) to SL and a National Science Foundation Graduate Research Fellowship (174530) to ALH. We thank E. Shortt for technical assistance. This work was adapted from protocols in Herneisen et al. (2020) and Harding et al. (2020).

Competing interests

The authors declare no conflicts or competing interests.

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简介

[摘要]弓形虫是一种单细胞真核寄生虫,慢性感染四分之一的全球人口。近年来,表型筛选确定了阻断寄生虫的化合物复制。解开此类化合物干扰的途径和分子机制需要目标解卷积。在寄生虫中,这种去卷积是通过化学基因组学实现的方法——例如,定向进化,然后是全基因组测序或全基因组淘汰赛屏幕。作为一种蛋白质组学替代方案,可以直接探测之间的物理相互作用化合物和蛋白质,热蛋白质组分析 (TPP),也称为细胞热位移分析(CETSA),最近作为一种识别活细胞中小分子-靶标相互作用的方法出现,多种生物体中的细胞提取物,包括单细胞真核病原体。配体结合诱导热稳定性转变——稳定或破坏蛋白质的构象变化以响应配体——可以通过质谱法 (MS) 测量。细胞与不同的配体浓度升高并加热,导致蛋白质热变性。可溶性蛋白质是使用多重定量 MS 提取和定量,产生数千个热变性配置文件。结合配体的蛋白质可以通过它们的化合物依赖性来识别热位移。此处提供的协议可用于识别配体-靶标相互作用并评估环境或遗传扰动对弓形虫蛋白质组热稳定性的影响和其他真核病原体。

图文摘要:
用于顶复体寄生虫T. gondii 中目标识别的热蛋白质组分析。

[背景]目标解卷积是通过鉴定的大量化合物的主要挑战表型筛选。化学基因组学方法,例如定向进化或药物筛选,已经已成为真核寄生虫目标识别的首选工具(Paquet等人,2017 年;Cowell等人,2018 年;卢斯等人。, 2018; 罗森伯格等。, 2019; 哈丁等人。, 2020)。这种方法需要培养长期在复合处理下的寄生虫和宿主细胞,并经常识别通路受小分子而不是目标本身的间接影响。相比之下,几种蛋白质组学过去十年开发的方法直接识别化合物和蛋白质之间的相互作用目标(McClure 和 Williams,2018 年;Conway等人,2021 年)。例如,丰富互动蛋白质可以用衍生化合物进行亲和纯化,然后进行质量分析光谱(MS)。然而,这些方法需要专门的化学反应并引入一个接头和其他化学基团添加到感兴趣的化合物中,这可能会影响其行为。热蛋白质组分析 (TPP),也称为细胞热位移分析 (CETSA),提供了一个可以以多种形式进行的无标记方法,以保护细胞生理学,包括原位(Dai et al. , 2019; Mateus et al. , 2020b)。与目标的交互由蛋白质热分布的化合物依赖性变化。细胞或细胞提取物用混合并加热以引起热变性。去除聚集的蛋白质,并溶解通过 MS 对蛋白质进行定量以生成每种蛋白质的熔解曲线。TPP最近确定顶复体寄生虫恶性疟原虫(Dziekan)中抗寄生虫化合物的靶标等。, 2019; 卢等人。, 2020) 和弓形虫(Herneisen et al ., 2020),以及在锥虫利什曼原虫(Corpas-Lopez et al. , 2019) 。TPP 的应用超越了目标反卷积(Becher等人,2018 年;Dai等人,2018 年和2019; 斯里达兰等人。, 2019; 马特乌斯等人。, 2020b)。可能会改变蛋白质状态和稳定性从构象变化、翻译后修饰、改变的定位以及与其他蛋白质和生物分子,如代谢物和核酸。例如,我们执行了对缺乏线粒体 DegP2 的寄生虫进行 TPP,以根据这种蛋白酶的损失(Harding等,2020)。遗传扰动与功能蛋白质组相结合分析处于早期阶段(Mateus等人,2020a),可能特别适合绘制寄生虫蛋白质组的未注释部分。虽然 TPP 主要在哺乳动物系统中进行,但它正在扩展到其他生物(Corpas-Lopez等人,2019 年;Dziekan等人,2019 年;Volkening等人,2019 年;Lu等人,2020 年;赫内森等人。, 2020; 哈丁等人。, 2020; 贾扎布等人。, 2020)。我们相信这种方法对对真核寄生虫的研究特别好,它们的进化分歧使情况复杂化通过基因组注释或生物信息学分析识别分子途径。为此,我们提供详细的协议,描述我们为生物体弓形虫开发的热分析管道。下面,我们确定了选择适合研究人员的 TPP 工作流程的关键考虑因素生物学问题。遵循分步指南。

关键字:热蛋白组分析, 细胞热迁移实验, 弓形虫, 寄生虫 , 蛋白质组学

实验类型:在这个协议中,我们通过选择材料和处理来对步骤进行分层。TPP 可以在现场进行寄生虫或寄生虫裂解物,并通过在 10 个温度范围内熔化样品(温度范围)或超过 10 个在单一温度下熔化的化合物浓度范围(浓度范围)。这些变化产生了程序中描述的四种排列,如(B) 裂解物温度范围实验,(C) 寄生虫温度范围实验,(D) 裂解液浓度范围实验和(E)寄生虫浓度范围实验。每个实验有优点也有缺点(Franken等人,2015 年;Dai等人,2019 年;Mateus等人,2020b)。为了例如,使用活细胞的实验更具生理性,但结合了直接和间接影响。裂解物实验可以更直接地识别配体-蛋白质相互作用,但细胞丢失划分也可能导致非生理相互作用。浓度范围实验产生更多信息丰富的热剖面,但如果热挑战可能会错过真正的相互作用温度不理想(太低或太高),蛋白质组的整体覆盖率降低由于全局变性。

