ABSTRACT: This project evaluates the immune regulating properties of maple syrup extract in spleen samples utilizing an LPS induced peritonitis mouse model.
Project description:Data-independent acquisition (DIA) is a promising method for quantitative proteomics. Library-based DIA database searching against project-specific data-dependent acquisition (DDA) spectral libraries is the gold standard. These libraries are constructed using material-consuming pre-fractionation two dimensional DDA analysis. The alternative to this is library-free DIA analysis. Limited sample amounts restrict the use of fractionation to build spectral libraries for post-translational modifications (PTMs) DIA analysis. We present the use of gas-phase fractionation (GPF) DDA data to improve the depth of library-free DIA identification for the phosphoproteome, called GPF-DDA hybrid DIA. This method fully utilizes the remnants of samples post-DIA analysis and leverages both library-based and -free DIA database searching. GPF-DDA hybrid DIA analyzes phosphopeptides surplus sample after DIA analysis using a number of DDA injections with each scanning different mass-to-charge (m/z) windows, instead of preforming traditional off-line fractionation-based DDA. The GPF-DDA data is integrated into the library-free DIA database search to create a hybrid library, enhancing phosphopeptide identification. Two GPF-DDA injections proved to increase 18 % phosphopeptide and 13 % phosphosite identification in HEK293 cell lines, while five injections resulted in up to 28 % phosphopeptide and 21 % phosphosite increases compared to library-free DIA analysis alone. We used GPF-DDA hybrid DIA phosphoproteomics to characterize lung tissue upon direct (smoke induced) and indirect (sepsis induced) acute lung injury (ALI) in mice. The differentially expressed phosphosites (DEPsites) in direct ALI were found in proteins related to mRNA processing and RNA. DEPsites in indirect ALI were enriched in proteins related to microtubule polymerization, positive regulation of microtubule polymerization and fibroblast migration. This study demonstrates that GPF-DDA hybrid DIA analysis workflow can indeed promote depth of DIA analysis of phosphoproteome and could be extended to DIA analysis of other PTMs.
Project description:In the LPS-induced innate immune response, the expression level of IL-10 in the spleen of mice was significantly increased. The expression of this key anti-inflammatory cytokine is regulated by local sympathetic projections; however, the cell type in which it is expressed is unknown. Therefore, in this study, we used the LPS-induced mouse spleen model to analyze the spatial transcriptome, and found that IL10+ cells were mainly B cells. In addition, single-cell sequencing analysis of B cells isolated from the spleen of mice also showed that they expressed IL10. These results collectively suggest that B cells are the predominant cell type expressing IL10 in mouse spleen.
Project description:Ageing is characterized by the progressive lowering of the acute innate immune response and the progressive up-regulation of low-grade inflammation (i.e. inflammaging). At the cellular level, telomeres are considered as a mark of ageing as their length is progressively shortened. However, the links between telomeres and innate immune ageing remain undefined. Using a proteomic approach conducted on laboratory mice, we tested whether the experimental triggering of an acute innate response is deleterious for telomere maintenance, and whether this is dependent of age. To do so, we challenged young and old mice using bacterial lipopolysaccharide (LPS) and measured individuals’ end-points of telomere length and proteomic profiles in spleen tissue.
Project description:Mass spectrometry-based proteomics is constantly challenged by the presence of contaminant background signals. In particular, protein contaminants from reagents and sample handling are almost impossible to avoid. For data-dependent acquisition (DDA) proteomics, an exclusion list can be used to reduce the influence of protein contaminants. However, protein contamination has not been evaluated and is rarely addressed in data-independent acquisition (DIA). How protein contaminants influence proteomic data is also unclear. In this study, we established new protein contaminant FASTA and spectral libraries that are applicable to all proteomic workflows and evaluated the impact of protein contaminants on both DDA and DIA proteomics. We demonstrated that including our contaminant libraries can reduce false discoveries and increase protein identifications, without influencing the quantification accuracy in various proteomic software platforms. With the pressing need to standardize proteomic workflow in the research community, we highly recommend including our contaminant FASTA and spectral libraries in all bottom-up proteomic data analysis. Our contaminant libraries and a step-by-step tutorial to incorporate these libraries in various DDA and DIA data analysis platforms can be valuable resources for proteomic researchers, freely accessible at https://github.com/HaoGroup-ProtContLib.
