Proteomes最新文献

Breast cancer (BC) is a major global health issue, affecting a significant proportion of the female population and contributing to high rates of mortality. One of the primary challenges in the treatment of BC is the disease's heterogeneity, which can lead to ineffective therapies and poor patient outcomes. Spatial proteomics, which involves the study of protein localization within cells, offers a promising approach for understanding the biological processes that contribute to cellular heterogeneity within BC tissue. To fully leverage the potential of spatial proteomics, it is critical to identify early diagnostic biomarkers and therapeutic targets, and to understand protein expression levels and modifications. The subcellular localization of proteins is a key factor in their physiological function, making the study of subcellular localization a major challenge in cell biology. Achieving high resolution at the cellular and subcellular level is essential for obtaining an accurate spatial distribution of proteins, which in turn can enable the application of proteomics in clinical research. In this review, we present a comparison of current methods of spatial proteomics in BC, including untargeted and targeted strategies. Untargeted strategies enable the detection and analysis of proteins and peptides without a predetermined molecular focus, whereas targeted strategies allow the investigation of a predefined set of proteins or peptides of interest, overcoming the limitations associated with the stochastic nature of untargeted proteomics. By directly comparing these methods, we aim to provide insights into their strengths and limitations and their potential applications in BC research.

Protein phosphorylation is a key post-translational modification (PTM) that is a central regulatory mechanism of many cellular signaling pathways. Several protein kinases and phosphatases precisely control this biochemical process. Defects in the functions of these proteins have been implicated in many diseases, including cancer. Mass spectrometry (MS)-based analysis of biological samples provides in-depth coverage of phosphoproteome. A large amount of MS data available in public repositories has unveiled big data in the field of phosphoproteomics. To address the challenges associated with handling large data and expanding confidence in phosphorylation site prediction, the development of many computational algorithms and machine learning-based approaches have gained momentum in recent years. Together, the emergence of experimental methods with high resolution and sensitivity and data mining algorithms has provided robust analytical platforms for quantitative proteomics. In this review, we compile a comprehensive collection of bioinformatic resources used for the prediction of phosphorylation sites, and their potential therapeutic applications in the context of cancer.

Cellular interactions within the bone marrow microenvironment modulate the properties of subsets of leukemic cells leading to the development of drug-resistant phenotypes. The intercellular transfer of proteins and organelles contributes to this process but the set of transferred proteins and their effects in the receiving cells remain unclear. This study aimed to detect the intercellular protein transfer from mouse bone marrow stromal cells (OP9 cell line) to human T-lymphoblasts (CCRF-CEM cell line) using nanoLC-MS/MS-based shotgun proteomics in a 3D co-culture system. After 24 h of co-culture, 1513 and 67 proteins from human and mouse origin, respectively, were identified in CCRF-CEM cells. The presence of mouse proteins in the human cell line, detected by analyzing the differences in amino acid sequences of orthologous peptides, was interpreted as the result of intercellular transfer. The transferred proteins might have contributed to the observed resistance to vincristine, methotrexate, and hydrogen peroxide in the co-cultured leukemic cells. Our results suggest that shotgun proteomic analyses of co-cultured cells from different species could be a simple option to get a preliminary survey of the proteins exchanged among interacting cells.

Invasive ductal carcinoma (IDC) is the most common histological subtype of malignant breast cancer (BC), and accounts for 70-80% of all invasive BCs. IDC demonstrates great heterogeneity in clinical and histopathological characteristics, prognoses, treatment strategies, gene expressions, and proteomic profiles. Significant proteomic determinants of the progression from intraductal pre-invasive malignant lesions of the breast, which characterize a ductal carcinoma in situ (DCIS), to IDC, are still poorly identified, validated, and clinically applied. In the era of "6P" medicine, it remains a great challenge to determine which patients should be over-treated versus which need to be actively monitored without aggressive treatment. The major difficulties for designating DCIS to IDC progression may be solved by understanding the integrated genomic, transcriptomic, and proteomic bases of invasion. In this review, we showed that multiple proteomics-based techniques, such as LC-MS/MS, MALDI-ToF MS, SELDI-ToF-MS, MALDI-ToF/ToF MS, MALDI-MSI or MasSpec Pen, applied to in-tissue, off-tissue, BC cell lines and liquid biopsies, improve the diagnosis of IDC, as well as its prognosis and treatment monitoring. Classic proteomics strategies that allow the identification of dysregulated protein expressions, biological processes, and interrelated pathway analyses based on aberrant protein-protein interaction (PPI) networks have been improved to perform non-invasive/minimally invasive biomarker detection of early-stage IDC. Thus, in modern surgical oncology, highly sensitive, rapid, and accurate MS-based detection has been coupled with "proteome point sampling" methods that allow for proteomic profiling by in vivo "proteome point characterization", or by minimal tissue removal, for ex vivo accurate differentiation and delimitation of IDC. For the detection of low-molecular-weight proteins and protein fragments in bodily fluids, LC-MS/MS and MALDI-MS techniques may be coupled to enrich and capture methods which allow for the identification of early-stage IDC protein biomarkers that were previously invisible for MS-based techniques. Moreover, the detection and characterization of protein isoforms, including posttranslational modifications of proteins (PTMs), is also essential to emphasize specific molecular mechanisms, and to assure the early-stage detection of IDC of the breast.

