Introduction: Sample preparation is a key step of the proteomics workflow, determining the overall reproducibility and robustness of the analysis. Although previous studies have assessed the performance of different sample preparation protocols, the majority of them focused on HeLa cell line as model. Also, there is a scarcity of proteomic optimization workflows tailored for macrophages – a model cell line for studies involving the immune system, inflammation, atherosclerosis and obesity.

Methods: THP-1 monocytes were cultured as usual, then differentiated to macrophages with 200 nM phorbol myristate (PMA), followed by incubation at 37 °C and 5% CO₂ for three days. Adherent macrophages were lysed with SDC buffer (100 mM ammonium bicarbonate, 1% SDC and cOmplete protease inhibitor cocktail). The resulting proteins were quantified and digested following in-solution, in-gel or Filter-Aided Sample Preparation (FASP) protocols, then analyzed by nanoLC-MS/MS (ThermoScientific EASY-nLC 1200 coupled to an Orbitrap Fusion Lumos mass spectrometer) in a data-dependent acquisition mode, in 104 min runs. HeLa protein digest standard was acquired from ThermoFisher and analyzed by nanoLC-MS/MS under the same conditions as for the macrophage digests. For both sample types, we analyzed at least three replicates. Raw files were processed in MaxQuant then analyzed using Perseus, String, and WebGestalt.

Results: Key differences in protein composition between HeLa and differentiated THP-1 (macrophages) cell lines can be visualized (Figure 1). HeLa cell"s proteome shows enrichment in biological processes related to DNA replication, telomerase activity and transcription. The most enriched proteins are related to the nucleus cellular compartment and biological processes – all characteristic of its origin as a cervical cancer cell line. On the other hand, an analysis of macrophages" proteome reveals their role in immunity and infection, with enrichment in proteins associated with cellular responses to bacterial infection or inflammation, and a relationship with NADPH oxidase complex, indicating an important role in oxidative stress.

Conclusions: The striking differences between the proteome of HeLa and differentiated THP-1 cells reinforce the need for an optimized sample preparation protocol for macrophages. Our comprehensive assessment of digestion workflows for macrophages identified proteins specific to this cell line, enabling further studies to understand the role of these immune cells in inflammatory conditions.