Search Results (10)

Acinetobacter baumannii is a multidrug-resistant bacterium that has gained significant attention in recent years due to its involvement in a growing number of hospital-acquired infections. The World Health Organization has classified it as a critical priority pathogen, underscoring the urgent need for new therapeutic strategies. Post-translational modifications (PTMs), such as phosphorylation, play essential roles in various bacterial processes, including antibiotic resistance, virulence or biofilm formation. Although proteomics has increasingly enabled their characterization, the identification of phosphorylated peptides remains challenging, primarily due to the enrichment procedures. In this study, we focused on characterizing serine, threonine, and tyrosine phosphorylation in the A. baumannii ATCC 17978 strain. We optimized three parameters for phosphopeptide enrichment using titanium dioxide (TiO₂) beads (number of enrichment fractions between the phosphopeptides and TiO₂ beads, the quantity peptides and type of loading buffer) to determine the most effective conditions for maximizing phosphopeptide identification. Using this optimized protocol, we identified 384 unique phosphorylation sites across 241 proteins, including 260 novel phosphosites previously unreported in A. baumannii. Several of these phosphorylated proteins are involved in critical bacterial processes such as antimicrobial resistance, biofilm formation or pathogenicity. We discuss these proteins, focusing on the potential functional implications of their phosphorylation. Notably, we identified 34 phosphoproteins with phosphosites localized at functional sites, such as active sites, multimer interfaces, or domains important for structural integrity. Our findings significantly expand the current phosphoproteomic landscape of A. baumannii and support the hypothesis that PTMs, particularly phosphorylation, play a central regulatory role in its physiology and pathogenic potential. Full article

Protein phosphorylation is one of the most common and important post-translational modifications (PTMs) and is highly involved in various biological processes. Ideal adsorbents with high sensitivity and specificity toward phosphopeptides with large coverage are therefore essential for enrichment and mass spectroscopy-based phosphoproteomics analysis. In this study, a newly designed IMAC adsorbent composite was constructed on the graphene matrix coated with mesoporous silica. The outer functional 3D-network layer was prepared by free radical polymerization of the phosphonate-functionalized vinyl imidazolium salt monomer and subsequent metal immobilization. Due to its unique structural feature and high content of Ti⁴⁺ ions, the resulting phosphonate-immobilized adsorbent composite G@mSiO₂@PPFIL-Ti⁴⁺ exhibits excellent performance in phosphopeptide enrichment with a low detection limit (0.1 fmol, tryptic β-casein digest) and superior selectivity (molar ratio of 1:15,000, digest mixture of β-casein and bovine serum albumin). G@mSiO₂@PPFIL-Ti⁴⁺ displays high tolerance to loading and elution conditions and thus can be reused without a marked decrease in enrichment efficacy. The captured phosphopeptides can be released globally, and mono-/multi-phosphopeptides can be isolated stepwise by gradient elution. When applying this material to enrich phosphopeptides from human lung cell lysates, a total of 3268 unique phosphopeptides were identified, corresponding to 1293 phosphoproteins. Furthermore, 2698 phosphorylated peptides were found to be differentially expressed (p < 0.05) between human lung adenocarcinoma cells (SPC-A1) and human normal epithelial cells (Beas-2B), of which 1592 were upregulated and 1106 were downregulated in the cancer group. These results demonstrate the material’s superior enrichment efficiency in complex biological samples. Full article

Toxoplasma gondii, an obligate intracellular parasite, has the ability to invade and proliferate within most nucleated cells. The invasion and destruction of host cells by T. gondii lead to significant changes in the cellular signal transduction network. One important post-translational modification (PTM) of proteins is phosphorylation/dephosphorylation, which plays a crucial role in cell signal transmission. In this study, we aimed to investigate how T. gondii regulates signal transduction in definitive host cells. We employed titanium dioxide (TiO₂) affinity chromatography to enrich phosphopeptides in the small intestinal epithelia of cats at 10 days post-infection with the T. gondii Prugniuad (Pru) strain and quantified them using iTRAQ technology. A total of 4998 phosphopeptides, 3497 phosphorylation sites, and 1805 phosphoproteins were identified. Among the 705 differentially expressed phosphoproteins (DEPs), 68 were down-regulated and 637 were up-regulated. The bioinformatics analysis revealed that the DE phosphoproteins were involved in various cellular processes, including actin cytoskeleton reorganization, cell necroptosis, and MHC immune processes. Our findings confirm that T. gondii infection leads to extensive changes in the phosphorylation of proteins in the cat intestinal epithelial cells. The results of this study provide a theoretical foundation for understanding the interaction between T. gondii and its definitive host. Full article

