Background: Bacille Calmette-Guerin (BCG) is currently the only available vaccine against tuberculosis (TB) and comprises a heterogeneous family of sub-strains with genotypic and phenotypic differences. The World Health Organization (WHO) affirms that the characterization of BCG sub-strains, both on genomic and proteomic levels, is crucial for a better comprehension of the vaccine. In addition, these studies can contribute in the development of a more efficient vaccine against TB. Here, we combine two-dimensional electrophoresis (2DE) and mass spectrometry to analyse the proteomic profile of culture filtrate proteins (CFPs) from M. bovis BCG Moreau, the Brazilian vaccine strain, comparing it to that of BCG Pasteur. CFPs are considered of great importance given their dominant immunogenicity and role in pathogenesis, being available for interaction with host cells since early infection. Results: The 2DE proteomic map of M. bovis BCG Moreau CFPs in the pH range 3 - 8 allowed the identification of 158 spots corresponding to 101 different proteins, identified by MS/MS. Comparison to BCG Pasteur highlights the great similarity between these BCG strains. However, quantitative analysis shows a higher expression of immunogenic proteins such as Rv1860 (BCG1896, Apa), Rv1926c (BCG1965c, Mpb63) and Rv1886c (BCG1923c, Ag85B) in BCG Moreau when compared to BCG Pasteur, while some heat shock proteins, such as Rv0440 (BCG0479, GroEL2) and Rv0350 (BCG0389, DnaK), show the opposite pattern. Conclusions: Here we report the detailed 2DE profile of CFPs from M. bovis BCG Moreau and its comparison to BCG Pasteur, identifying differences that may provide relevant information on vaccine efficacy. These findings contribute to the detailed characterization of the Brazilian vaccine strain against TB, revealing aspects that may lead to a better understanding of the factors leading to BCG's variable protective efficacy against TB.

Overexpression of human epidermal growth factor receptor-2 (HER-2) occurs in 20% of all breast cancer subtypes, especially those that present the worst prognostic outcome through a very invasive and aggressive tumour. HCC-1954 (HER-2+) is a highly invasive, metastatic cell line, whereas MCF-7 is mildly aggressive and non-invasive. We investigated membrane proteins from both cell lines that could have a pivotal biological significance in metastasis. Membrane protein enrichment for HCC-1954 and MCF-7 proteomic analysis was performed. The samples were analysed and quantified by mass spectrometry. High abundance membrane proteins were confirmed by Western blot, immunofluorescence, and flow cytometry. Protein interaction prediction and correlations with the Cancer Genome Atlas (TCGA) patient data were conducted by bioinformatic analysis. In addition, β1 integrin expression was analysed by Western blot in cells upon trastuzumab treatment. The comparison between HCC-1954 and MCF-7 membrane-enriched proteins revealed that proteins involved in cytoskeleton organisation, such as HER-2, αv and β1 integrins, E-cadherin, and CD166 were more abundant in HCC-1954. β1 integrin membrane expression was higher in the HCC-1954 cell line resistant after trastuzumab treatment. TCGA data analysis showed a trend toward a positive correlation between HER-2 and β1 integrin in HER-2+ breast cancer patients. Differences in protein profile and abundance reflected distinctive capabilities for aggressiveness and invasiveness between HCC-1954 and MCF-7 cell line phenotypes. The higher membrane β1 integrin expression after trastuzumab treatment in the HCC-1954 cell line emphasised the need for investigating the contribution of β1 integrin modulation and its effect on the mechanism of trastuzumab resistance.

