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Mouse liver proteome fractionation using leptin-based cartridges.

ABSTRACT: This study is aimed at the evaluation of leptin-based cartridges fractionation for glycoprotein enrichment of mouse liver protein shotgun proteomic profile.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Liver

SUBMITTER: Giangiacomo Beretta

LAB HEAD: Giangiacomo Beretta

PROVIDER: PXD025097 | Pride | 2021-07-30

REPOSITORIES: Pride

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			Action	DRS
	19-02-07-19P04-NONtotal-01.mzML	Mzml
	19-02-07-19P04-NONtotal-01.raw	Raw
	19-02-07-19P04-NONtotal-02.mzML	Mzml
	19-02-07-19P04-NONtotal-02.raw	Raw
	19-02-07-19P04-total-01.mzML	Mzml

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Publications

DDASSQ: An open-source, multiple peptide sequencing strategy for label free quantification based on an OpenMS pipeline in the KNIME analytics platform.

Svecla Monika M Garrone Giulia G Faré Fiorenza F Aletti Giacomo G Norata Giuseppe Danilo GD Beretta Giangiacomo G

Proteomics 20210801 16

In this study we investigated the performance of a computational pipeline for protein identification and label free quantification (LFQ) of LC-MS/MS data sets from experimental animal tissue samples, as well as the impact of its specific peptide search combinatorial approach. The full pipeline workflow was composed of peptide search engine adapters based on different identification algorithms, in the frame of the open-source OpenMS software running within the KNIME analytics platform. Two differ ...[more]

PMID: 34312990

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Low-Density Lipoprotein Receptor fuels the cellular cholesterol-mTORC1 axis required for CD8+ T cell activation

Project description:In this study, the contribution of cholesterol uptake by the low-density-lipoprotein receptor (LDLR) in the metabolic reprogramming of activated T cells was investigated at proteome level by mass-spectrometry-assisted shotgun proteomics.

2022-09-20 | PXD030365 | Pride

Identification of regulatory networks and crosstalk factors in brown adipose tissue and liver of a cold-exposed cardiometabolic mouse model

Project description:oActivationf brown adipose tissue (BAT) has gained central attention due to its known ability to dissipate energy and counteract cardiometabolic diseases (CMDs). The cold exposure on BAT activation and the induction of thermogenesis have been extensively studied, but reports on the effects at the proteomic level in metabolic tissues of animal models mimicking CMD in humans are lacking.

2024-08-16 | PXD051640 | Pride

Regional differences in the small intestinal proteome of control mice and of mice lacking lysosomal acid lipase

Project description:Background: Despite recent studies investigating the involvement of single cells in regional differences of the small intestine (SI), the metabolic contribution of the duodenum, jejunum, and ileum to the overall intestinal metabolism is still unclear. Basic procedures and main findings: Using an untargeted proteomics approach, we identified similarities and differences between the three intestinal tracts of C57BL/6J mice and found that proteins highly abundant in the mouse ileum correlated with high ileal expression of the corresponding genes in humans. Consistent with human data, we detected increasing abundance of lysosomal acid lipase (LAL) in C57BL/6J mice from the duodenum to ileum. LAL is the only enzyme known to degrade triacylglycerols and cholesteryl esters within the lysosome. Lipid accumulation in various organs along with gastrointestinal disturbances and malabsorption are typical features of patients and mice with LAL deficiency. We previously demonstrated that macrophages massively infiltrate the SI of Lal-deficient (KO) mice, especially the duodenum. To identify potential mechanisms behind the intestinal lipid accumulation and infiltration of immune cells, we used untargeted proteomics. Our results revealed a general inflammatory response and a common lipid-associated macrophage phenotype in all three intestinal segments of Lal KO mice, accompanied by higher expression of GPNMB and increased concentrations of circulating sTREM2. However, only duodenal macrophages activated a metabolic switch from lipids to other pathways such as glycolysis, TCA cycle, and oxidative phosphorylation to meet their high energy demand. Unexpectedly, these pathways were downregulated in the jejunum and ileum of Lal KO mice. Conclusion: Our results provide new insights into the process of absorption in control mice and the basis for novel markers of LAL-D and/or systemic inflammation in LAL-D.

2024-02-20 | PXD048378 | Pride

Transcriptome and proteome analysis in LUHMES cells overexpressing alpha-synculein

Project description:LUHMES cells share many characteritics with human dopamingeric neurons in the substantia nigra, the cells whose demise is responsible for the motor symptoms in Parkinson’s disease (PD). LUHMES cells can therefore be used bona fide as a model to study pathophysiological processes involved in PD. Previously, we showed that LUHMES cell degenerate after six days upon overexpression of wild type alpha-synuclein. In the present study we performed a transcriptome and proteome expression analysis in alpha-synuclein-overexpressing cells and GFP-expressing control cells in order to identify genes and proteins that are differentially regulated upon overexpression of alpha-synuclein. The analysis was performed four days after the initiation of alpha-synuclein or GFP overexpression, before the cells died in order to identify processes that preceded cell death.

