Whole-cell MALDI-TOF-massespektrometri er en nøjagtig og hurtig metode til at analysere forskellige måder makrofagaktivering

Summary

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.

Abstract

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.

Introduction

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.

Protocol

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: The preparation of the biological samples: control macrophages and macrophages stimulated with different agonists. The analysis of each type of sample with technical replicates by whole-cell MALDI-TOF MS. The bioinf…

Representative Results

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).<…

Discussion

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 10^5</…