治疗条件:我们对细胞外寄生虫进行了热分析实验,以避免添加宿主蛋白质组的复杂性和化合物渗透性和宿主的混杂效应代谢。通常对细胞内寄生虫进行复合治疗;然而用于热分析实验的适当化合物浓度应通过以下方式确定使用细胞外寄生虫进行检测。热分析实验应模拟测定条件,如尽可能接近。考虑因素包括化合物到达所需的时间扩散和结合平衡,发生平衡的缓冲液,以及平衡温度(例如,室温与 37°C)。哺乳动物研究经常进行孵化在 PBS 中(Reinhard等人,2015 年;Savitski等人,2014 年;Franken等人,2015 年)。我们使用了缓冲区组成类似于宿主细胞质的离子组成(Herneisen等人2020 年;Harding等人2020)。缓冲液应不含血清,这会淹没可通过 MS 量化的寄生虫信号。我们的目标是每个参考样品处理 25 µg 蛋白质,根据我们的经验,这相当于来自1×10 7 IRH菌株的细胞外寄生虫的材料。我们主题浓度范围样品到至少两个不同的热挑战温度;我们发现 54°C 58°C 可以工作同时仍然提供足够的蛋白质组覆盖范围。热挑战温度可能需要针对每个实验进行优化;数据分析部分提供了进一步的评论。

裂解条件

最终裂解缓冲液组合物应包含 0.5-1% IGEPAL CA-630(也称为 NP-40),其在溶解膜蛋白而不重新溶解聚集蛋白之间提供平衡(莱因哈德等人2015 年)。对于大多数实验,裂解缓冲液应包含蛋白酶抑制剂(和可选的磷酸酶抑制剂,取决于实验的重点)和 benzonase

SP3 净化之前消化核酸。如果认为感兴趣的化合物会影响蛋白酶、磷酸酶或核酸结合活性,则应省略这些补充剂直到分离可溶性蛋白和聚集蛋白之后。我们的裂解缓冲液具有离子

组成类似于 PBSHerneisen等人2020 年)和细胞内样缓冲液(Harding等人2020 年),取决于应用程序。缓冲液的离子组成(例如ATP 和代谢物的存在)可以显着影响蛋白质的熔解行为(Lim等人2018 年;Sridharan等人2019 年)。寄生虫裂解物的浓度也会影响熔解行为;因此,计算裂解前的寄生虫数量,并对寄生虫数量使用一致的裂解缓冲液体积。收获后,寄生虫应至少在洗涤缓冲液中重悬一次裂解缓冲液(但不含去污剂)的成分以稀释细胞培养污染物,例如血清蛋白质。

材料和试剂

1. T12.5 烧瓶(例如Corning Falcon Tissue Culture Flasks,目录号:29185-298

2. T175烧瓶(例如,蜂星。®过滤器帽细胞培养烧瓶,目录号:82050-872

3. 15 厘米培养皿(例如Corning Falcon ®组织培养皿,目录号:25383-103

4. Corning ® 150 ml 瓶顶真空过滤器,0.22 µm 孔径 13.6 cm 2 CA 膜(Corning,

目录号:430624

5. 50 ml锥形管(Corning,目录号:430829

6.人包皮成纤维细胞(HFF)细胞(ATCC,目录号:SCRC-1041

7.弓形虫细胞系(RH例如ATCC 50838 PRA-319

8. T. gondii过滤器(Whatman Pop-Top Swin-Lok 塑料过滤器支架,用于 47 毫米膜

过滤器尺寸,例如VWR 目录号:28163-089,使用 GE Healthcare Whatman Nuclepore

亲水膜 3 5 µm 圆,目录号:111112 111113

9. 细胞刮刀(Corning ® Small Cell Scraper,目录号:3010

10. 蛋白质低结合管(例如Eppendorf TM LoBind微量离心管,1.5 ml Thermo FisherScientific,目录号:13698794

11. 8 PCR 管(例如Genesee Scientific,目录号:27.125 U

12. 厚壁聚碳酸酯开顶超速离心管(0.2 ml7 × 20 mmBeckman Coulter,目录号:343775

13.蛋白质低结合96孔板(Eppendorf,目录号:951032905

14.注射器20毫升(BD Biosciences,目录号:302830

15.疏水Sera-Mag Speed BeadsGE Healthcare,目录号:65152105050250~50mg/ml,保持在 4°C 直至使用)

16.亲水Sera-Mag Speed BeadsGE Healthcare,目录号:45152105050250~50mg/ml,保持在 4°C 直至使用)

17. DMEMThermo Fisher Scientific,目录号:11965118,在使用前保持在4°C

18. 美国产新生小牛血清,热灭活,无菌过滤,适用于细胞培养(SigmaAldrich,目录号:N4762-500ML,保持在 -80°C 直至使用)

19. 10 mg/ml 庆大霉素(Life Technologies,目录号:15710072,室温)

20. 200 mM L-谷氨酰胺(Life Technologies,目录号:25030081,保持在-20°C直至使用)

21. 250 U/µl benzonaseSigma Aldrich,目录号:E1014-25KU-20°C 储存)

22. 100× Halt Protease Inhibitor CocktailLife Technologies,目录号:87786

23. IGEPAL ® CA-630粘稠液体(Sigma Aldrich公司,目录号:I3021-50ML

24. 适用于组织培养的 10× PBS例如VWR,目录号:45001-130

25. DC蛋白质测定(Bio-Rad,目录号:5000116

26. 三(2-羧乙基)膦(TCEPPierce,目录号:20490;保持在 -20°C 直至使用)

27.甲硫代磺酸甲酯(MMTSThermo Fisher Scientific,目录号:23011,保留4°C)

28. 乙醇,Pure 200proofHPLC/分光光度级(Sigma-Aldrich,目录号:459828-1L)

29.测序级胰蛋白酶(例如Promega,目录号:V5113,保持在-80°C直至使用)

30. 三乙基碳酸氢铵缓冲液 1.0 MpH 8.5Sigma-Aldrich,目录号:T7408-

100ML,保持在4°C

31. Pierce Quantitative Fluorometric Peptide AssayThermo Fisher Scientific,目录号:

23290,在使用前保持在 4°C

32. TMT10plex Isobaric Label Reagent SetThermo Fisher Scientific,目录号:90110,保留-20°C 直至使用)

33. 50% 羟胺(Thermo Fisher Scientific,目录号:90115

34. Pierce pH 分馏试剂盒(Thermo Fisher Scientific,目录号:84868,保持在 4°C直到使用)

35. 超高效液相色谱 (UPLC)-MS 乙腈 (Thermo Fisher科学的,目录号:A9561

36. UHPLC-MS 水(Thermo Fisher Scientific,目录号:W81

37. Pierce 甲酸,LC-MS 级(Thermo Fisher Scientific,目录号:28905

38. DMEM + 3% CFS(见食谱)

39. PBS(见食谱)

40. 10% IGEPAL CA-630(也称为 NP-40)(见配方)

41. 10× CETSA 缓冲液(见配方)

42. CETSA 洗涤缓冲液(见配方)

43. CETSA 裂解缓冲液(见配方)

44. 1 M TCEP 库存溶液(见配方)

