Цельноклеточная MALDI-TOF масс-спектрометрии является точным и быстрым методом для анализа различных режимах активации макрофагов

Резюме

This protocol describes the use of whole-cell MALDI-TOF mass spectrometry on eukaryotic cells. Here, we illustrate the accuracy of this technique by analyzing the multiple activation states of macrophages in response to their microenvironment.

Аннотация

MALDI-TOF is an extensively used mass spectrometry technique in chemistry and biochemistry. It has been also applied in medicine to identify molecules and biomarkers. Recently, it has been used in microbiology for the routine identification of bacteria grown from clinical samples, without preparation or fractionation steps. We and others have applied this whole-cell MALDI-TOF mass spectrometry technique successfully to eukaryotic cells. Current applications range from cell type identification to quality control assessment of cell culture and diagnostic applications. Here, we describe its use to explore the various polarization phenotypes of macrophages in response to cytokines or heat-killed bacteria. It allowed the identification of macrophage-specific fingerprints that are representative of the diversity of proteomic responses of macrophages. This application illustrates the accuracy and simplicity of the method. The protocol we described here may be useful for studying the immune host response in pathological conditions or may be extended to wider diagnostic applications.

Введение

Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight Mass Spectrometry (MALDI-TOF MS) is a popular mass spectrometry technique to study biological samples. Using a laser beam and an energy-absorbing matrix allows a soft ionization process: the evaporation and genesis of large mostly single-charged biomolecules. This process is called desorption/ionization, justifying the acronym MALDI. These ions are then accelerated by application of voltage and enter a TOF analyzer that allows the separation of these ions and the quantification of their respective masses¹.

MALDI-TOF MS has been extensively used in biology, chemistry, and medicine to identify molecules and biomarkers^2-4 or to monitor post-translational modifications on proteins^5,6. Recently, several groups applied MALDI-TOF MS to the identification of microorganisms from clinical samples^7,8. This microbiological application is now used routinely in the clinical settings. Whole cell MALDI-TOF has many advantages compared to classical applications of MALDI-TOF MS. Samples containing whole cells are directly processed, avoiding time consuming steps to fractionate or separate large amounts of material. Moreover, no characterization of the various peaks is needed: the whole spectrum is considered as a fingerprint of the sample, and matching algorithms compare the tested spectrum with a database of reference spectra.

We and others have applied this whole-cell analysis technique to eukaryotic cells. Many applications may be derived from this technique: (1) identify the main cell types from a mixed sample^9-11; (2) assess the viability of cell cultures over time (including quality control industrial applications)¹²; (3) monitor activation states of a single cell type¹³; (4) assess the malignant transformation of a clinical sample^14,15.

Here, we describe the use of whole-cell MALDI-TOF MS to explore the various polarization phenotypes of macrophages in response to cytokines or heat-killed bacteria. Macrophages play a pivotal role in the immune response to microbial pathogens. They detect infectious agents in the tissues through pattern recognition receptors able to detect conserved microbial patterns, such as lipopolysaccharide (LPS)¹⁶. Macrophages are professional antigen-presenting cells that interact with T cells to mount the adaptive immune response. T cells influence macrophages by releasing cytokines that either reinforce or regulate the microbicidal activity of macrophages. By analogy to the Th1/Th2 lymphocyte polarization, inflammatory, microbicidal, and tumoricidal macrophages have been classified into M1 macrophages and immunoregulator macrophages as M2 macrophages^17-19. The term M1 refers to the classical activation of macrophages by type I cytokines, such as interferon (IFN)-γ and tumor necrosis factor (TNF), or bacterial products, such as LPS^18,20-23, whereas macrophages activated by alternative pathways (interleukin (IL)-4, IL-10, Transforming Growth Factor-β1 are considered M2 macrophages^19,24,25. The high phenotypic and functional plasticity of macrophages in response to their microenvironment renders these macrophages useful to analyze subtle changes by a MALDI-TOF MS approach.

протокол

In the present protocol, the whole-cell MALDI-TOF technique is used to obtain a mass spectrum considered as a fingerprint of the sample. A bioinformatic analysis allowed the comparison and the classification of these fingerprints. There were three main parts in this protocol:

1. The preparation of the biological samples: control macrophages and macrophages stimulated with different agonists.
2. The analysis of each type of sample with technical replicates by whole-cell MALDI-TOF MS.
3. The bioinformatics analysis of raw data.

