Dataset Information

Assessment of protein extraction and digestion efficiency of well-established shotgun protocols for heart proteomics

ABSTRACT: In this study, we evaluated three well-established sample preparation methods for shotgun proteomics, named: i) UREA; ii) sodium dodecyl sulfate (SDS); and iii) filter-aided sample preparation (FASP) protocols. We focused our analysis on protein extraction and digestion from heart left ventricle (LV) tissue.The three methods were compared by relative quantification using dimethyl labeling.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Rattus Norvegicus (rat)

TISSUE(S): Heart

SUBMITTER: Thiago Verano-Braga

LAB HEAD: Thiago Verano-Braga

PROVIDER: PXD012591 | Pride | 2019-05-21

REPOSITORIES: Pride

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Assessment of protein extraction and digestion efficiency of well-established shotgun protocols for heart proteomics.

Rodrigues-Ribeiro Lucas L Melo-Braga Marcella Nunes MN Kjeldsen Frank F Gómez-Mendoza Diana Paola DP Verano-Braga Thiago T

Analytical biochemistry 20190511

Ischemic heart disease is the leading cause of deaths worldwide. Thus, understanding the molecular mechanisms underlying disease progression is needed. Due to heart importance and lack of studies evaluating different sample preparation methods for heart proteomics, we compared three well-established protocols in shotgun proteomics using dimethyl label quantitation to allow relative quantitation. The tested methods for the analysis of left ventricle (LV) tissue were: i) in-solution digestion (ISD ...[more]

PMID: 31085165

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Project description:We have used genome editing to generate inactivating deletion mutations in all three copies of the dicer (hdcr) gene present in the human cell line 293T. As previously shown in murine ES cells lacking Dicer function, hDcr-deficient 293T cells are severely impaired for the production of mature microRNAs (miRNAs). Nevertheless, RNA-induced silencing complexes (RISCs) present in these hDcr-deficient cells are readily programmed by transfected, synthetic miRNA duplexes to repress mRNAs bearing either fully or partially complementary targets, including targets bearing incomplete seed homology to the introduced miRNA. Using these hDcr-deficient 293T cells, we demonstrate that human pre-miRNA processing can be effectively rescued by ectopic expression of the Drosophila Dicer 1 protein, but only in the presence of the PB isoform of Loquacious (Loqs-PB), the fly homolog of the hDcr co-factor TRBP. In contrast, Drosophila Dicer 2, even in the presence of its co-factors Loqs-PD and R2D2, was unable to support human pre-miRNA processing. Interestingly, although ectopic Drosophila Dicer 1/Loqs-PB or hDcr both rescued pre-miRNA processing effectively in these hDcr-deficient cells, there were significant differences in the ratio of the miRNA isoforms that were produced, especially in the case of miR-30 family members, and we also noted differences in the relative expression level of miRNAs versus passenger strands for a subset of human miRNAs. These data demonstrate that the mechanisms underlying the accurate processing of pre-miRNAs are largely, but not entirely, conserved between mammalian and insect cells. Series includes three datasets of total small RNA reads from wild type and Dicer negative 293T cells. Also included are total small RNA reads of the Dicer-negative cell line NoDice(4-25) transfected with a vector expressing human Dicer, Drosophila Dicer1, or mock transfected. RISC-associated small RNAs identified by ribonucleoprotein immunoprecipitation (RIP-Seq) in wild type 293T, Dicer-negative NoDice(4-25) line, and NoDice(4-25) transfected with either hsa-miR-155 or kshv-miR-K12-11 miRNA duplexes. The final data series are PAR-CLIP libraries which identified microRNA targets in untransfected NoDice(4-25) cells and NoDice(4-25) cells transfected with either hsa-miR-92a, hsa-miR-155, kshv-miR-K12-11 miRNA duplexes

Dataset Information

Assessment of protein extraction and digestion efficiency of well-established shotgun protocols for heart proteomics

Publications

Assessment of protein extraction and digestion efficiency of well-established shotgun protocols for heart proteomics.

Similar Datasets

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