Protein phosphorylation is an essential post-translational modification that regulates multiple cellular processes. Due to their low stoichiometry and ionization efficiency, it is critical to efficiently enrich phosphopeptides for phosphoproteomics. Several phosphopeptide enrichment methods have been reported; however, few studies have comprehensively compared different TiO₂-based phosphopeptide enrichment methods using complex proteomic samples. Here, we compared four TiO₂-based phosphopeptide enrichment methods that used four non-phosphopeptide excluders (glutamic acid, lactic acid, glycolic acid, and DHB). We found that these four TiO₂-based phosphopeptide enrichment methods had different enrichment specificities and that phosphopeptides enriched by the four methods had different physicochemical characteristics. More importantly, we discovered that phosphopeptides had a higher deamidation ratio than peptides from cell lysate and that phosphopeptides enriched using the glutamic acid method had a higher deamidation ratio than the other three methods. We then compared two phosphopeptide fractionation methods: ammonia- or TEA-based high pH reversed-phase (HpH-RP). We found that fewer phosphopeptides, especially multi-phosphorylated peptides, were identified using the ammonia-based method than using the TEA-based method. Therefore, the TEA-based HpH-RP fractionation method performed better than the ammonia method. In conclusion, we comprehensively evaluated different TiO₂-based phosphopeptide enrichment and fractionation methods, providing a basis for selecting the proper protocols for comprehensive phosphoproteomics. Full article

Small membrane-derived extracellular vesicles have been proposed as participating in several cancer diseases, including breast cancer (BC). We performed a phosphoproteomic analysis of breast cancer-derived small extracellular vesicles (sEVs) to provide insight into the molecular and cellular regulatory mechanisms important for breast cancer tumor progression and metastasis. We examined three cell line models for breast cancer: MCF10A (non-malignant), MCF7 (estrogen and progesterone receptor-positive, metastatic), and MDA-MB-231 (triple-negative, highly metastatic). To obtain a comprehensive overview of the sEV phosphoproteome derived from each cell line, effective phosphopeptide enrichment techniques IMAC and TiO₂, followed by LC-MS/MS, were performed. The phosphoproteome was profiled to a depth of 2003 phosphopeptides, of which 207, 854, and 1335 were identified in MCF10A, MCF7, and MDA-MB-231 cell lines, respectively. Furthermore, 2450 phosphorylation sites were mapped to 855 distinct proteins, covering a wide range of functions. The identified proteins are associated with several diseases, mostly related to cancer. Among the phosphoproteins, we validated four enzymes associated with cancer and present only in sEVs isolated from MCF7 and MDA-MB-231 cell lines: ATP citrate lyase (ACLY), phosphofructokinase-M (PFKM), sirtuin-1 (SIRT1), and sirtuin-6 (SIRT6). With the exception of PFKM, the specific activity of these enzymes was significantly higher in MDA-MB-231 when compared with MCF10A-derived sEVs. This study demonstrates that sEVs contain functional metabolic enzymes that could be further explored for their potential use in early BC diagnostic and therapeutic applications. Full article

Interleukin-33 (IL-33), a member of the IL-1 superfamily cytokines, is an endogenous danger signal and a nuclear-associated cytokine. It is one of the essential mediators of both innate and adaptive immune responses. Aberrant IL-33 signaling has been demonstrated to play a defensive role against various infectious and inflammatory diseases. Although the signaling responses mediated by IL-33 have been previously reported, the temporal signaling dynamics are yet to be explored. To this end, we applied quantitative temporal phosphoproteomics analysis to elucidate pathways and proteins induced by IL-33 in THP-1 monocytes. Employing a TMT labeling-based quantitation and titanium dioxide (TiO₂)-based phosphopeptide enrichment strategy followed by mass spectrometry analysis, we identified and quantified 9448 unique phosphopeptides corresponding to 3392 proteins that showed differential regulation. Of these, 171 protein kinases, 60 phosphatases and 178 transcription factors were regulated at different phases of IL-33 signaling. In addition to the confirmed activation of canonical signaling modules including MAPK, NFκB, PI3K/AKT modules, pathway analysis of the time-dependent phosphorylation dynamics revealed enrichment of several cellular processes, including leukocyte adhesion, response to reactive oxygen species, cell cycle checkpoints, DNA damage and repair pathways. The detailed quantitative phosphoproteomic map of IL-33 signaling will serve as a potentially useful resource to study its function in the context of inflammatory and pathological conditions. Full article