Helicobacter pylori infection affects over 50% of the world’s population and leads to chronic inflammation and gastric disorders, being the main pathogen correlated to gastric cancer development. Increasing antibiotic resistance levels are a major global concern and alternative treatments are needed. Soybean peptides and other compounds might be an alternative in the treatment to avoid, eradicate and/or control symptoms of H. pylori infection. This study aimed to characterize a lunasin-enriched soybean extract (LSE) using proteomics tools and to evaluate its antioxidant, anti-inflammatory and antibacterial properties against H. pylori infection. By LC–MS/MS analysis, 124 proteins were identified, with 2S albumin (lunasin and large-chain subunits) being the fourth most abundant protein (8.9%). Lunasin consists of 44 amino acid residues and an intramolecular disulfide bond. LSE at a low dose (0.0625 mg/mL) reduced ROS production in both H. pylori-infected and non-infected AGS gastric cells. This led to a significant reduction of 6.71% in the levels of pro-inflammatory interleukin (IL)-8. LSE also showed antibacterial activity against H. pylori, which can be attributed to other soybean proteins and phenolic compounds. Our findings suggest that LSE might be a promising alternative in the management of H. pylori infection and its associated symptoms.

Background/Objectives: Tarin exhibits immunomodulatory and antiproliferative properties against several tumor cell lines. Nano-encapsulation in liposomes enhances its therapeutic potential by improving protein stability, bioavailability, and sustained release. Previous studies demonstrated that nano-encapsulated tarin induces cell cycle arrest, migration inhibition, apoptosis, and autophagy in triple-negative breast cancer cells; however, the molecular mechanisms underlying these effects remain poorly understood. To investigate the proteomic response elicited by nano-encapsulated tarin, MDA-MB-231 cells were treated for 24 and 48 h. Methods: Intracellular proteins were extracted, digested with trypsin, and analyzed by label-free LC-2D-MS/MS using HDMSE acquisition. Differentially expressed proteins were identified and quantified using the Progenesis QI platform, and then functional classification and pathway enrichment analyses were performed. Results: A total of 2818 proteins were identified, of which 2150 displayed time-dependent modulation following treatment. After 24 h, cells exhibited an adaptive stress response profile characterized by increased DNA repair proteins (CHEK1, CDK12), migration/remodeling factors (LAMA4, CTTN, A2M), and immune/cell cycle regulators (PER2, HLA-B), while antioxidant proteins (SOD1, GPX1) and BRCA1 were reduced, indicating oxidative stress and DNA damage. After 48 h, the proteomic profile shifted toward cell death, with increased PARK7, OPA1, ATL3, and CASP8 expression, disruption of DNA repair and cell cycle regulators (CHEK1, CDK12, MSH6, KIF2C), and decreased migration-related proteins (LAMA4, CTTN, ITGB3, A2M). Conclusions: Nano-encapsulated tarin promotes a time-dependent transition from early adaptive stress responses to apoptosis, autophagy, cell cycle disruption, and loss of migratory capacity. These findings provide novel insights into the molecular mechanisms underlying tarin antitumoral activity and support its potential as a promising therapeutic strategy against triple-negative breast cancer.

Reliable quantitative proteomic profiling is essential for understanding drug-induced molecular responses in cancer cells. Efficient extraction and digestion of proteins, especially membrane-associated proteins, remain major analytical challenges. Herein, we validated a surfactant-assisted protein extraction and digestion workflow for comprehensive proteome profiling of MDA-MB-231 cell, to establish a baseline reference for drug-response studies at different time points (0, 24, and 48 h). Total proteins were extracted using acid-labile surfactant - RapiGest™ SF - and trypsinized, followed by two-dimensional liquid chromatography coupled to high-definition data-independent mass spectrometry (2D-LC-HDMSE). Data are available via ProteomeXchange with identifier PXD076864. Label-free quantitative analysis identified 2,818 proteins, achieving performance comparable or superior to urea-based and filter-aided sample preparation (FASP) protocols under similar chromatographic conditions. The employed workflow was able to discriminate distinct cellular stages associated with culture establishment and progression over 48h. This temporal proteomic profiling enabled the mapping of protein expression modulated during adhesion, cytoskeletal reorganization, metabolic adaptation, and high-density growth. Data indicate that at high-density culture, intrinsic pathways for immunogenicity are downregulated, a typical evading mechanism of tumor masses and metastatic niches. This study provides a high-depth, reproducible, and MS-compatible strategy to characterize MDA-MB-231 baseline proteome, which may be a reference for drug-response studies.