45. 200 mM MMTS 原液(见配方)

46. 缓冲液 A(见食谱)

47. 缓冲液 B(见食谱)

 

设备

1. CO 2培养箱(Thermo Fisher Scientific Forma Steri-Cycle 370,目录号:370

2. 临床台式离心机(Eppendorf,型号:Centrifuge 5810R,目录号:022625101)

3. 微量离心机(Eppendorf,型号:Centrifuge 5424R [已停产],替代品包括离心机 5425/5425 R)

4.微型离心机(VWR Galaxy Mini Centrifuge,目录号:37000-700

5.血细胞计数器(VWR Counting Chamber,目录号:1517O-173

6. 热循环仪(Bio-Rad C1000 Touch TM Thermal Cycler with Dual 48/48 Fast Reaction模块目录号:1851148 和带有 96-Deep Bio-Rad C1000 Touch TM热循环仪井反应模块,目录号:1851197

7. 台式超速离心机(Beckman Ultra MAX [已停产],替代品包括 OptimaMAX-XP Optima MAX-TL)

8.热混合器(Eppendorf,型号:ThermoMixer C,目录号:53820000231.5mlSmartBlock,目录号:5360000038

9.磁性支架(Invitrogen Dynamag 2,目录号:12321D

10. 真空离心机(Savant TM

通用 SpeedVac TM真空系统,目录号:SPD111V 和,目录号:UV5450

11.冻干机(Labconco FreeZone Triad Freeze Dryer,目录号:794001030

12. Orbitrap 质谱仪(Thermo Fisher Scientific Q Exactive HFX [已停产] Exploris 480,目录号:BRE725533),带有可选的 FAIMS Pro 接口(Thermo Fisher科学,目录号:FMS02-10001

13. MS 耦合 LC 系统(Thermo Fisher Scientific EASY-nLC 1200,目录号:LC140)使用 Acclaim PepMap 100 75 µm × 2 µm nanoViper 捕集柱(Thermo Fisher Scientific,目录号:164946)和 PepMap RSLC C18 3 µm100A75 µm × 15 cm 分析柱(Thermo Fisher Scientific目录号:ES900

14. Pierce 甲酸,LC-MS 级(Life Technologies,目录号:28905

15. UPLC-MS 乙腈(Thermo Fisher Scientific,目录号:A9561

16. UPLC-MS 水(Thermo Fisher Scientific,目录号:W81

软件

1. Proteome Discoverer2.4 版(Thermo Fisher Scientific

2. R4.0 或更高版本:https : //cran.r-project.org/

3. Tidyverse 包,1.3 版:https ://cran.r-project.org/web/packages/tidyverse/index.html

4. TPP 包,3.12 版:https : //bioconductor.org/packages/TPP/

程序

该协议假设读者熟悉弓形虫寄生虫和宿主细胞繁殖。为了标准评论,见 Roos等人。(1994) Jacot等人。(2020)

A. 寄生虫收获

1. 2 × 10 7 -5 × 10 7 RH 速殖子融合的 HFF 感染 T175 烧瓶或 15 厘米培养皿每个,相当于来自一个完全溶解的 T12.5 烧瓶中的寄生虫,在检测前 40-48 小时。应感染足够的T175 以收获 4 × 10 8寄生虫用于测定。产量可能会有所不同取决于宿主细胞年龄、寄生虫菌株和治疗;根据我们的经验,3-4 15 厘米的盘子通常足以达到这个数量的寄生虫。

2. 当寄生虫从单层中完全溶解后,刮掉烧瓶并收集培养基含有细胞外寄生虫。完全裂解的单层含有丰富的细胞外寄生虫和少数剩余的附着宿主细胞。去除宿主细胞碎片将培养基通过 3 µm 过滤器放入每个烧瓶或培养皿中的一个 50 ml 锥形瓶中。

3. 通过在室温下以 1,000 × g离心10 分钟来浓缩寄生虫溶液在带有摆动斗转子的离心机中的温度。丢弃上清液。重新暂停寄生虫沉淀在 1 ml 洗涤缓冲液(不含去污剂、抑制剂酶的裂解缓冲液)中并将寄生虫悬浮液转移到 1.5 ml 蛋白质低结合管中。

4. 1:500 稀释寄生虫悬液并使用血细胞计数器计数。

5.在室温下以 1,000 × g离心寄生虫10 分钟。丢弃上清液。

6. 根据所需的处理,进入(B)部分裂解物温度范围实验,(C) 寄生虫温度范围实验,(D) 裂解物浓度范围实验,或(E)寄生虫浓度范围实验

B. 裂解液温度范围实验

1. 寄生虫裂解

一种。每 2 × 10 7 个寄生虫在 100 µl 裂解缓冲液中重悬寄生虫悬浮液(见裂解背景中的考虑因素)。应收获足够的寄生虫 (4 × 10 8 )至少 1.1 ml 裂解液,少量过量以解决移液错误

下面的步骤。湾 允许裂解在冰上进行 15 分钟,偶尔通过移液混合。

2. 复合处理一种。在裂解缓冲液中制备 2 倍所需终浓度的化合物稀释液和含有等量 DMSO(或适当的载体)的载体溶液。等分 550 µl将每种溶液倒入 1.5 ml 蛋白质低结合管中。湾  550 µl 寄生虫裂解物与 550 µl 2x 化合物或载体溶液混合,然后轻轻移液器混合。该化合物现在处于所需的最终浓度。

C。将 100 µl 含有载体或化合物的寄生虫悬液分装到 10 个标记的 PCR 中对应于预期熔化温度的管(见下文)。

d. 让溶液在室温或 37°C 下平衡至少 5 分钟(参见背景中的治疗注意事项)。

3. 热挑战一种。使用带 PCR 管的微型离心机简要收集管底部的液体适配器。离心大约 3 秒。

  PCR 管以适当的方向放置在热循环仪上,使管与裂解物匹配所需的温度。

注意:应提前启动热循环仪程序,使孔处于加入管子时的温度;精确的顺序取决于温度可以由用户的热循环仪实现的梯度。我们使用了熔化温度分别为 37°C41°C43°C47°C50°C53°C56°C59°C63°C 67°C穿过 48 孔热循环仪中的两个 PCR 试管。

