Project description:We established a spectral library for data-independent analysis of human plasma samples, using a pooled plasma sample from Danish adults. The samples were prepared for proteome analysis using an in-house developed protocol consuming one microliter of plasma per sample, and the tryptic digest and subsequent C18 purification was performed in 96-well plates. The spectral library consists of plasma which was analyzed neat (i.e. non-treated), and top 14 depleted. The samples were subjected to high pH reversed-phase peptide fractionated using C18 tips (8 fraction) and a HPLC system (20 fraction). The resulting peptides were analyzed on a Q Exactive with a capflow reversed-phase C18 LC-MS/MS setup in data-dependent mode. The final spectral library contains 21788 precursors and 1000 proteins (both at FDR<0.01).
Project description:Lameness due to femoral head separation (FHS) is a production and welfare issue in commercial poultry. FHS is an idiopathic disorder, which is attributed to a myriad of factors but in order to improve bone health, broiler breeders must be prognosed for disease susceptibility and selected against FHS using biomarkers. Proteins from plasma of blood, which can be obtained using minimally invasive methods represent an ideal, rich source of biomarkers which might be different in susceptible or affected birds. The peptide and proteins in plasma of healthy (HLTH) and affected birds (FHS) were compared using Matrix assisted laser desorption ionization mass spectrometry (MALDI-TOF-MS) and Liquid chromatography and tandem mass spectrometry (LC-MS/MS). The peptide profile of HLTH and FHS were compared using ClinPro tools and the differentially expressed peptides were isolated by Reverse phase liquid chromatography fractionation (C18-RP-HPLC) and identified using peptide mass fingerprinting. Peptides derived from fibrinogen precursor and fetuin were reduced in FHS birds. Based on the proteomic analysis, proteins such as Gallinacin 10, Apolipoprotein A-1 and Hemoglobin chains are elevated in FHS while Alpha 1-acid glycoprotein is reduced in FHS birds. Our study shows that bodyweight, lipid profile and the above mentioned proteins could be useful as a biomarker for improvement of bone health. These proteins indicate that blood lysis, antimicrobial defense and lipid disorder but lack of an inflammatory response might be consequential to FHS.
Project description:Ionizing radiation (IR) therapy for malignant tumors can damage adjacent tissues, leading to severe wound complications. Plasma-derived exosome treatment has recently emerged as a safe and impactful cell-free therapy. Herein, we aimed to determine whether plasma-derived exosomes could improve the healing of post-radiation wound. Rat plasma-derived exosomes (RP-Exos) were locally injected on cutaneous wounds created on the backs of irradiated rats and boosted the healing process as well as the deposition and remodeling of the extracellular matrix with collagen formation. Subsequently, the effects of RP-Exos were further evaluated on irradiated fibroblasts in vitro. The results suggested that exosomes promoted fibroblast proliferation, migration, cell cycle progression, and cell survival. Moreover, transcriptome sequencing, analysis, and quantitative polymerase chain reaction validation were performed to identify the underlying molecular mechanisms. RP-Exos enhanced the expression of cell proliferation and radioresistance-related genes, and yet downregulated ferroptosis pathway in irradiated fibroblasts. Inhibition of ferroptosis by RP-Exos was further confirmed through colorimetric assay, fluorescence probe and flow cytometry in ferroptosis-induced fibroblasts. Our results suggest that RP-Exos regulate cell proliferation and ferroptosis in radiated fibroblasts, thereby boosting the healing of radiated wounds. These findings support plasma-derived exosomes as a potential therapeutic method for post-radiation wound complications.
