Otimização da extração proteica de Mycobacterium tuberculosis: avaliação de tampões de extração para aplicação em cromatografia líquida com espectrometria de massas (LC-MS)

Abstract

Mycobacterium tuberculosis is the etiological agent of tuberculosis (TB), one of the infectious diseases responsible for the highest number of deaths worldwide. It is an obligate intracellular bacterium capable of infecting host macrophages. In addition to the expression of proteins associated with pathogenicity, it possesses survival mechanisms such as a rigid, lipid-rich cell wall. While this cell wall contributes to the structural stability of the microorganism, it also represents a major challenge for protein extraction methods, requiring the use of effective and safe extraction buffers to overcome the rigidity and impermeability of the M. tuberculosis cell wall. The present study aimed to compare the efficiency of guanidine hydrochloride (GdnHCl)- and urea-based extraction buffers for application in Liquid Chromatography-Mass Spectrometry (LC-MS) proteomic analyses. Clinical isolates were cultivated in supplemented Middlebrook 7H9 medium. Cell lysis was performed by vortexing with 0.5 mm zirconia beads using denaturing buffers containing GdnHCl or urea, in assays conducted in triplicate. Protein extracts were quantified by Qubit fluorimetry, followed by reduction, alkylation, and enzymatic digestion with trypsin. The resulting peptides were desalted by solid-phase extraction (SPE) and lyophilized. Proteomic analyses were carried out by liquid chromatography coupled to mass spectrometry (LC-MS) using an Orbitrap system with a label-free approach for data acquisition and analysis. The GdnHCl-based buffer yielded higher protein concentrations, with greater mean values across all replicates (1.71 μg/μL) (p < 0.05) compared to the urea-based buffer (0.788 μg/μL). Furthermore, it was possible to identify 1482 proteins with the GdnHCl buffer, demonstrating better chromatographic performance, evidenced by a greater number of peaks. The higher chaotropic potential of GdnHCl compared to urea may account for its enhanced ability to promote protein denaturation and solubilization. In conclusion, the GdnHCl-based buffer combined with the exclusive use of 0.5 mm zirconia beads constitutes a biosafe and efficient method for protein extraction from M. tuberculosis, with suitable applicability for LC- MS/MS-based proteomic analyses.