C。允许变性发生 3 分钟。

d. 快速从热循环仪中取出管子,放在冰上 5 分钟。

e. 使用小型离心机,在管底部短暂收集蒸发的液体PCR管适配器。离心大约 3 秒。

F。继续执行下面描述的步骤,F. 可溶性和聚集蛋白的分离

C. 寄生虫温度范围实验

1. 复合处理一种。制备 2 倍所需终浓度的化合物溶液和载体溶液用等量的 DMSO(或适当的载体)。等分 550 µl 的每种溶液到 1.5 ml 蛋白质低结合管中。湾  550 µl 寄生虫悬浮液与 550 µl 2x 化合物或载体溶液混合并轻轻但彻底地移液混合。该化合物现在处于所需的最终专注。

C。将 100 µl 含有载体或化合物的寄生虫悬液分装到 10 个标记的 PCR 中对应于预期熔化温度的管(见下文)。

d. 让化合物在室温或 37°C 下与寄生虫平衡至少 5 分钟(参见背景中的治疗注意事项)。

2. 热挑战一种。使用小型离心机,在管底部短暂收集蒸发的液体PCR管适配器。离心大约 3 秒。湾  PCR 管以适当的方向放置在热循环仪上,使管与裂解物匹配所需的温度。

注意:应提前启动热循环仪程序,使孔处于加入管子时的温度;精确的顺序取决于温度热循环仪可实现的梯度。我们使用的熔化温度为37°C, 41°C, 43°C, 47°C, 50°C, 53°C, 56°C, 59°C, 63°C, 67°C 分为两个 PCR条形管和 48 孔热循环仪。

C。允许变性发生 3 分钟。

d. 快速从热循环仪中取出管子,放在冰上 5 分钟。

3. 寄生虫裂解一种。使用带 PCR 管的微型离心机,简要收集管中蒸发的液体适配器。离心大约 3 秒。湾 向每个管中加入 20 µl 6× 裂解缓冲液,轻轻移液器混合(见处理背景中的考虑因素)。让寄生虫在冰上裂解至少 15 分钟。

C。继续执行下面描述的步骤,F. 可溶性和聚集蛋白的分离

D. 裂解液浓度范围实验

1. 寄生虫裂解一种。每 2 × 10 7 个寄生虫在 100 µl 裂解缓冲液中重悬寄生虫悬浮液(见裂解背景中的考虑因素)。应收获足够的寄生虫 (4 × 10 8 )至少 1.1 ml 裂解液,少量过量以解决移液错误下面的步骤。湾 允许裂解在冰上进行 15 分钟,偶尔通过移液混合。

2. 复合处理一种。制备十个浓度的化合物的稀释系列,包括单独的载体,在裂解缓冲液中所需终浓度的 2 倍。等分 110 µl 2x 化合物溶液放入 PCR 管中。

注意:我们建议确保在所有车辆中保持相同的车辆浓度通过将稀释系列制备成含有赋形剂浓度的裂解缓冲液来制备样品等于最高化合物浓度。

  110 µl 寄生虫裂解物分装到含有 2x 化合物溶液的 PCR 管中。

化合物现在处于最终所需浓度,每管中的体积为 220

微升。

C。使用多道移液器轻轻混合裂解液并将一半体积 (110 µl) 转移到

另一组 PCR 管。现在有两组 10 个管,每管 110 µl

d. 让溶液在室温或 37°C 下平衡至少 5 分钟(参见

背景中的治疗注意事项)。

3. 热挑战

一种。使用带 PCR 管的微型离心机简要收集管底部的液体适配器。离心大约 3 秒。

 为了诱导热变性,将一组管放在预热至54°C 和预热至 58°C 的深井热循环仪中的另一组管(参见背景中的考虑因素)。

注意:应提前启动热循环仪程序,使孔处于添加管时的温度。

C。允许变性发生 3 分钟。

d. 快速从热循环仪中取出管子,放在冰上 5 分钟。

e. 继续执行下面描述的步骤,F. 可溶性和聚集蛋白的分离

E. 寄生虫浓度范围实验

1. 复合处理

一种。制备浓度范围为 2 倍的 10 种化合物溶液,包括赋形剂裂解缓冲液中所需的最终浓度。将 110 µl 2x 化合物溶液分装到一个PCR管。

注意:我们建议确保在所有车辆中保持相同的车辆浓度通过将稀释系列制备成含有赋形剂浓度的裂解缓冲液来制备样品等于最高化合物浓度。  110 µl 寄生虫悬液分装到含有 2x 化合物的 PCR 管中解决方案。化合物现在处于最终所需的浓度,每个化合物的体积

管为 220 µl

C。使用多道移液器轻轻混合寄生虫悬浮液并转移一半体积 (110 µl) 到另一组 PCR 管。现在有两组 10 管,110

µl 复合溶液中的寄生虫。

d. 让化合物在室温或 37°C 下与寄生虫平衡至少 5 分钟(参见背景中的治疗注意事项)。

2. 热挑战

一种。使用带有 PCR 管适配器的微型离心机,简单地收集管中的液体。离心大约 3 秒。

 为了诱导热变性,将一组管放在预热至54°C 和预热至 58°C 的深井热循环仪中的另一组管(参见背景中的考虑因素)。

注意:应提前启动热循环仪程序,使孔处于添加管时的温度。

C。允许变性发生 3 分钟。

d. 快速从热循环仪中取出管子,放在冰上 5 分钟。

3. 寄生虫裂解

一种。使用带 PCR 管的微型离心机,简要收集管中蒸发的液体适配器。离心大约 3 秒。

 向每个管中加入 20 µl 6× 裂解缓冲液,轻轻移液器混合(见处理背景中的考虑因素)。让寄生虫在冰上裂解至少 15 分钟。

C。继续执行下面描述的步骤,F. 可溶性和聚集蛋白的分离

F. 可溶性和聚集蛋白的分离下面,我们描述了用于分离可溶性蛋白质的两种分离方法。为了更多信息,请参见注释 1

1.超速离心法

一种。将热挑战的裂解物(约 100 µl 的体积)转移到超速离心机中在冰床上预冷的管子。

注意:这些试管的最小体积为 100 µl。使用较低的容量有不平衡的风险离心机转子。

 将试管装入台式超速离心机(例如BeckmanUltra MAX) 冷却至 4°CTLA-100 转子最多可安装 20 个管子,足以满足温度范围实验的两种处理条件或两种挑战温度浓度范围实验。管子必须适当平衡以避免转子和超速离心机损坏。