Project description:Data-independent acquisition (DIA) is a promising technique for the proteomic analysis of complex protein samples. A number of studies have claimed that DIA experiments are more reproducible than data-dependent acquisition (DDA), but these claims are unsubstantiated since different data analysis methods are used in the two methods. Data analysis in most DIA workflows depends on spectral library searches, whereas DDA typically employs sequence database searches. In this study, we examined the reproducibility of the DIA and DDA results using both sequence database and spectral library search. The comparison was first performed using a cell lysate and then extended to an interactome study. Protein overlap among the technical replicates in both DDA and DIA experiments was 30% higher with library-based identifications than with sequence database identifications. The reproducibility of quantification was also improved with library search compared to database search, with the mean of the coefficient of variation decreasing more than 30% and a reduction in the number of missing values of more than 35%. Our results show that regardless of the acquisition method, higher identification and quantification reproducibility is observed when library search was used.
Project description:Aim: This study aims to evaluate the impact of experimental workflow on fecal metaproteomic observations, including the recovery of small and antimicrobial proteins often overlooked in metaproteomic studies. The overarching goal is to provide guidance for optimized metaproteomic experimental design, considering the emerging significance of the gut microbiome in human health, disease, and therapeutic interventions. Methods: Mouse feces were utilized as the experimental model. Fecal sample pre-processing methods (differential centrifugation and non-differential centrifugation), protein digestion techniques (in-solution and filter-aided), data acquisition modes (data-dependent and data-independent, or DDA and DIA) when combined with parallel accumulation-serial fragmentation (PASEF), and different bioinformatic workflows were assessed. Results: We showed that, in DIA-PASEF metaproteomics, the library-free search using protein sequence database generated from DDA-PASEF data achieved better identifications than using the generated spectral library. Compared to DDA, DIA-PASEF identified more microbial peptides, quantified more proteins with fewer missing values, and recovered more small antimicrobial proteins. We did not observe any obvious impacts of protein digestion methods on both taxonomic and functional profiles. However, differential centrifugation decreased the recovery of small and antimicrobial proteins, biased the taxonomic observation with a marked overestimation of Muribaculum species, and altered the measured functional compositions of metaproteome. Conclusion: This study underscores the critical impact of experimental choices on metaproteomic outcomes and sheds light on the potential biases introduced at different stages of the workflow. The comprehensive methodological comparisons serve as a valuable guide for researchers aiming to enhance the accuracy and completeness of metaproteomic analyses.
Project description:9 rabbit spleen label free proteomics studies.In order to study some drugs on animal innate immunity, we built rabbit models, using rabbit immune organs - spleen made mass spectrometry based label free proteomics studies. Finally, it is expected to find the target of the drug and the mechanism of action.
Project description:total RNA from mouse (male c57BL/6) spleen labeled with Cy3 vs total RNA from mouse (male c57BL/6) B cells treated with Lipopolysaccharide (LPS) labeled with Cy5- time course with repeats Keywords: ordered
Project description:Comparison between gene expression profiles of splenic stroma from wild type and lymphotoxin beta receptor knockout mice. The goal was to identify a set of genes which expression in splenic stroma is under lymphotoxin control and which can potentially be important for proper stroma development and function in secondary lymphoid organs. Total RNA isolated from mechanically separated stroma and splenocytes of wild type and LTbR-KO mice, as well as cultured spleen stroma cells from wild type mice. technical replicate - extract: A,B technical replicate - extract: C,D technical replicate - extract: E,F technical replicate - extract: G,H
Project description:Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer, and deeper proteome coverage is needed for its molecular characterization. We present comprehensive library of targeted mass spectrometry assays specific for TNBC and demonstrate its applicability. Proteins were extracted from 105 TNBC tissues and digested. Aliquots were pooled, fractionated using hydrophilic chromatography and analyzed by LC-MS/MS in data-dependent acquisition (DDA) parallel accumulation-serial fragmentation (PASEF) mode on timsTOF Pro LC-MS system. 16 individual lysates were analyzed in data-independent acquisition (DIA)-PASEF mode. Hybrid library was generated in Spectronaut software and covers 244,464 precursors, 168,006 peptides and 11,564 protein groups (FDR = 1%). Application of our library for pilot quantitative analysis of 16 tissues increased identification numbers in Spectronaut 18.5 and DIA-NN 1.8.1 software compared to library-free setting, with Spectronaut achieving the best results represented by 190,310 precursors, 140,566 peptides, and 10,463 protein groups. In conclusion, we introduce assay library that offers the deepest coverage of TNBC proteome to date. The TNBC library is available via PRIDE repository (PXD047793).