Photoreceptor cells are highly susceptible to oxidative-stress-induced damage due to their high metabolic rate. Oxidative stress plays a key role in driving pathological events in several different ocular diseases, which lead to retinal degeneration and ultimately blindness. A growing number of studies have been performed to understand downstream events caused by ROS induced oxidative stress in photoreceptor cells; however, the underlying mechanisms of ROS toxicity are not fully understood. To shed light on ROS induced downstream pathological events, we employed a tandem mass tag (TMT) labelling-based quantitative mass-spectrometric approach to determine proteome changes in 661W photoreceptor cells following oxidative stress induction via the application of different concentrations of H₂O₂ at different time points. Overall, 5920 proteins were identified and quantified, and 450 differentially expressed proteins (DEPs) were identified, which were altered in a dose and time dependent manner in all treatment groups compared to the control group. These proteins were involved in several biological pathways, including spliceosome and ribosome response, activated glutathione metabolism, decreased ECM-receptor interaction, oxidative phosphorylation, abnormally regulated lysosome, apoptosis, and ribosome biogenesis. Our results highlighted ECM receptor interaction, oxidative phosphorylation and spliceosome pathways as the major targets of oxidative stress that might mediate vascular dysfunction and cellular senescence.

Escherichia coli represents the main cause of diarrhoea in pigs. Saliva can provide information about the pathophysiology of diseases and be a source of biomarkers. We aimed to identify changes in the salivary proteome of pigs with diarrhoea caused by E. coli. Saliva samples were collected from 10 pigs with this disease and 10 matched healthy controls. SDS-PAGE (1DE) and two-dimensional gel electrophoresis (2DE) were performed, and significantly different protein bands and spots were identified by mass spectrometry. For validation, adenosine deaminase (ADA) was measured in 28 healthy and 28 diseased pigs. In 1DE, increases in lipocalin and IgA bands were observed for diseased pigs, whereas bands containing proteins such as odorant-binding protein and/or prolactin-inducible protein presented decreased concentrations. Two-dimensional gel electrophoresis (2DE) results showed that saliva from E. coli animals presented higher expression levels of lipocalin, ADA, IgA and albumin peptides, being ADA activity increased in the diseased pigs in the validation study. Spots containing alpha-amylase, carbonic anhydrase VI, and whole albumin were decreased in diseased animals. Overall, pigs with diarrhoea caused by E. coli have changes in proteins in their saliva related to various pathophysiological mechanisms such as inflammation and immune function and could potentially be biomarkers of this disease.

Although numerous studies support a dose-effect relationship between Endocrine disruptors (EDs) and the progression and malignancy of tumors, the impact of a chronic exposure to non-lethal concentrations of EDs in cancer remains unknown. More specifically, a number of studies have reported the impact of Aldrin on a variety of cancer types, including prostate cancer. In previous studies, we demonstrated the induction of the malignant phenotype in DU145 prostate cancer (PCa) cells after a chronic exposure to Aldrin (an ED). Proteins are pivotal in the regulation and control of a variety of cellular processes. However, the mechanisms responsible for the impact of ED on PCa and the role of proteins in this process are not yet well understood. Here, two complementary computational approaches have been employed to investigate the molecular processes underlying the acquisition of malignancy in prostate cancer. First, the metabolic reprogramming associated with the chronic exposure to Aldrin in DU145 cells was studied by integrating transcriptomics and metabolomics via constraint-based metabolic modeling. Second, gene set enrichment analysis was applied to determine (i) altered regulatory pathways and (ii) the correlation between changes in the transcriptomic profile of Aldrin-exposed cells and tumor progression in various types of cancer. Experimental validation confirmed predictions revealing a disruption in metabolic and regulatory pathways. This alteration results in the modification of protein levels crucial in regulating triacylglyceride/cholesterol, linked to the malignant phenotype observed in Aldrin-exposed cells.