C。使用超速离心机在 4°C 下以 100,000 × g离心样品20 分钟。到计算合适的转速,使用转子半径规格和在线计算器例如https://www.beckman.com/centrifuges/rotors/calculator

d. 轻轻取下转子,注意不要打扰管子,并立即转移管冰。如果有条件,请在寒冷的房间里工作。

e. 去除上清液体积约 80% 的顶部并转移到预冷的蛋白质中低结合管。重要的是不要破坏含有聚集蛋白质和膜质部分。

F。继续下一部分,F. 使用 SP3 协议进行蛋白质清理和消化。

2、滤板法

一种。用 100 µl 1× 裂解缓冲液(含有化合物/处理液,如果适用)预先润湿滤板。将滤板放在 96 孔板的顶部。在摆动桶中以 500 × g离心离心 5 分钟,直到溶液通过过滤器进入 96 孔板。丢弃解决方案。湾 将滤板放在干净的蛋白质低结合或聚丙烯 96 孔板上。转移

热激发裂解物 (~100 µl) 到平衡的滤板和离心机在 500 ×g 4°C 下离心 5 分钟以将可溶性蛋白质与聚集体分离。可溶性蛋白通过通过过滤器进入 96 孔板。

C。将可溶性部分从 96 孔板转移到蛋白质低结合管。体积可溶性蛋白质溶液相对于输入体积减少,应进行测量在下一步之前,G. 蛋白质清理和消化

G. 使用 SP3 协议进行蛋白质净化和消化

1. 量化蛋白质丰度

一种。确定参考样品中的蛋白质浓度(温度范围为 37°C实验和浓度范围实验的最低化合物浓度)使用蛋白质定量测定,例如DC蛋白质测定(Bio-Rad),根据制造商的说明。稀释溶液应包含感兴趣的化合物或车辆,如果适用,因为它可能会显着改变吸光度读数。大量的在此步骤中确定的蛋白质将用于计算用于检测的 SP3 珠的量样品净化和胰蛋白酶添加消化。我们通常量化 20-60 µg 的可溶性参考样品中的蛋白质。以下步骤假设参考中的产量为 50 µg样本相应地调整体积以减少蛋白质含量。湾 将与参考样品中 50 µg 蛋白质相对应的体积转移到新的蛋白质低结合管。将相同体积的剩余样品转移至低蛋白也绑定管子。用裂解缓冲液将体积提高到 100 µl

2. 减少半胱氨酸

一种。向每个样品中加入 0.5 µl 1 M TCEP 溶液。TCEP 的浓度现在是 5 mM。湾 将样品在 55°C 下孵育 10 分钟,例如,在加热块或热混合器上。

3. 烷基化半胱氨酸

55°C 中取出管子,让它们冷却至室温。添加 7.54 µl 200mM MMTS 库存溶液使浓度达到 15 mM。允许反应发生室温下 10 分钟。注意:替代方案中的碘乙酰胺 (IAA) 烷基化黑暗的。我们更喜欢 MMTS 进行溶液内消化,因为它的反应速度快、稳定性好且较低

非特异性烷基化(Müller Winter2017),这可以增加肽的数量MS 分析后的鉴定。烷化剂的选择将决定搜索对半胱氨酸的修饰,MMTS 的甲硫基 (+45.988 Da) 或氨基甲酰甲基 (+57.021Da) IAA

4. 使用 SP3 协议清理样本(Hughes2019)。有关更多信息,请参阅注意2.

一种。制备足够的疏水性和亲水性 Sera-Mag 珠,浓度为 50 µg/µl,用于 1:10相对于参考样品的珠子/蛋白质 (wt/wt) 比率。例如,要处理 10样品与 50 µg 参考样品,制备 5 mg 珠子。

一。在 1.5 毫升管中,将 50 微升 50 毫克/毫升疏水珠与 50 微升 50mg/ml 亲水珠。

ii. 将珠子放在磁性架上,让它们分开。使用 P200 移液器

取出并丢弃上清液。

三、在 100 µl MS 级水中清洗珠子。将珠子放在磁性架上,然后

再次丢弃上清液。

四、在 100 µl MS 级水中重悬珠子,最终浓度为 50 µg/µl

 向每个样品中加入 10 µl 50 µg/µl 珠子。珠子/蛋白质 (wt/wt) 比率现在至少为

10:1

C。通过添加 4 倍体积的 100% HPLC 级乙醇将蛋白质结合到珠子上。为了

例如,将 100 µl 样品与 0.5 µl TCEP7.54 µl MMTS 10 µl Sera-Mag 磁珠,加入 472 µl 100% 乙醇。注:蛋白质溶液现为80%按体积计乙醇。我们发现这个比例对于弓形虫蛋白的结合是最佳的到 Sera-Mag 珠子。

d. 将试管放入热混合器中,让蛋白质与珠子聚集,然后在 24°C 下以 1,000 rpm 的速度振荡至少 10 分钟。珠子在结合时应该结块蛋白质。

e. 将管子放在磁性架上,让珠子分开,这需要大约 30 秒。将上清液丢弃到适合 80% 的废物流中乙醇。

F。用 180 µl 80% 乙醇清洗珠子 3 次,可通过稀释制备HPLC 级乙醇与 HPLC 级水。每次,让珠子磁性与溶液分离 30 秒,并将上清液处理为适当的废物溪流。

注意:在最后的漂洗中,尽可能多地去除乙醇洗涤液,以尽量减少酶消化步骤中的残留物。我们去除几乎所有的液体将珠子以 16,000 × g 离心 30 秒,然后将 P200 P10 尖端双重堆叠以去除上清液。

5. 将蛋白质消化成肽

一种。在 50 mM TEAB 中以 1:50 (wt/wt) 蛋白质:胰蛋白酶的比例制备胰蛋白酶消化溶液。

为要处理的样品数量准备足够的储备溶液,例如20 个样品对于具有控制和处理条件的温度范围实验以及 10熔化温度各一个。湾 向每个管中加入 35 µl 消化液。用尖端轻轻将珠子移入液体中移液器,但避免移液珠子,因为它们很粘。

C。将试管放入加热至 37°C 的热混合器中,并以 1,000 rpm 的速度摇动过夜(16-18 小时)。

d. 在室温下以16,000 × g离心管1 分钟以沉淀珠子和收集蒸发的液体。

e. 将管子放在磁性架上,让珠子分离 30 秒。转移将含有消化肽的水性上清液转移到新的蛋白质低结合管中。肽被洗脱后,样品可以在液氮中速冻并在冷凝器温度为 -80°C 且腔室压力约为 0 毫巴的冻干机。肽通常在四小时或更短的时间内冻干成粉末。冻干肽可在 -80°C 下保存数月。

