Quantitative whole-cell MALDI-TOF MS fingerprints distinguishes human monocyte sub-populations activated by distinct microbial ligands

Damien Portevin, Valentin Pflüger, Patricia Otieno, René Brunisholz, Guido Vogel, Claudia Daubenberger

Conventionally, human monocyte sub-populations are classified according to surface marker expression into classical (CD14(++)CD16(-)), intermediate (CD14(++)CD16(+)) and non-classical (CD14(+)CD16(++)) lineages. The involvement of non-classical monocytes, also referred to as proinflammatory monocytes, in the pathophysiology of diseases including diabetes mellitus, atherosclerosis or Alzheimer's disease is well recognized. The development of novel high-throughput methods to capture functional states within the different monocyte lineages at the whole cell proteomic level will enable real time monitoring of disease states. We isolated and characterized (pan-) monocytes, mostly composed of classical CD16(-) monocytes, versus autologous CD16(+) subpopulations from the blood of healthy human donors (n = 8) and compared their inflammatory properties in response to lipopolysaccharides and M.tuberculosis antigens by multiplex cytokine profiling. Following resting and in vitro antigenic stimulation, cells were recovered and subjected to whole-cell mass spectrometry analysis. This approach identified the specific presence/absence of m/z peaks and therefore potential biomarkers that can discriminate pan-monocytes from their CD16 counterparts. Furthermore, we found that semi-quantitative data analysis could capture the subtle proteome changes occurring upon microbial stimulation that differentiate resting, from lipopolysaccharides or M. tuberculosis stimulated monocytic samples. Whole-cell mass spectrometry fingerprinting could efficiently distinguish monocytic sub-populations that arose from a same hematopoietic lineage. We also demonstrate for the first time that mass spectrometry signatures can monitor semi-quantitatively specific activation status in response to exogenous stimulation. As such, this approach stands as a fast and efficient method for the applied immunology field to assess the reactivity of potentially any immune cell types that may sustain health or promote related inflammatory diseases.