Dataset Information

Proteomic analysis of subchloroplastic compartments

ABSTRACT: Arabidopsis thaliana chlorosplasts extraction followed by subchloroplastic compartments fractionation (envelope, stroma, thylakoids). Afer SDS-PAGE separation and trypsin digestion, analysis of all gel bands twice by LC-MS/MS on a LTQ-FT

INSTRUMENT(S): LTQ FT

ORGANISM(S): Arabidopsis Thaliana (mouse-ear Cress)

TISSUE(S): Leaf

SUBMITTER: Hesse Anne-Marie

LAB HEAD: Christophe Bruley

PROVIDER: PXD000136 | Pride | 2016-12-23

REPOSITORIES: Pride

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Publications

hEIDI: An Intuitive Application Tool To Organize and Treat Large-Scale Proteomics Data.

Hesse Anne-Marie AM Dupierris Véronique V Adam Claire C Court Magali M Barthe Damien D Emadali Anouk A Masselon Christophe C Ferro Myriam M Bruley Christophe C

Journal of proteome research 20160912 10

Advances in high-throughput proteomics have led to a rapid increase in the number, size, and complexity of the associated data sets. Managing and extracting reliable information from such large series of data sets require the use of dedicated software organized in a consistent pipeline to reduce, validate, exploit, and ultimately export data. The compilation of multiple mass-spectrometry-based identification and quantification results obtained in the context of a large-scale project represents a ...[more]

PMID: 27560970

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Project description:Human hepatoma HepG2 cells (American Type Culture Collection; HB-8065) were grown in 75 ml tissue cell culture flasks in Eagle's minimal essential medium (EMEM) augmented with 10% fetal bovine serum at 37°C in a humidified atmosphere with 5% CO2. For treatment with fatty acids or analogues, the cells were seeded at a density of 2 × 106 cells in a T25-cm2 flask for 24 h. Each FA or analogue was added to the culture medium as a fatty acid-bovine serum albumin (BSA) complex (2.5:1, FA:BSA) to give the desired final concentration. The controls in these experiments were HepG2 cells with BSA alone. After 24 h treatment, the media was collected, cells were rinsed twice with PBS and cells were harvested for analysis. To each cell suspension prior to lipid extraction a standard mixture of 25 µg C15:0 PE, C17:0 PC and C71:1 TAG was added as standards. Extraction of lipids was performed according to the Folch method. For metabolomics analysis, the lipid extracts were resuspended in chloroform/methanol, 2:1 (v/v).All analyses were carried out using an Agilent 1200 Series HPLC, ACE 5 C8-300 column (2.1 x 100 mm) and linear gradient elution at a flow rate of 0.1 mL/min. Mobile phase A and B consisted of 0.1% formic acid; 10 mM ammonium acetate in H2O and 0.1% formic acid; 10 mM ammonium acetate in ACN/isopropanol (50/50; v/v), respectively. The injection volume was 4 µL. Separation of metabolites was achieved at the following gradient: T=0 min: 30% B; T=1 min: 30% B; T=25 min: 100% B; T=45 min: 100% B; T=47 min: 30% B; and T=60 min: 30% B (re-equilibration). The HPLC system was directly coupled to a Bruker Soalrix 70 Hybrid FTMS instrument equipped with electrospray ionization source (ESI) (Bruker Daltonics). The system was controlled by HyStar v.3.4.8.0 software. MS data was collected with resolving power of 78,000 (at m/z 400) in positive or negative mode under following conditions: a capillary voltage of (+/-) 4,500 V and an end plate offset of -500 V. The dry temperature was set at 180°C. Dry gas flow was maintained 4 L/min. Acquisition range was 244-1,800 m/z with 0.2 s ion accumulation time. LC-MS data was converted into mzXML format using CompassXport v. 3.0.6 and processed by mzMine v.2.10 [25] or XCMS data analysis software. Data processing involved mass detection, chromatographic peak detection and deconvolution, isotopic peaks grouping, normalization and peak alignment. Metabolite data were mean-centered and unit-variance scaled to remove the offsets and adjust the importance of high and low abundance metabolites to an equal level. Significantly altered metabolites were defined by a fold change (FC) >2 and p<0.05. Principal component analysis (PCA) and hierarchical clustering analysis (HCA) of signature metabolites altered in compounds treated cells compared to control were performed in the Metaboanalyst web portal (www.metaboanalyst.ca).

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