H. 串联质量标签标记样品多路复用是使用同量异位质量标签进行的,这些标签可在 10-

plex 16-plex 格式(Werner等人2014 年;Li等人2020 年)。我们保持TMT 10-的工作库存plex 试剂,浓度为 6.66 µg/µl(每 15 µl 100 µg),在 -80°C 的乙腈中保存 3-6 个月。我们以 2:1 (wt/wt) TMT:peptide 进行标记(Zecha2019)。应在标记之前选择 TMT 标记方案。每个温度或浓度用一个 TMT 通道标记。执行两个完整的 10 重标记反应每个实验: 10 熔化温度与载体和化合物的温度范围实验或 10 个化合物浓度在两个温度下熔化以进行浓度-范围实验。我们在顺序标记时观察到报告离子干扰(Brenes等人2019 年)。因此,推荐使用 1所示的标记方案。

 

1. 推荐的温度和浓度范围的 TMT 标记策略实验。t 1 /c 1是指实验中的最低温度或浓度。

 

1. 使用 Pierce 荧光肽测定法量化肽丰度在开始之前,量化参考样品中的肽数量(温度为 37°C范围实验和浓度范围的最低化合物浓度实验)根据制造商的说明使用 Pierce 荧光肽测定法指示。如果肽已被冻干,则重悬在 35 µl 50 mM TEAB pH 8.5 中。1:20稀释通常足以将样品置于标准曲线的范围内,例如0.5 µl样品 + 9.5 µl 水。对于标准的全蛋白质组 TMT 反应,使用 25-50 µg 的肽在 35 µl 50 mM TEAB 中,根据需要稀释样品。使用等量和稀释

非参考样本。以下步骤适用于含有 50 µg 的样品35 µl 中的肽。

2. TMT标记反应

一种。在室温下平衡 TMT 试剂 3 分钟。

注意:记录该批次的试剂批号和同位素校正。此信息可用于在 Proteome Discoverer 中创建自定义定量方法,以纠正用于天然碳同位素产生的同位素杂质。

  TMT 试剂以 13,000 × g离心1 分钟,然后将每个小瓶重新悬浮在 120 µl

100% MS 级乙腈。创建 15 µl 等分试样并在 -80°C 下储存长达 6 个月。

注意:如果长时间不使用重新悬浮的 TMT 试剂,请冻干

试剂并以粉末形式储存于 -20°C

C。将 15 µl TMT 试剂 (100 µg) 添加到参考样品(35 µl 50 mM 中的 50 µg 蛋白质)

茶)。如果使用更多或更少的肽输入,保持最终的体积/体积比

乙腈(TMT 试剂)到水性缓冲液(TEAB 缓冲液)。例如,如果仅标记

35 µl TEAB 中加入 25 µg 肽,在 7.5 µl 7.5 µl 100% 中加入 50 µg TMT 试剂

乙腈使最终组合物达到 30% vol/vol 乙腈。

d. 将管以 13,000 × g离心30 秒以收集液体。

e. 将管子放入热混合器中,并在室温下以 400 rpm 的速度摇动 60 分钟。

F。每 50 µl 反应加入 3.2 µl 5% 羟胺,淬灭未反应的 TMT 试剂。

将试管放入 ThermoMixer 中,并在室温下以 400 rpm 的速度摇动 15 分钟。

G。将样品混合在 1.5 ml 蛋白质低结合管中。使用相同的移液器吸头

转移以避免因与新表面接触而丢失肽。音量现在应该大约为 530 µl

H。在液氮中快速冷冻汇集的样品并冻干直至干燥。注:样品体积也可以通过真空离心减少。

一世。干燥样品可在 -80°C 下储存数月。

3. 脱盐分馏

应在 MS 数据采集之前对 TMT 标记的样品进行分级以减少分离MS 分析期间的干扰。我们进行高 pH 反相肽分级分离使用 HPLC例如,使用 Shimadzu LC-20AD;参见 Herneisen等人2020)或 Pierce High pH反相肽分馏试剂盒根据制造商的说明,我们已经发现提供了对弓形虫蛋白质组的等效覆盖,并且还可以作为脱盐步骤。我们将样品分成八个部分用于 LC-MS。馏分可以冻干并在 -80°C 下无限期保存。

一、质谱数据采集

数据采集方法高度依赖于设施。至少,TMT 标记的样本应该使用足够的分辨率来分辨报告离子并使用长梯度获得分离复杂的肽混合物并减少共分离干扰。在这里,我们描述

我们的 Exploris 480 轨道阱的数据采集协议与 FAIMS Pro 接口耦合到一个Easy-nLC 1200 系统。

1. 样品重悬和进样

A. 将每个冻干部分重悬在缓冲液 A 中至估计浓度为 0.5-1 µg/微升。我们通常将每个样品重新悬浮在 25 µl 中。确保冻干物是完全溶解;它可能有助于彻底清洗管子的侧面并收集

通过以 16,000 × g离心1 分钟来分离液体。

B. 将每个重悬部分转移到自动进样器管中。一旦样本重悬后,应保存在 4°C

C. 注入 0.5-1 µg 肽用于 MS 分析(通常为 1-2 µl)。属于同一类的样品TMT 标记实验可以顺序注射(,组份)。我们执行不同 TMT 标记实验之间的空白进样以减少残留。

2. LC 梯度

我们的样品在 1 描述的 90 分钟梯度内分离。梯度包括可选的 12 分钟跷跷板用于色谱柱维护。我们的 LC 系统包括一个商业捕获柱(Acclaim PepMap 100 75 µm × 2 µm nanoViper)连接到 15 cm商业分析柱(PepMap RSLC C18 3 µm100A75 µm × 15 cm)。

3. MS 采集设置

带有 FAIMS Pro 界面的轨道阱 Exploris 480 的方法参数总结在2。根据我们的经验,在 -50 -65 的补偿电压之间交替产生对弓形虫蛋白质组的最佳报道。ddMS 2分辨率 30,000 已优化对于 TurboTMT 扫描选项(Bekker-Jensen等人2020 年);选择不使用此功能的用户设置应选择更高的分辨率。

