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Proteomic study of baicalein effects on LPS-primed BV2 microglia

ABSTRACT: To comprehensively study the intracellular signaling changes and explore the underlying mechanisms of baicalein-mediated microglial responses, we employed systemic proteomic approach, using target-free SWATH mass spectra (SWATH-MS), focusing on intracellular proteins. BV2 cells were treated with LPS followed by the addition of vehicle or baicalein and they were cultured for a total of 48 hours before being collected for proteomic analysis.

INSTRUMENT(S): TripleTOF 6600

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Cell Culture, Microglial Cell Activation

DISEASE(S): Retinal Degeneration

SUBMITTER: Li PAN

LAB HEAD: Chi-Wai Do

PROVIDER: PXD030447 | Pride | 2022-02-14

REPOSITORIES: Pride

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Red blood cells protein profile is modified in breast cancer patients

Project description:We characterized the proteomic composition of RBCs from 53 breast cancer patients (stages I-III and IV), compared with 33 healthy donors. For that, we performed two different proteomic analyses (LC-MS/MS in DDA mode-shotgun- and a SWATH-MS) using the Triple TOF 6600 technology.

2023-01-17 | PXD030936 | Pride

Proteomic profiling of NCI60 cell lines from Cancer Cell Line Encyclopedia

Project description:This ArrayExpress record contains meta-data and results of quantitative analysis of cell lines from the NCI-60 panel using pressure cycling technology (PCT) and SWATH-mass spectrometry. Each cell line was analyzed in duplicate. Raw data files are available at the EMBL-EBI protemics data archive (PRIDE) at accession PXD003539 (http://www.ebi.ac.uk/pride/archive/projects/PXD003539). Since the record here does not include the raw data files and hence there is no need to explicitly link individual replicate to a raw file, each sample is only listed once in the ArrayExpress samples table for clarity.

2017-01-27 | E-PROT-2 | biostudies-arrayexpress

Reanalysis of PXD009319: "An alliance of gel-based and gel-free proteomic techniques displays substantial insight into the proteome of a virulent and an attenuated Histomonas meleagridis strain"

Project description:The current work aimed to detect and identify significantly differentially expressed proteins between a virulent and an attenuated Histomonas meleagridis strain which were in vitro co-cultivated with Escherichia coli DH5alpha. This was done by applying 2D-DIGE and sequential window acquisition of all theoretical spectra (SWATH) MS as tools on the two well-defined strains. In the gel-based experiments, 49 identified protein spots were found to be differentially expressed, of which 37 belonged to the in vitro cultivated virulent strain and 12 to the attenuated one. The most frequently identified proteins in the virulent strain take part in cytoskeleton formation, carbohydrate metabolism and adaptation to stress. However, post-translationally modified or truncated ubiquitous cellular proteins such as actin and GAPDH were identified as upregulated in multiple gel positions. This indicated their contribution to processes not related to cytoskeleton and carbohydrate metabolism, such as fibronectin or plasminogen binding. On the other hand, proteins involved in cytoskeleton, cell division and cytoskeleton organization were frequently observed in the attenuated strain. The findings of the gel-based studies were supplemented by the gel-free SWATH MS analysis, which identified and quantified 42 significantly differentially regulated proteins. In this case proteins with peptidase activity, metabolic proteins and actin-regulating proteins were the most frequent findings in the virulent strain, while proteins involved in hydrogenosomal carbohydrate metabolism dominated the results in the attenuated one.

2023-03-11 | PXD034844 | Pride

Bull sperm SWATH-MS-based proteomics reveals link between high fertility and energy production, motility structures, and sperm-oocyte interaction. Quantitation

Project description:Prediction of male or semen fertility potential remains a persistent challenge that has yet to be fully resolved. This work analyzed several in vitro parameters and proteome of spermatozoa in bulls cataloged as high (HF; n=5) and low field (LF; n=5) fertility after more than a thousand artificial inseminations. Sperm motility was evaluated by Computer-Assisted Sperm Analysis. Sperm viability, mitochondrial membrane potential (MMP), and reactive oxygen species (mROS) of spermatozoa were assessed by flow cytometry. Proteome was evaluated by SWATH-MS procedure. Spermatozoa of HF bulls showed significantly higher total motility than the LF group (41.4% vs. 29.7%). Rates of healthy sperm (live, high MMP, and low mROS) for HF and LF bull groups were 49% and 43%, respectively (p > 0.05). Spermatozoa of HF bulls showed higher presence of differentially abundant proteins (DAPs) related to both energy production (COX7C), mainly OXPHOS pathway, and to the development of structures linked with the motility process (TPPP2, SSMEM1 and SPAG16). Furthermore, we observed that EQTN, together with other DAPs related to the interaction with the oocyte, were overrepresented in HF bull spermatozoa. The biological processes related to protein processing, catabolism, and protein folding were found to be overrepresented in LF bull sperm in which the HSP90AA1 chaperone was identified as the most DAP

2023-10-18 | PXD042286 | Pride

Robust and High-Throughput Analytical Flow Proteomics Analysis of Cynomolgus Monkey and Human Matrices with Zeno SWATH Data Independent Acquisition