Prepare sterile solutions for cell isolation and culture. Prepare and store all reagents at 4 °C

1. Preparation of Human Monocytes

1. Prepare cell culture medium. Add 55 ml of human serum AB+ or fetal bovine serum (FBS) and 5 ml of the antibiotic solution (penicillin at 10,000 UI/ml and streptomycin at 10,000 μg/ml; final concentration of 100 UI/ml for penicillin and 100 μg/ml for streptomycin) to RPMI 1640 medium (500 ml).
   1. Isolate peripheral blood mononuclear cells (PBMCs) from leukopacks (leukocyte concentrates).
      1. Prepare 50 ml tubes containing 15 ml Ficoll. Dilute the blood in saline (vol/vol, 1/10). Deposit 30 ml diluted PBMCs on Ficoll as previously described²⁶.
      2. Centrifuge at 700 x g for 20 min. Recover PBMCs at the interface between Ficoll (density of 1.077 g/ml) and diluted plasma. Dilute PBMCs in culture medium and centrifuge at 300 x g for 5 min.
   2. Prepare CD14+ monocytes from PBMCs using magnetic beads coated with anti-CD14 antibodies.
      Note: Keep products and cells at 4 °C until monocyte obtention.
      1. Prepare running buffer consisting of phosphate-buffered saline (PBS) pH 7.2, 0.5% bovine serum albumin (BSA) and 2 mM ethylenediaminetetraacetic acid (EDTA).
      2. Gently dissociate pelleted PBMCs (10⁷ cells per assay) into 80 μl of running buffer.
      3. Add 20 μl CD14 MicroBeads to PBMCs. Mix and incubate PBMCs for 15 min. Wash PBMCs with 5 ml of running buffer and centrifuge at 300 x g for 5 min. Gently dissociate pelleted PBMCs into 500 μl of running buffer for 10⁸ PBMCs.
         Note: use 500 μl of running buffer for PBMC concentrations lower than 10⁸ PBMCs.
      4. Proceed to magnetic separation.
         Note: for less than 2 x 10⁸ PBMCs, use MS column; for more than 2 x 10⁸ PBMCs and less than 2 x 10⁹ PBMCs, use LS column.
      5. Rinse precolumn and column with running buffer (500 μl for MS column or 3 ml for LS column). Add PBMCs into the precolumn (wait for unlabeled cells to pass through the column). Rinse 3 times with running buffer (500 μl for MS column or 3 ml for LS column). Remove the precolumn.
      6. Place the column on 15 ml tubes. Add running buffer (1 ml for MS column or 5 ml for LS column) on the column and elute CD14+ cells by applying a pressure on the column.
      7. Centrifuge the eluate at 300 x g for 5 min. Discard the supernatant. Wash monocyte pellet with 10 ml of culture medium.
         Note: Analyze the purity of monocyte preparation using anti-CD14 antibodies and flow cytometry (classically higher than 95%).
2. Differentiation of monocytes into macrophages.
   1. Incubate monocytes (10⁶ monocytes per well in 6 well plates) in 3 ml of culture medium containing 10% human serum AB+ at 37 °C. After 4 days, replace the culture medium containing human serum by 3 ml culture medium containing 10% FBS for 3 additional days.
   2. Identify the obtained cell population as monocyte-derived macrophages (MDMs) by flow cytometry (see Figure 1).
      1. Replace the culture medium by 3 ml PBS. Scrape MDMs with a rubber policeman and collect MDM suspensions. Centrifuge at 400 x g for 5 min. Add PBS containing 2% BSA to pelleted MDMs and gently agitate cell suspension. Adjust the cell concentration (10⁶ MDMs in 200 ml PBS) and incubate at 4 °C.
