Autophagy inhibition through small-molecule inhibitors is one of the approaches to increase the efficiency of radiotherapy in oncological patients. A new inhibitor—Lys05—with the potential to accumulate within lysosomes and to block autophagy was discovered a few years ago. Several studies have addressed its chemosensitizing effects but nothing is known about its impact in the context of ionizing radiation (IR). To describe its role in radiosensitization, we employed radioresistant human non-small cell lung carcinoma cells (H1299, p53-negative). Combined treatment of H1299 cells by Lys05 together with IR decreased cell survival in the clonogenic assay and real-time monitoring of cell growth more than either Lys05 or IR alone. Immunodetection of LC3 and p62/SQSTM1 indicated that autophagy was inhibited, which correlated with increased SQSTM1 and decreased BNIP3 gene expression determined by qRT-PCR. Fluorescence microscopy and flow cytometry uncovered an accumulation of lysosomes. Similarly, transmission electron microscopy demonstrated the accumulation of autophagosomes confirming the ability of Lys05 to potentiate autophagy inhibition in H1299 cells. We report here for the first time that Lys05 could be utilized in combination with IR as a promising future strategy in the eradication of lung cancer cells.

Mass spectrometry (MS) is a powerful and sensitive method often used for the identification of phosphoproteins. However, in phosphoproteomics, there is an identified need to compensate for the low abundance, insufficient ionization, and suppression effects of non-phosphorylated peptides. These may hamper the subsequent liquid chromatography–mass spectrometry/mass spectrometry (LC–MS/MS) analysis, resulting in incomplete phosphoproteome characterization, even when using high-resolution instruments. To overcome these drawbacks, we present here an effective microgradient chromatographic technique that yields specific fractions of enriched phosphopeptides compatible with LC–MS/MS analysis. The purpose of our study was to increase the number of identified phosphopeptides, and thus, the coverage of the sample phosphoproteome using the reproducible and straightforward fractionation method. This protocol includes a phosphopeptide enrichment step followed by the optimized microgradient fractionation of enriched phosphopeptides and final LC–MS/MS analysis of the obtained fractions. The simple fractionation system consists of a gas-tight microsyringe delivering the optimized gradient mobile phase to reversed-phase microcolumn. Our data indicate that combining the phosphopeptide enrichment with the microgradient separation is a promising technique for in-depth phosphoproteomic analysis due to moderate input material requirements and more than 3-fold enhanced protein identification.