Project description:<p>Clinical metabolic phenotyping employs metabolomics and lipidomics to detect and measure thousands of metabolites and lipids within human samples. This approach aims to identify metabolite and lipid changes between phenotypes (e.g. disease status) that aid understanding of biochemical mechanisms driving the phenotype. Sample preparation is a critical step in clinical metabolic phenotyping: it must be reproducible and give a high extraction yield of metabolites and lipids. Here, we assessed the extraction of polar metabolites from human urine and polar metabolites and lipids from human plasma for analysis by ultra-high-performance liquid chromatography-mass spectrometry (UHPLC-MS) metabolomics and lipidomics. We evaluated several monophasic (urine and plasma) and biphasic (plasma) extractions, and we also tested alterations to (a) solvent-biofluid incubation time and temperature during monophasic extraction, and (b) phase partitioning time during biphasic extraction. Extracts were analysed by three UHPLC-MS assays: (i) HILIC for urine and plasma, (ii) C18 aqueous reversed phase for urine, and (iii) C18 reversed phase for plasma lipids, and the yield and reproducibility of each method was assessed. For HILIC UHPLC-MS plasma and urine analysis, monophasic 50:50 methanol:acetonitrile had the most detected putatively-identified polar metabolites. If lipid removal from the plasma polar HILIC extract is required, then the biphasic methanol/chloroform/water method is recommended. For C18 (aqueous) UHPLC-MS urine analysis, 50:50 methanol:water had high reproducibility and yield. For C18 UHPLC-MS plasma lipidomics, monophasic 100% isopropanol had the highest detection response of all annotated lipid classes. Increasing monophasic incubation time and temperature had little benefit on metabolite and lipid yield and reproducibility.</p>
Project description:Plasma samples of patients diagnosed with CCM. Samples were run with a standard extraction (Plate 1 5x) and then again through a Phree Kit (Phree Kit Plate) to remove phospholipids. Data was acquired using a Bruker Maxis Impact and C18 RP-UHPLC using positive and negative polarity of LC-MS/MS.
Project description:We performed LC-MSMS analysis using both CID and ETD for the identification of endogenous peptides. Endogenous peptides were extracted from mouse AtT 20 cells by acidified methanol method and all large molecules including proteins were removed by centrifugation. The supernatant containing endogenous peptides was freeze-dried. For LC-MSMS analysis, extracted peptides were resuspended and injected to Ultimate 3000 HPLC system and analysed on LTQ Orbitrap XL mass spectrometer. A 60 min gradient from 2% acetonitrile to 50% acetonitrile, both containing 0.1% formic acid was used to separate peptides on C18 column.The LTQ-Orbitrap mass spectrometer was operated in data-dependent mode, automatically switching between MS and MS/MS acquisition for the three most abundant peaks in a given MS spectrum. A chosen precursor ion was first fragmented by CID and ETD. Data processing: The raw data files were processed with Proteome Discoverer 1.3. The CID and ETD spectra were then written to Mascot generic files. OMSSA (version 2.1.9) was used and b- and y- ions were selected for CID data, and c-, y- and z- ions were used for ETD. The spectra were searched by setting the parent ion mass accuracy to +/- 0.02 Da. For fragment ions, the mass tolerance was set to +/- 0.4 Da. For the genome-wide peptide search, the mouse genomic sequence (NCBI build 37.61) was directly translated in its 6 reading frames, and used for spectral searching. No enzymatic cleavage was taken into account during the database searches. No variable PTMs were included.
Project description:Samples were subjected to LC–MS analysis using a dual pressure LTQ-Orbitrap Elite mass spectrometer (Thermo Fisher Scientific) connected to an electrospray ion source (Thermo Fisher Scientific) as recently described (PMID: 23017020). Peptide separation was carried out on an EASY nLC-1000 system (Thermo Fisher Scientific) equipped with a RP-HPLC column (75 μm × 30 cm) packed in-house with C18 resin (ReproSil-Pur C18–AQ, 1.9 μm resin, Dr. Maisch GmbH). A step-wise gradient from 95% solvent A (0.1% formic acid) and 5% solvent B (80% acetonitrile, 0.1% formic acid) to 50% solvent B over 60 min at a flow rate of 0.2 μl/min was used. Data acquisition mode was set to obtain one high resolution MS scan in the FT part of the mass spectrometer at a resolution of 240,000 full width at half-maximum (at m/z 400) followed by 20 MS/MS scans (TOP20) in the linear ion trap of the most intense ions using rapid scan speed. Unassigned and singly charged ions were excluded from analysis. Dynamic exclusion duration was set to 30 seconds.