Integrative top-down proteomics is an analytical approach that fully addresses the breadth and complexity needed for effective and routine assessment of proteomes. Nonetheless, any such assessments also require a rigorous review of methodology to ensure the deepest possible quantitative proteome analyses. Here, we establish an optimized general protocol for proteome extracts to improve the reduction of proteoforms and, thus, resolution in 2DE. Dithiothreitol (DTT), tributylphosphine (TBP), and 2-hydroxyethyldisulfide (HED), combined and alone, were tested in one-dimensional SDS-PAGE (1DE), prior to implementation into a full 2DE protocol. Prior to sample rehydration, reduction with 100 mM DTT + 5 mM TBP yielded increased spot counts, total signal, and spot circularity (i.e., decreased streaking) compared to other conditions and reduction protocols reported in the literature. The data indicate that many widely implemented reduction protocols are significantly 'under-powered' in terms of proteoform reduction and thus, limit the quality and depth of routine top-down proteomic analyses.

Toxoplasma gondii is an obligate intracellular apicomplexan that causes toxoplasmosis in humans and animals. Central to its dissemination and pathogenicity is the ability to rapidly divide in the tachyzoite stage and infect any type of nucleated cell. Adaptation to different cell contexts requires high plasticity in which heat shock proteins (Hsps) could play a fundamental role. Tgj1 is a type I Hsp40 of T. gondii, an ortholog of the DNAJA1 group, which is essential during the tachyzoite lytic cycle. Tgj1 consists of a J-domain, ZFD, and DNAJ_C domains with a CRQQ C-terminal motif, which is usually prone to lipidation. Tgj1 presented a mostly cytosolic subcellular localization overlapping partially with endoplasmic reticulum. Protein-protein Interaction (PPI) analysis showed that Tgj1 could be implicated in various biological pathways, mainly translation, protein folding, energy metabolism, membrane transport and protein translocation, invasion/pathogenesis, cell signaling, chromatin and transcription regulation, and cell redox homeostasis among others. The combination of Tgj1 and Hsp90 PPIs retrieved only 70 interactors linked to the Tgj1-Hsp90 axis, suggesting that Tgj1 would present specific functions in addition to those of the Hsp70/Hsp90 cycle, standing out invasion/pathogenesis, cell shape motility, and energy pathway. Within the Hsp70/Hsp90 cycle, translation-associated pathways, cell redox homeostasis, and protein folding were highly enriched in the Tgj1-Hsp90 axis. In conclusion, Tgj1 would interact with a wide range of proteins from different biological pathways, which could suggest a relevant role in them.

Staphylococcus aureus is one of the major community-acquired human pathogens, with growing multidrug-resistance, leading to a major threat of more prevalent infections to humans. A variety of virulence factors and toxic proteins are secreted during infection via the general secretory (Sec) pathway, which requires an N-terminal signal peptide to be cleaved from the N-terminus of the protein. This N-terminal signal peptide is recognized and processed by a type I signal peptidase (SPase). SPase-mediated signal peptide processing is the crucial step in the pathogenicity of S. aureus. In the present study, the SPase-mediated N-terminal protein processing and their cleavage specificity were evaluated using a combination of N-terminal amidination bottom-up and top-down proteomics-based mass spectrometry approaches. Secretory proteins were found to be cleaved by SPase, specifically and non-specifically, on both sides of the normal SPase cleavage site. The non-specific cleavages occur at the relatively smaller residues that are present next to the -1, +1, and +2 locations from the original SPase cleavage site to a lesser extent. Additional random cleavages at the middle and near the C-terminus of some protein sequences were also observed. This additional processing could be a part of some stress conditions and unknown signal peptidase mechanisms.