Dataset Information

Lactococcus lactis protein turnover

ABSTRACT: Triplicate chemostat cultivations were carried out with L. lactis subsp. lactis IL1403 at specific growth rates 0.1 and 0.5 h-1 on a chemically defined medium, where both, glucose and lysin (Lys) were jointly limiting the growth. After reaching the steady physiological state, medium was switched to heavy Lys (13C615N2-Lys) containing medium and incorporation of heavy amino acid into biomass was observed over the time course. Samples were collected before the medium switch (0 h) and 0.5; 1.5; 5; and 10 h after the medium switch. Cells were lysed in SDS lysis buffer and digested with trypsin according to Filter Aided Sample Preparation protocol (FASP). LC-MS/MS analysis were performed on Agilent 1200 series nanoflow system connected to a LTQ Orbitrap mass-spectrometer equipped with a nanoelectrospray ion source. Raw data files were analyzed with the MaxQuant software package (version 1.2.7.4). Generated peak lists were searched using the Andromeda search engine (built into MaxQuant) against L. lactis database (downloaded 28.06.2012 from http://www.genome.jp/kegg/genes.html). MaxQuant searches were performed with full tryptic specificity, a maximum of two missed cleavages and a mass tolerance of 0.5 Da for fragment ions. Carbamidomethylation of cysteine was set as a fixed modification and methionine oxidation and protein N-terminal acetylation were set as variable modification. The required false discovery rate (FDR) was set to 1% both for peptide and protein levels and the minimum required peptide length was set to six amino acids. In addition, "Match between runs" option with a time window of 1.5 min was allowed.

INSTRUMENT(S): LTQ Orbitrap

ORGANISM(S): Lactococcus Lactis Subsp. Lactis Il1403

SUBMITTER: Liisa Arike

LAB HEAD: Liisa Arike

PROVIDER: PXD000494 | Pride | 2014-01-28

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	120803_04_Orbi1_TT_SER_G240_T280_P0_F99_1.raw	Raw
	120803_05_Orbi1_TT_SER_G240_T280_P0_F99_2.raw	Raw
	120803_10_Orbi1_TT_SER_G240_T280_P1_F99_1.raw	Raw
	120803_11_Orbi1_TT_SER_G240_T280_P1_F99_2.raw	Raw
	120803_17_Orbi1_TT_SER_G240_T280_P2_F99_2.raw	Raw

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Project description:We used phosphoproteomics to compare the responses of the ERK1/2 inhibitors, SCH772984 and GDC0994, and the MKK1/2 inhibitor, trametinib. These are compared with responses to the MKK1/2 inhibitor, selumetinib (AZD6244), previously measured by our lab in the same metastatic melanoma cell line. In three replicate experiments, we quantified a total of 12,805 class I phosphosites on 3,819 proteins in the trametinib-SCH772984-DMSO experiment, and 7,074 class I phosphosites on 2,453 in the GDC0994-SCH772984-DMSO experiment. This included 466 phosphosites that reproducibly decreased in response to at least one inhibitor in the trametinib-SCH772984-DMSO experiment and 414 phosphosites in the GDC0994-SCH772984-DMSO experiment. The results demonstrate linearity in signaling through the MAP kinase pathway. By comparing multiple inhibitors targeted to multiple tiers of protein kinases in the MAPK pathway, we gain insight into regulation and new targets of the oncogenic BRAF driver pathway in cancer cells, and a useful approach for evaluating the specificity of drugs and drug candidates. SILAC Experimental Design Experiment 1 Replicate 1: Heavy – DMSO, Medium – SCH772984, Light – Trametinib Replicate 2: Heavy – SCH772984, Medium – Trametinib, Light – DMSO Replicate 3: Heavy – Trametinib, Medium – DMSO, Light – SCH772984 SILAC Experimental Design Experiment 2 Replicate 1: Heavy – DMSO, Medium – SCH772984, Light – GDC0994 Replicate 2: Heavy – SCH772984, Medium – GDC0994, Light – DMSO Replicate 3: Heavy – GDC0994, Medium – DMSO, Light – SCH772984 File List 1. Zipped MaxQuant search results folder containing index and output folders for each raw file, ‘combined’ output folder, and mqpar.xml MaxQuant search parameters file 2. Individual raw files of phosphopeptide-enriched ERLIC fractions 3. Zipped MaxQuant version used for analysis 4. FASTA file containing Uniprot human identifications 5. Instructions for viewing annotated spectra

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Lactococcus lactis protein turnover

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