      2. Add 10 ml of anti-CD14 antibodies and 10 ml of anti-CD68 antibodies to MDM suspension and incubate at 4 °C for 20 min in the dark.
         Note: these antibodies must be labeled with two different fluorochromes consistent with flow cytometry analysis. For example, anti-CD14 antibodies may be conjugated with phycoerythrin coupled (for monocytes staining) and anti-CD68 antibodies with Alexa Fluor 647 coupled (for macrophages staining).
      3. Centrifuge MDMs at 400 x g for 5 min. Remove supernatants. Gently dissociate the cell pellet and incubate MDMs in 250 ml 3% paraformaldehyde (PFA) for 15 min at room temperature. Centrifuge PFA-fixed MDMs at 400 x g for 5 min. Wash MDMs with 3 ml PBS and centrifuge MDMs at 400 x g for 5 min. Gently dissociate MDM pellet in 400 ml PBS.
      4. Analyze the differentiation of monocytes (that express CD14 but not CD68) into monocytes (that express CD68 but not CD14). Note: classically more than 95% of cells are MDM.
3. 18 hr stimulation of MDMs.
   1. Replace the culture medium of adherent MDMs by fresh 3 ml culture medium (containing 10% FBS).
   2. To induce a M1 or M2 polarization, use the following human recombinant cytokines: IFN-γ and TNF for M1 polarization, and IL-4 for M2 polarization. M1 macrophages are also obtained by stimulation with LPS. Note that stock-solutions of cytokines are conserved at -80 °C and that the stock-solution of LPS is conserved at -20 °C.
      1. Dilute the stock-solution of IFN-gγ (1 mg/ml) at 1/100 in culture medium to obtain a dilution of 10 ng/ml. Add 6 μl of this IFN-γ dilution to MDMs to obtain a final IFN-γ concentration of 20 ng/ml.
      2. Dilute the stock-solution of TNF (1 mg/ml) at 1/100 in culture medium to obtain a dilution of 10 ng/ml. Add 6 μl of this TNF dilution to MDMs to obtain a final TNF concentration of 20 ng/ml.
      3. Dilute the stock-solution of IL-4 (1 mg/ml) at 1/100 in culture medium to obtain a dilution of 10 ng/ml. Add 6 μl of this IL-4 dilution to MDMs to obtain a final IL-4 concentration of 20 ng/ml.
      4. Add 3 μl of the stock-solution of LPS (1 mg/ml) to MDMs to obtain a final LPS concentration of 1 μg/ml.
   3. To stimulate MDMs with bacteria, use heat-killed bacteria. Wash living bacteria with PBS and heat them at 95 °C for 1 hr. Use Orientia tsutsugamushi (strain Kato (CSUR R163), Mycobacterium bovis (Bacillus Calmette-Guérin, BCG CIP strain 671203) and Coxiella burnetii (Nine Mile in phase I) were because these pathogens are known to infect macrophages. Thus, prepare a O. tsutsugamushi suspension at 10⁹ bacteria/ml. Add 50 μl to MDMs . Repeat the same step for M. bovis and C. burnetii.
4. Preparation of biological samples for MALDI-TOF MS.
   1. Wash stimulated MDMs with PBS without Ca²⁺ or Mg²⁺ (cells may agglutinate in the presence of Ca²⁺ or Mg²⁺). Scrape MDMs with a rubber policeman and collect MDM suspensions. Centrifuge MDMs. Wash again MDMs in PBS without Ca²⁺ or Mg²⁺ at 400 x g for 5 min to discard FBS contamination.
   2. Adjust the cell concentration (2 x 10⁶ MDMs per assay). Centrifuge MDMs at 400 x g for 5 min and discard supernatants. Collect cell pellets in 20 μl of PBS without Ca²⁺ or Mg²⁺.
   3. Analyze samples immediately or store them in PBS at -80 °C before analysis.

