Increased oxidative stress would lead to an increase in the expression of antioxidant enzymes, such as GSTP1 and SOD2. Some antioxidant enzymes, such as PRDX6, were reported to be decreased during oxidative stress, and thus, their reduced expression in alcohol-fed female mice could suggest a state of enhanced oxidative stress. These changes in protein expression are consistent with an increased state of oxidative stress in female mice than that in males after ethanol exposure. Other significantly changed proteins between male and female mice are shown in Fig. 4. Together, the data presented herein suggested relative lower metabolic reactions, higher oxidative stress processes and higher cell development processes in the liver of female mice compared to male mice in response to chronic alcohol feeding and these differences may help to clarify the basis of why the female gender is more susceptible to alcohol. Protein–protein interaction studies, which are crucial for understanding many biological processes, are not being performed to a satisfactory extent at present. Most often, protein–protein interactions are determined by researches only for very specific biological processes, and global protein–protein interaction networks of only few model organisms have been investigated on the basis of medium- or high-throughput experiments. The B2H and yeast two-hybrid systems are the most commonly used tools to study protein– protein interactions. They are powerful techniques, but intrinsically carry major limitations. A large caveat is that the screening is far from physiological conditions, with a high rate of Varenicline false-positive and -negative results. To increase the number of genes encoding potentially interactive protein partners, the two-hybrid system was modified to incorporate 3 different genes, allowing Indacaterol independent expression and interaction of mycobacterial proteins in Escherichia coli. This three-hybrid system was used for the RD1 complex of M. tuberculosis. However, this method can decipher only tri-protein complexes, establishing that its reliability does not reach global and complex protein–protein interactions and it must be supported by other techniques. There is also a dedicated two-hybrid assay, called the mycobacterial protein fragment complementation assay, which is based on reconstitution of murine dihydrofolate reductase and allows investigation of protein–protein interactions in M. smegmatis host. This method presents a clear advantage of studying protein complex formation under physiological conditions and was successfully implemented both for soluble as well as for membrane proteins. In a different study, computer analysis of the interactome was used to analyse communication between a drug environment and proteins involved in resistance to them to identify the most plausible paths that triggered the emergence of drug resistance. Here we propose a single-epitope affinity purification technique combined with LC–MS/MS as a screening method for studying protein–protein interactions specifically in Mycobacterium. To determine the most efficient epitope, we designed 4 constructs containing 4 different fusion tags to be tested with targeted proteins. For further experiments, we selected FLAG, haemagglutinin, protein A and enhanced green fluorescent protein epitopes.We employed a localization and affinity purification method coupled with tandem mass spectrometry, an efficient tool to investigate protein–protein interactions in living cells under close-to-physiological conditions.