To investigate the biological role of protein phosphorylation in human nonfunctional pituitary neuroendocrine tumors (NF-PitNETs), proteins extracted from NF-PitNET and control tissues were analyzed with tandem mass tag (TMT)-based quantitative proteomics coupled with TiO₂ enrichment of phosphopeptides. A total of 595 differentially phosphorylated proteins (DPPs) with 1412 phosphosites were identified in NF-PitNETs compared to controls (p < 0.05). KEGG pathway network analysis of 595 DPPs identified nine statistically significant signaling pathways, including the spliceosome pathway, the RNA transport pathway, proteoglycans in cancer, SNARE interactions in vesicular transport, platelet activation, bacterial invasion of epithelial cells, tight junctions, vascular smooth muscle contraction, and protein processing in the endoplasmic reticulum. GO analysis revealed that these DPPs were involved in multiple cellular components (CCs), biological processes (BPs), and molecule functions (MFs). The kinase analysis of 595 DPPs identified seven kinases, including GRP78, WSTF, PKN2, PRP4, LOK, NEK1, and AMPKA1, and the substrate of these kinases could provide new ideas for seeking drug targets for NF-PitNETs. The randomly selected DPP calnexin was further confirmed with immunoprecipitation (IP) and Western blot (WB). These findings provide the first DPP profiling, phosphorylation-mediated molecular network alterations, and the key kinase profiling in NF-PitNET pathogenesis, which are a precious resource for understanding the biological roles of protein phosphorylation in NF-PitNET pathogenesis and discovering effective phosphoprotein biomarkers and therapeutic targets and drugs for the management of NF-PitNETs. Full article

Phosphopeptide enrichment is a commonly used sample preparation step for investigating phosphorylation. TiO₂-based enrichment has been demonstrated to have excellent performance both for large amounts of complex and for small amounts of simple samples. However, it has not yet been studied for complex samples in the nanogram range. Our objective was to develop a methodology applicable for complex samples in the low nanogram range, useful for mass spectrometry analysis of tissue microarrays. The selectivity and performance of two stationary phases (TiO₂ nanoparticle-coated monolithic column and spin tip filled with TiO₂ microspheres) and several loading solvents were studied. Based on this study, we developed an effective and robust method, based on a spin tip with a non-conventional 50 mM citric acid-based loading solvent. It gave excellent results for phosphopeptide enrichment from samples containing a few nanograms of a complex protein mixture. Full article

Microcystins are potent hepatotoxins that have become a global health concern in recent years. Their actions in at-risk populations with pre-existing liver disease is unknown. We tested the hypothesis that the No Observed Adverse Effect Level (NOAEL) of Microcystin-LR (MC-LR) established in healthy mice would cause exacerbation of hepatic injury in a murine model (Lepr^db/J) of Non-alcoholic Fatty Liver Disease (NAFLD). Ten-week-old male Lepr^db/J mice were gavaged with 50 μg/kg, 100 μg/kg MC-LR or vehicle every 48 h for 4 weeks (n = 15–17 mice/group). Early mortality was observed in both the 50 μg/kg (1/17, 6%), and 100 μg/kg (3/17, 18%) MC-LR exposed mice. MC-LR exposure resulted in significant increases in circulating alkaline phosphatase levels, and histopathological markers of hepatic injury as well as significant upregulation of genes associated with hepatotoxicity, necrosis, nongenotoxic hepatocarcinogenicity and oxidative stress response. In addition, we observed exposure dependent changes in protein phosphorylation sites in pathways involved in inflammation, immune function, and response to oxidative stress. These results demonstrate that exposure to MC-LR at levels that are below the NOAEL established in healthy animals results in significant exacerbation of hepatic injury that is accompanied by genetic and phosphoproteomic dysregulation in key signaling pathways in the livers of NAFLD mice. Full article

We created different TiO₂-based coatings on silicon nanowires (SiNWs) by using either thermal metallization or atomic layer deposition (ALD). The fabricated surfaces were characterized by X-ray photoelectron spectroscopy (XPS), energy dispersive X-ray spectroscopy (EDX), and reflectivity measurements. Surfaces with different TiO₂ based coating thicknesses were then used for phosphopeptide enrichment and subsequent detection by laser desorption/ionization mass spectrometry (LDI-MS). Results showed that the best enrichment and LDI-MS detection were obtained using the silicon nanowires covered with 10 nm of oxidized Ti deposited by means of thermal evaporation. This sample was also able to perform phosphopeptide enrichment and MS detection from serum. Full article