表二带有 FAIMS Pro 接口的 Orbitrap Exploris 480 MS 采集设置

数据分析

A.    使用 Proteome Discoverer 进行蛋白质定量

采集 MS 数据后,使用多种分析管道中的任何一种对 RAW 文件进行处理,以 MS/MS 扫描和报告离子丰度获得蛋白质定量。本协议描述了使用 Proteome Discoverer 2.4 软件进行的数据处理。替代方案是其他地方有记录(Perez-Riverol等人2014 年;Franken等人2015 年)。

1. 通过创建新的研究和分析将数据加载到 Proteome Discoverer 2.4

一种。选择适合所用仪器的处理和共识工作流程用于数据采集和基于报告离子的定量方法。我们使用常见的Thermo Fisher Q Exactive 为我们的轨道阱平台提供的模板。

 选择 TMT 10-plex 作为量化方法。我们创建自定义量化方法带有特定批次的更正,但默认的量化方法就足够了。

C。添加 RAW 光谱文件作为分数(如果遵循此处的协议,则每添加 8 RAW 文件

实验)。我们分别分析每组分数。例如,车辆温度范围实验的处理将与复合治疗。

2. 调整处理工作流程中的设置以符合实验。我们使用默认Minora Feature DetectorSpectrum Selector Percolator 节点的设置(严格目标 FDR 0.01,基于 q 值,宽松的 FDR 0.05)。重大调整Sequest 搜索引擎节点包括

一种。输入正确的蛋白质数据库(对于刚地弓形虫RH 菌株,最新版本的GT1 注释的蛋白质 *.fasta,可以在https://toxodb.org/toxo/app/downloads/Current_Release/TgondiiGT1/fasta/data/ )

 选择所需的动态修改。我们在 M 上使用了氧化 (+15.995 Da)

S/T/Y 上的磷酸化 (+79.966 Da),以及 N 末端的乙酰化 (+42.011 Da)蛋白质。包括额外的动态修改会增加搜索空间,但根据蛋白质处理步骤中使用的条件,这可能是常见的做法。

C。选择合适的静态修饰:肽 N 上的 TMT 6-plex (+229.163 Da)

末端和 K,以及 C 上的甲硫基 (+45.988 Da)。注意使用其他烷化剂例如IAA)将需要对半胱氨酸进行替代修饰。对于超级困惑SILAC,见注 3

3. 调整Consensus Workflow中的设置,启用熔解的下游处理

曲线:一种。仅使用独特的肽段进行定量。 关闭缩放。

C。对于温度范围实验,将 Normalization Mode 设置为 none;重要的是不要按通道标准化丰度,因为蛋白质丰度在全球范围内下降熔化温度。

d. 对于浓度范围实验,可选择将标准化模式设置为无。数据可以 TPP R 包中标准化(见下一节)。我们也选择了正常化 Proteome Discoverer 中并放弃 TPP 包中的标准化。

e. 可选择调整共同隔离阈值或平均报告者 S/N 阈值。降低这些阈值可能会增加量化,但会降低数据质量。

4. 分析完成后,将蛋白质水平定量导出为 *.txt 文件。

B. 曲线拟合

曲线拟合是使用 TPP R 包执行的,该包已被广泛记录(Franken等人,2015 年;Childs等人,2019 年;Kurzawa等人,2020 年)。最近,替代热已经提出了蛋白质组分析数据分析包(Dziekan等人,2020 年),并且用户可以开发自己的自义归一化和曲线拟合方法。输出文件来自必须修改 Proteome Discoverer 以匹配 TPP 包的输入格式。

表 S1-S4代表来自 Proteome Discoverer 的示例输出。

表 S5-S9显示流线型表用作 TPP 包的输入,

表 S10是代表输出。

S1。温度范围,细胞,从 Proteome Discoverer 2.4 软件复制 1 个输出。

S2。温度范围、细胞、Proteome Discoverer 2.4 软件的重复输出 2

S3。温度范围,裂解物,复制 Proteome Discoverer 2.4 软件的 1 个输出。

S4。温度范围、裂解物、Proteome Discoverer 2.4 软件的复制 2 输出。

S5。温度范围,单元,将 1 个修整输入复制到 TPP R 封装。

S6。温度范围,电池,将 2 个修整输入复制到 TPP R 封装。

S7。温度范围,裂解物,将 1 个修整输入复制到 TPP R 包。

S8。温度范围,裂解物,将 2 个修整输入复制到 TPP R 包。

S9。指定实验、条件和重复的示例配置表用于 TPP R 包中的曲线拟合。

S10TPP R 包的示例输出。

C. 预期结果在典型的温度范围实验中,我们检测到 3,000 多种蛋白质,其中约 80% 具有足以进行曲线拟合的量化值。我们的其他蛋白质组学实验鉴定了 4,600-4,800 种蛋白质,表明热分析方法固有的热挑战

减少蛋白质组覆盖。我们在生物副本中进行实验。 2A揭示复制计算出的蛋白质熔解温度的变异性。为了生成参考数据集,我们对属于弓形虫RH/TIR1 菌株的活寄生虫或裂解物进行热分析并使用 SILAC 对样本进行超融合(Harding等人,2020 年;Herneisen等人,2020 年);见注 4。使用协议F 部分中描述的过滤板方法分离聚集体。作为对于其他生物体(Jarzab等人,2020 年)观察到,蛋白质组范围内的热稳定性在裂解物比细胞中的多(图 2B)。为了在们的分析中包括热稳定蛋白质,我们计算了曲线下的数值面积 (AUC) 使用梯形规则(图 2C)。相比之下

熔化温度,这需要至少 50% 的热变性,AUC 度量可以是对具有完整热曲线的所有蛋白质进行计算。图 2D显示了两者之间的关系熔化温度和 AUC。这样的参考数据集可用于选择温度范围和热挑战涉及化合物处理的实验的温度。表 3总结了分布来自两组不同实验的寄生虫和裂解物的熔化温度。首先在 37-67°C 的温度范围内实验融化的寄生虫或裂解物并分离可溶性通过超速离心从聚集体中分离蛋白质(Herneisen等,2020)。第二个实验,此处展示的是在 41-73°C 的温度范围内融化的寄生虫或裂解物并分离用滤板聚合。研究人员可以参考与他们最相似的分布预期的工作流程。检测依赖于化合物的热稳定性,浓度范围实验应在略高于目标蛋白质的熔解温度下进行载体处理条件(Franken等人,2015 年)。在蛋白质靶点未知的情况下,我们选择在对应于中位数的两个温度下执行热挑战和熔化分布的四分之三温度。然而,熔化温度往往取决于蜂窝环境。图 2EF显示了熔解温度和 AUC 值通过基于 MS LOPIT 方法(Barylyuk等,2020)通过亚细胞分配进行分层。一些亚细胞结构,如微管蛋白细胞骨架和蛋白酶体的 20S 亚基,证明特别耐热;检测属于的蛋白质的化合物依赖性热位移这些子结构需要很高的热挑战温度。相比之下,蛋白质细胞核和核仁往往容易沉淀,并使用中值热挑战温度会导致这些亚结构中蛋白质的定量和覆盖率不佳。其他细胞器对细胞制备特别敏感;例如,组件的60S 核糖体在细胞中共熔,但在裂解物中表现出不同的熔解曲线。因此,研究人员应该利用他们对化合物作用机制的观察和预测来选择最合适的热分析参数。

