Global analysis of circulating immune cells by matrix-assisted laser desorption ionization time-of-flight mass spectrometry

Abstract

Background: MALDI-TOF mass spectrometry is currently used in microbiological diagnosis to characterize bacterial populations. Our aim was to determine whether this technique could be applied to intact eukaryotic cells, and in particular, to cells involved in the immune response.

Methodology/principal findings: A comparison of frozen monocytes, T lymphocytes and polymorphonuclear leukocytes revealed specific peak profiles. We also found that twenty cell types had specific profiles, permitting the establishment of a cell database. The circulating immune cells, namely monocytes, T lymphocytes and polymorphonuclear cells, were distinct from tissue immune cells such as monocyte-derived macrophages and dendritic cells. In addition, MALDI-TOF mass spectrometry was valuable to easily identify the signatures of monocytes and T lymphocytes in peripheral mononuclear cells.

Conclusions/significance: This method was rapid and easy to perform, and unlike flow cytometry, it did not require any additional components such as specific antibodies. The MALDI-TOF mass spectrometry approach could be extended to analyze the cell composition of tissues and the activation state of immune cells.

Figure 1. MALDI-TOF MS spectra of monocytes.

Human monocytes (10⁶ cells per assay) were collected in 10 µl of PBS, and 1 µl was deposited on the MALDI target. Representative MALDI-TOF MS spectra are shown for A, freshly isolated and B, frozen monocytes.

Figure 2. MALDI-TOF MS spectra of circulating cells.

T lymphocytes (A), PMNs (B) and RBCs (C) were isolated from a healthy blood donor. Representative MALDI-TOF MS spectra are shown.

Figure 3. Gel view representation of monocytes and T lymphocytes.

Monocytes and T lymphocytes were isolated from a healthy blood donor. MALDI-TOF MS spectra were analyzed using the ClinProTools software, and the spectra are presented in Gel View representation. Representative spectra are shown with m/z values on the x-axis and the peak intensity (in arbitrary units) on the y-axis. Major differences between monocytes and T lymphocytes are indicated by arrowheads.

Figure 4. Reproducibility of MALDI-TOF MS signatures.

Monocytes (in green), T lymphocytes (in red) and PMNs (in blue) were isolated from ten healthy blood donors. MALDI-TOF MS spectra were analyzed using the ClinProTools software and 2D Peak Distribution View. The relative intensities of the two peaks automatically selected were homogenous among blood donors, and the ellipses represent the standard deviation within each cell population (monocytes, T lymphocytes and PMNs, respectively).

Figure 5. Hierarchical clustering of circulating cells.

Monocytes, T lymphocytes, PMNs and RBCs were isolated from a healthy blood donor. MALDI-TOF MS spectra were analyzed using MeV software. A conventional value of +1 was assigned to the m/z values of spectra (in red) and −1 to m/z positions without peaks (in green).

Figure 6. Dendrogram of 22 eukaryotic cell types.

MALDI-TOF MS was performed on 22 cell types with at least 20 spectra per cell type. An averaged spectrum for each cell type was added to the database using the BioTyper software and the dendrogram creation method.

Figure 7. Efficiency of the database.

A MALDI-TOF MS spectrum of unknown cells (here, monocytes from a blood donor) was compared to the averaged spectrum of monocytes generated from the database using the BioTyper software. The score indicates the identification of the tested cell population as monocytes.