2022-05-19 | PXD028322 | Pride

Reproducibility, specificity and accuracy of DIA quantification - DDA analysis

Project description:Data dependent acquisition (DDA) is the method of choice for mass spectrometry based proteomics discovery experiments, data-independent acquisition (DIA) is steadily becoming more important. One of the most important requirement to perform a DIA analysis is the availability of spectral libraries for the peptide identification and quantification. Several researches were already conducted regarding the creation of spectral libraries from DDA analyses and obtaining identifications with these in DIA measurements. But so far only few experiments were conducted, to estimate the effect of these libraries on the quantitative level. In this work we created a spike-in gold standard dataset with known contents and ratios of proteins in a complex sample matrix. With this dataset, we first created spectral libraries using different sample preparation approaches with and without sample prefractionation on peptide and protein level. Two different search engines were used for protein identification. In total, five different spike-in states were compared with DIA analyses, comparing eight different spectral libraries generated by varying approaches and one library free method, as well as one default DDA analysis. Not only the number of identifications on peptide and protein level in the spectral libraries and the corresponding analyses was inspected, but also the number of expected and identified significant quantifications and their ratios were thoroughly examined. We found, that while libraries of prefractionationed samples are generally larger, the actually yielded identifications are not increased compared to repetitive non-fractionated measurements. Furthermore, we show that the accuracy of the quantifications is also highly dependent on the applied spectra library and also whether the peptide or protein level is analysed. Overall, the reproducibility and accuracy of DIA is superior to DDA in all analysed approaches.

2019-11-08 | PXD012986 | Pride

A primary human T-cell spectral library to facilitate large scale quantitative T-cell proteomics

Project description:Comprehensive spectral libraries are essential in sequential window acquisition of all theoretical mass spectra (SWATH-MS) based high throughput proteomic studies. Even though SWATH-MS assays provide robust quantitative proteomics data its applications to human T-cell studies are limited by the lack of a human T-cell spectral library. To address this resource gap, we generated a high-quality spectral library containing data for 3,941 unique proteins from primary human T-cells across genetically unrelated donors. SWATH-MS analysis of 18 primary T-cell samples using the new human T-cell spectral library identified and quantified 3,022 proteins at 1% FDR, whereas the larger Pan-human spectral library identified and quantified 2,794 proteins, with only 34% overlap. Combining the two libraries resulted in 4,061 proteins, covering ~50% of proteins in immune-related pathways. Overall, this data suggests DDA-MS is suited to discovery projects through to its enhanced sensitivity and SWATH-MS is suited to high-throughput projects.

2020-10-19 | PXD019446 | Pride

Simulated phosphopeptide spectral library for confident site localization

Project description:We have developed a new workflow to unambiguously localize phosphorylation sites on proteins. We demonstrate that spectral matching of phosphopeptide datasets against a library of the well-simulated spectra provided higher sensitivity for confident site localization than other tested programs. To computationally simulate tandem mass spectra representing all possible singly phosphorylated forms of a peptide, characteristic fragment ions are predicted from ions of their dephosphorylated form generated by beam-type collision-induced dissociation.

2015-03-17 | PXD000474 | Pride

Data independent acquisition mass spectrometry in severe Rheumatic Heart Disease (RHD) identifies a proteomic signature showing ongoing inflammation and effectively classifying RHD cases.

Project description:Rheumatic heart disease (RHD) remains a major source of morbidity and mortality in developing countries. A deeper insight into the pathogenetic mechanisms underlying RHD could provide opportunities for drug repurposing, guide recommendations for secondary penicillin prophylaxis, and/or inform development of near-patient diagnostics. We performed quantitative proteomics using Sequential Windowed Acquisition of All Theoretical Fragment Ion Mass Spectrometry (SWATH-MS) to screen protein expression in 215 African patients with severe RHD, and 230 controls. A machine learning (ML) approach was applied to feature selection among the 366 proteins quantifiable in at least 40% of samples, using the Boruta wrapper algorithm. The case-control differences and contribution to AUC of the ROC for each of the 56 proteins identified by the Boruta algorithm were calculated by Logistic Regression adjusted for age, sex and BMI. Adiponectin, complement component C7 and fibulin-1, a component of heart valve matrix, were each higher in cases when compared with controls. Ficolin-3, a protein with calcium-independent lectin activity that activates the complement pathway, was lower in cases than controls. The top six biomarkers from the Boruta analyses conferred an AUC of 0.90 indicating excellent discriminatory capacity between RHD cases and controls.

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Reproducibility, specificity and accuracy of DIA quantification - OpenSWATH

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PCT-SWATH kidney tissues - Rapid mass spectrometric conversion of tissue biopsy samples into permanent quantitative digital proteome maps

Project description:DDA and SWATH analysis of human kidney tissues. Data set used for PCT-SWATH paper. PCT-SWATH provides the first method to generate a digital map representing the proteome of biopsy level clinical samples from which thousands of proteins can be accurately quantified with a high degree of reproducibility across sample sets.

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