2.弓形虫蛋白质组的熔化行为。(A)熔融温度的再现性在完整细胞熔解实验的两个重复中定量的蛋白质的数量。(B)分布在完整细胞和裂解液的复制品中定量的蛋白质的平均熔解温度温度范围实验。(C)曲线下面积 (AUC) 是如何计算的描述使用蛋白质 CDPK1 的梯形规则进行数值积分。(D)之间的关系两个值都可用的蛋白质的平均解链温度和 AUC。点与较浅的灰色阴影不太适合 S 型熔解曲线 (R 2 < 0.8)。(E)分布通过 Barylyuk 的 LOPIT 分配,细胞或(F)裂解物中的平均解链温度和 AUC等。(2020)。

3.弓形虫裂解物和完整细胞的熔解温度分布

两组不同的实验

笔记

1. 在热挑战和整体蛋白质变性之后,可溶性蛋白质从未折叠的蛋白质聚集体。最初的 CETSA 协议描述了在微型离心机中的离心以20,000 ×(贾法里等人。,2014),随后将其升温至100000 ×g在一个

超速离心机以提高 MS 分析的信噪比(Franken2015)。筛选板可用作替代品,具有更高的吞吐量(Mateus等人2018年)和 2020a;Dziekan等人。, 2020)。可溶性蛋白质与蛋白质分离后聚集体,样品可以在液氮中速冻并在 -80°C 下储存次

个月。

2. 使用改良的 SP3 清理和处理含有可溶性蛋白质的溶液

基于 Hughes等人的协议(2019),它提供了很好的高捕获和吞吐量

适用于稀释和低丰度 TPP 样品。不推荐蛋白质沉淀,因为

它会导致不均匀的样品损失,从而降低熔解曲线的质量。该协议有

针对弓形虫蛋白质样本进行了优化(Harding等人2020 年;Herneisen等人2020 年)和

洗脱时与 TMT 标记兼容。

3. 为了减少 MS 时间和运行间变异性,我们使用了超复杂的 TPP 实验

SILAC,如其他地方所述(Herneisen2020)。这种变化需要增长

TPP 实验之前,将寄生虫在重质和轻质 SILAC 培养基中进行 3 次传代。寄生虫

在不同培养基中生长的被视为生物副本并以相等的权重组合

在烷基化之前。源自重样品的量化值通过以下方式获得

寻找含有重精氨酸 (+10.008 Da) 和重赖氨酸-TMT6plex 的肽

Proteome Discoverer 中的 (+237.177 Da) 修改。

4. MS 蛋白质组学数据已通过以下方式存入 ProteomeXchange Consortium

PRIDE (Perez-Riverol et al ., 2019) 合作伙伴存储库,数据集标识符为 PXD024912

10.6019/PXD024912

食谱

1. DMEM + 3% CFS(用于常规寄生虫传代)每 500 毫升

5 毫升 200 毫米谷氨酰胺

500 微升 10 毫克/毫升庆大霉素

15 毫升小牛血清

通过瓶顶过滤器将补充的 DMEM 过滤到一个干净的玻璃瓶中

用洗涤剂洗过。

2. PBS

100 ml 10× 组织培养级 PBS

900 毫升去离子水

过滤灭菌并在室温下储存

3. 10% IGEPAL CA-630(也称为 NP-40)(50 毫升)

5 毫升 IGEPAL CA-630

45 毫升去离子水

4°C 保存 6 个月

4. 10× CETSA 缓冲液(1 L

50 mM NaCl1.42 M KCl10 mM MgCl 256 mM 葡萄糖、250 mM HEPES pH 7.2

2.922 克氯化钠

105.86 克氯化钾

2 克氯化镁2

10.1 克葡萄糖

59.575 HEPES

加入去离子水至 1 L 并用 KOH 调节 pH 7.2

对溶液进行无菌过滤并在 4°C 下储存

5. CETSA 洗涤缓冲液(1 毫升)

5 mM NaCl142 mM KCl1 mM MgCl 25.6 mM 葡萄糖、25 mM HEPES pH 7.2

100 µl 10× CETSA 缓冲液

900 微升去离子水

6. CETSA 裂解缓冲液 (1 ml)

100 µl 10× CETSA 缓冲液

80 微升 10% IGEPAL CA-360

10 µl Halt 蛋白酶抑制剂

1微升苯甲酸酶

809 µl 去离子水

7. 1 M TCEP 库存解决方案

1 TCEP HCl

3.489 毫升去离子水

-80°C 下储存为 500 µl 等分试样,在 -20°C 下储存为 20 µl 工作等分试样

8. 200 mM MMTS 库存溶液

200 毫克 MMTS

7.924 毫升异丙醇

4°C 下储存为 500 µl 等分试样

9. 缓冲液 A100 毫升)

MS 级水中的 0.1% 甲酸)

100 毫升 MS 级水

100 µl >99% 甲酸

超声 10 分钟

10. 缓冲液 B25 毫升)

80% 乙腈和 0.1% 甲酸)

20 毫升 MS 级乙腈

25 µl >99% 甲酸

5 毫升 MS 级水

超声 10 分钟

致谢

这项研究得到了 NIH 赠款 (1R01AI144369) 给 SL 和国家ALH 的科学基金会研究生研究奖学金 (174530)。我们感谢 E. Shortt技术援助。这项工作改编自 Herneisen等人的协议。(2020) 和哈丁。(2020)。

利益争夺

作者声明没有冲突或竞争利益。

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引用:Herneisen, A. L. and Lourido, S. (2021). Thermal Proteome Profiling to Identify Protein-ligand Interactions in the Apicomplexan Parasite Toxoplasma gondii. Bio-protocol 11(21): e4207. DOI: 10.21769/BioProtoc.4207.
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