2. Analysis of Macrophages by Whole-Cell Maldi-TOF MS

1. Preparation of CHCA matrix.
   1. Add 500 μl of acetonitrile, 250 μl 10% trifluoroacetic acid, and 250 μl of high-performance liquid chromatography (HPLC) water to a vial. Dilute 10 mg of CHCA in this solution to a final concentration of 10 mg/ml. Mix and sonicate for at least 20 min.
      Note: sonication will improve the saturation of the matrix but this step is not mandatory.
   2. Centrifuge at 13,000 x g for 5 min. Discard the pellet and keep the supernatant.
      Note: prepare the matrix solution just before use. A matrix solution that contains crystals does not allow a good ionization of sample molecules, and this may affect the quality of the spectra.
2. Preparation of MALDI steel target.
   1. Moisten the polished steel target with hot tap water. Rub with precision wipe paper. Add 70% ethanol and rub. Rinse with water by rubbing. Add 70% ethanol and rub with precision paper.
   2. Immerse the target in 70% ethanol and sonicate for at least 15 min. Cover the target with 500 μl to 1 ml of trifluoroacetic acid at 80%. Rub and wipe with precision paper. Rinse with HPLC water without rubbing. Dry the target at room temperature.
      Note: an improperly cleaned target may affect the quality of the spectra.
3. Preparation of deposits.
   1. Place the clean target on a horizontal and level support to obtain uniform deposits.
   2. Gently thaw MDM samples on ice. Note: rapid and vigorous thawing may alter samples, thus affecting the quality of the spectra. Homogenize MDMs (by pipetting back and forth the cell suspension) before deposition of 1 μl (containing approximately 1 x 10⁵ cells) on the MALDI target. Add 1 μl of the MALDI matrix solution on the sample. Avoid mixing spot with the pipette. Mixing spots with pipettes alters the quality of the spectra. Depose 12-16 samples/assay (technical replicates).
   3. Evaporate spontaneously at room temperature. Note: evaporation takes place gradually and leads to the formation of matrix/sample crystals. The deposits may be immediately analyzed or stored in the dark for several days before analysis (up to 2 weeks).
   4. User should control that the cytokines or heat-killed bacteria used in the experiment do not provide peaks within the range of stimulated macrophages' spectra. Here, we checked that the cytokines and heat-killed bacteria alone did not provide any signal within the studied range (0-20 kDa).
4. Mass spectrometer tuning and data acquisition.
   1. Insert the steel target containing samples in the mass spectrometer.
   2. Configure the mass spectrometer and run data acquisition. Use the default configuration for automated acquisition of the data.
   3. Note: a detailed view of the configuration of flexControl software is given in the appendix/Table 1. This may vary according to mass spectrometer and software used.

3. Bioinformatic Analysis

Note: the bioinformatic analysis was performed using the free and open source statistical analysis software R, along with specific analysis libraries (MALDIquant). R can be downloaded freely from its website http://cran.r-project.org/. A detailed description of the script is provided as supplementary material.

1. Loading and pretreatment of raw data.
   1. Store raw data on the computer associated with the mass spectrometer in multiple files and folders. Retrieve the root folder of the experiment with all subfolders. Copy root folder to personal computer for analysis.
   2. Use readBrukerFlexData and MALDIquant libraries to load and analyze raw data.
      Note: readBrukerFlexData allows the loading of raw data from the mass spectrometer into a specific object in R for further analysis. See MALDIquant description for further information²⁷.
   3. Analyze generated spectra.
      Note: each spectrum consists of a list of peaks with their respective masses and intensities (relative abundance). Use the square root of the intensities to enhance the graphical visualization of the spectra. Correct the background using a statistic-sensitive nonlinear peak-clipping algorithm for baseline estimation²⁷. Use a signal-to-noise ratio of 6 to detect peaks. Consider that the detected peaks are similar across spectra when the mass/charge (m/z) values are within a 2,000 ppm window.
2. Score definition and computation. Classify the spectra using a hierarchical clustering with a ward algorithm for agglomeration and a dissimilarity matrix based on the Jaccard distance.
   Note: the Jaccard index measures the similarity between Boolean sample sets (i.e. the presence/absence of a list of peaks).
3. Comparison of spectra and viewing.
   1. Analyze raw spectra plots. Note: x-axis represents the m/z ratio (in Daltons) and the y-axis represents the intensity (relative abundance).
   2. Assess the similarity between spectra by hierarchical clustering. Represent similarity (or divergence) as dendrogram.
   3. Assess the reproducibility by the mean of virtual gel-view representation. Note: virtual gel-view representation is a modified heatmap plot where relative abundance is color-coded with increasing intensities of blue.

Результаты

The aim of the present protocol is to demonstrate the accuracy of whole-cell MALDI-TOF MS to assess the responsiveness of macrophages to their microenvironment.

Figure 1 describe preparation of stimulated macrophages from blood samples. Figure 2 represents the analysis of monocytes and MDMs by flow cytometry. Note that monocytes expressed CD14 but not CD68 (Figure 1A). Conversely, MDMs expressed CD68 but not CD14 (Figure 1B).<...

Обсуждение

This protocol describes the use of MALDI-TOF-MS on eukaryotic whole cells. Here, we illustrate the accuracy of the method by analyzing the multiple activation states of macrophages in response to their microenvironment.

The success of the protocol relies on few critical steps. First, any solution contaminant may alter the spectra. For example, it is important to wash cells in PBS to remove culture medium and serum proteins before deposition on the target. A cell concentration of 1 x 10⁵