Dataset Information

Label free proteomics analysis of differentiating human muscle cells.

ABSTRACT: We have carried out systematic analysis of protein expression changes during differentiation of human myoblasts into myotubes over 6 days period in cell culture.

INSTRUMENT(S): LTQ FT

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Activated Skeletal Muscle Satellite Cell

SUBMITTER: Adelina Rogowska-Wrzesinska

LAB HEAD: Adelina Rogowska-Wrzesinska

PROVIDER: PXD018588 | Pride | 2020-05-20

REPOSITORIES: Pride

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Publications

PolySTest: Robust Statistical Testing of Proteomics Data with Missing Values Improves Detection of Biologically Relevant Features.

Schwämmle Veit V Hagensen Christina E CE Rogowska-Wrzesinska Adelina A Jensen Ole N ON

Molecular & cellular proteomics : MCP 20200518 8

Statistical testing remains one of the main challenges for high-confidence detection of differentially regulated proteins or peptides in large-scale quantitative proteomics experiments by mass spectrometry. Statistical tests need to be sufficiently robust to deal with experiment intrinsic data structures and variations and often also reduced feature coverage across different biological samples due to ubiquitous missing values. A robust statistical test provides accurate confidence scores of larg ...[more]

PMID: 32424025

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Project description:To provide insights into the role of Ankrd2 in the pathways controlling myogenic differentiation, the gene networks that are disturbed in Ankrd2 knockout or overexpressing primary cells were explored during differentiation. Ankrd2 plays an important role in myogenic differentiation by modulating the activity of its interacting partners (i.e p53). Since Ankrd2 binds to numerous transcription factors many signaling pathways remained to be explored to determine the functional significance of crosstalk between Ankrd2 and the activation of transcriptional programmes that regulate muscle remodeling. In contrast to previous gene expression studies in which we and others have studied the effect of Ankrd2 silencing in mouse differentiating C2C12 cells (Series GSE7542) and human differentiated CHQ5B muscle cells (Belgrano et al., 2011), we here used an in vivo experimental model in which the expression of Ankrd2 was completely abrogated. Furthermore, since Ankrd2 is known to be strongly upregulated in response to various stress stimuli, we studied the effect of Ankrd2 rescue and acute overexpression in KO and WT cells by adenoviral infection 20 hours prior to collection of cells. The pathways that change significantly are involved in distinct inflammatory response phases having NF-kB as a central node. Based on these results Ankrd2 is emerging for the first time as a repressor of immune and inflammatory responses. Gene expression profiling experiments have been performed using the SurePrint G3 Mouse 8x60K array by Agilent with 8 subarrays, each containing 39,430 60mer oligonucleotide probes for Entrez Gene RNAs, 16,251 for lincRNAs, 96 x 10 control probes, and 32 x 10 spike-in control probes. The Agilent platforms with one-color approach were selected to make possible the all-to all experiments comparison. The animal model, created and characterized in the laboratory of Dr.ML Bang, directed at knocking out Ankrd2 function was used. The adenovirus expressing Ankrd2-HA was produced using the AdEasy strategy (Agilent Technologies). Primary muscle cultures derived from wild type (WT) and Ankrd2-knockout (KO) mice infected with adenovirus expressing Ankrd2 or control GFP were collected at three different stages: proliferating, fusing and differentiated myoblasts. Three biological replicates per condition were included and a total of 36 microarray experiments were thus performed. First, the over-expression of Ankrd2 was checked by Western blot analysis. Then RNA was extracted from each sample and the relative transcriptional profiles were carried out using updated microarrays from Agilent technologies. Information from probe features was extracted from microarray scan data with the Feature Extraction Software (Agilent). Only arrays with at least 8/9 quality control metrics in range were used for the following data analyses. Intra-array normalization was performed with the Feature Extraction Software. Quantile inter-arrays normalization was performed using the Expander software (Sharan et al., 2003).

Project description:High-fat diet or exposure to saturated fatty acids affects skeletal muscle growth and function. The aim of the present study was to investigate the effect of palmitate on the transcriptomic profile of mouse C2C12 myoblasts. Global gene expression was evaluated using whole mouse genome oligonucleotide microarrays, and the results were validated through real-time PCR. A total of 4047genes were identified as differentially expressed, including 3492 down-regulated and 555 up-regulated genes during 48-h-exposure to palmitate (0.1 mmol/l). Functional classification showed the involvement of PA-regulated genes in several processes which regulate cell growth. In conclusion, palmitate modifies the expression of genes associated with: i) myoblast responsiveness to hormones and growth factors, ii) cytokine and growth factor expression, and iii) regulation of cell-cell cell-matrix communication. Such alterations can affect myoblast growth and differentiation, however further studies in this field are required. To whom the correspondence should be addressed: Dr K. Grzelkowska-Kowalczyk; e-mail: k_grzel_kow@poczta.fm, tel/fax: (48 22) 847 24 52 After scanning of hybridized microarrays, quantitation of slide images was performed using Feature Extraction Software (Agilent) using default parameters and the raw data were exported to GeneSpring GX 12 (Agilent, Santa Clara, CA) and log2 transformed. For identification of genes significantly altered in cell compared with the control normal gene set. Total detected entities were filtered by flags (detected, non detected) and error (coefficient of variation: CV < 50.0 percent) to remove very low signal entities and to select reproducible signal values of entities among the replicated experiments, respectively. In statistical analysis, separated for experiment with myoblasts treated with palmitate acid was used t-test unpaired (p < 0.05) with multiple testing correction: Benjamini-Hochberg <0.05, all significant changes over fold change 1.6 were selected.

Dataset Information

Label free proteomics analysis of differentiating human muscle cells.

Publications

PolySTest: Robust Statistical Testing of Proteomics Data with Missing Values Improves Detection of Biologically Relevant Features.

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