Project description:Modern mass spectrometers routinely allow deep proteome coverage in a single experiment. These methods are typically operated at nano and micro flow regimes, but they often lack throughput and chromatographic robustness, which is critical for large-scale studies. In this context, we have developed, optimized and benchmarked LC-MS methods combining the robustness and throughput of analytical flow chromatography with the added sensitivity provided by the Zeno trap across a wide range of cynomolgus monkey and human matrices of interest for toxicological studies and clinical biomarker discovery. SWATH data independent acquisition (DIA) experiments with Zeno trap activated (Zeno SWATH DIA) provided a clear advantage over conventional SWATH DIA in all sample types tested with improved sensitivity, quantitative robustness and signal linearity as well as increased protein coverage by up to 9-fold. Using a 10-min gradient chromatography, up to 3,300 proteins were identified in tissues at 2 µg peptide load. Importantly, the performance gains with Zeno SWATH translated into better biological pathway representation and improved the ability to identify dysregulated proteins and pathways associated with two metabolic diseases in human plasma. Finally, we demonstrate that this method is highly stable over time with the acquisition of reliable data over the injection of 1,000+ samples (14.2 days of uninterrupted acquisition) without the need for human intervention or normalization. Altogether, Zeno SWATH DIA methodology allows fast, sensitive and robust proteomic workflows using analytical flow and is amenable to large-scale studies. This work provides detailed method performance assessment on a variety of relevant biological matrices and serves as a valuable resource for the proteomics community.

2023-05-09 | PXD039414 | Pride

SWATH-MS identification of CXCL7, LBP, TGFβ1 and PDGFRβ as novel biomarkers in human systemic mastocytosis.

Project description:We carried out a novel clinical biomarker investigation of the blood of systemic mastocytosis patients versus healthy controls; utilizing a new and emerging technology platform Sequential Window Acquisition of all Theoretical fragment-ion spectra mass spectrometry (SWATH-MS). This is the first use of this novel proteomic technique in the study of mastocytosis. A Swath-MS based biomarkers dsicovery study for systemic mastocytosis with orthogonal validation of biomarkers via ELISA.

2022-05-20 | PXD030947 | Pride

Label free SWATH proteomics reveals sex-biased proteins potentially associated with sex expression and modification in dioecious C. grandis

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2019-07-29 | PXD011064 | Pride

APPLICATION OF A NOVEL PEPTIDE SPECTRAL LIBRARY USING SWATH ANALYSIS FOR THE QUANTITATION OF PROTEINS IN EX-DIAGNOSTIC CLINICAL SERUM SAMPLES FROM SHEEP

Project description:This project applies SWATH approach to analyse ex-diagnostic sheep serum samples from actual clinical cases submitted to The University of Queensland's School of Veterinary Science, Veterinary Laboratory Services (VLS) compared with serum of normal sheep.

2022-11-15 | PXD005077 | Pride

Comparison of fractionation proteomics for local SWATH library building

Project description:For data-independent acquisition by means of sequential window acquisition of all theoretical fragment ion spectra (SWATH), a reference library of data-dependent acquisition (DDA) runs is typically used to correlate the quantitative data from the fragment ion spectra with peptide identifications. The quality and coverage of such a reference library is therefore essential when processing SWATH data. In general, library sizes can be increased by reducing the impact of DDA precursor selection with replicate runs or fractionation. However, these strategies can affect the match between the library and SWATH measurement, and thus larger library sizes do not necessarily correspond to improved SWATH quantification. Here, three fractionation strategies to increase local library size were compared to standard library building using replicate DDA injection: protein SDS-PAGE fractionation, peptide high-pH RP-HPLC fractionation and MS-acquisition gas phase fractionation. The impact of these libraries on SWATH performance was evaluated in terms of the number of extracted peptides and proteins, the match quality of the peptides and the extraction reproducibility of the transitions. These analyses were conducted using the hydrophilic proteome of differentiating human embryonic stem cells.

2017-07-04 | PXD006190 | Pride

Reanalysis of PXD012038: "Molecular characterization of Histomonas meleagridis exoproteome with emphasis on protease secretion and parasite-bacteria interaction - SWATH dataset"

Project description:Exoproteome from parasitic protists constitutes of extracellular proteins which play a fundamental role in multifactorial host-parasite interactions. Lytic factors, especially secreted proteases in extracellular milieu, are capable to modulate tissue invasion, thereby aggravating host susceptibility. Despite the important role of exoproteins during infection, exoproteomic data on Histomonas meleagridis are non-existent. The present study employed traditional 1D-in-gel-zymography (1D-IGZ) and micro-LC-ESI-MS/MS (shotgun proteomics), to scrutinize H. meleagridis exoproteomes, obtained from a clonal virulent and attenuated strains. Both strains were maintained as mono-eukaryotic monoxenic culture with Escherichia coli. We demonstrated active in vitro secretion kinetics of proteases by both parasites, with widespread proteolytic activity ranging from 17 kDa to 120 kDa. Based on protease inhibitor-susceptibility tests, a predominant repertoire of cysteine proteolysis was present in the parasite exoproteomes, with stronger activity from virulent H. meleagridis. Shotgun proteomics, aided by customized database, identified 176 proteins including actin, potential moonlighting glycolytic enzymes, lytic molecules such as pore-forming proteins (PFPs) and proteases like cathepsin-L like cysteine protease. To quantify the exoproteomic differences between the virulent and the attenuated H. meleagridis cultures, a sequential window acquisition of all theoretical spectra mass spectrometric (SWATH-MS) approach was applied. Surprisingly results showed most of the exoproteomic differences to be of bacterial origin, involving metabolism and locomotion. By deciphering such molecular signatures, novel insights into an inherent complex in vitro protozoan- bacteria relationship was elucidated.

2023-03-11 | PXD034834 | Pride

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