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Escherichia coli proteome during stationary phase

ABSTRACT: The aim of this study was to determine the fate of ribosomes and r-proteins. In this respect, SILAC (Stable Isotope Labeled Amino acids in cell Culture) based experimental approach was used. E.coli cells were grown in MOPS medium supplemented with “heavy” labeled arginine (Arg10) and lysine (Lys8). At the mid-log phase, the culture was further supplemented with a 20-fold molar excess of “light” unlabeled arginine (Arg0) and lysine (Lys0), divided into 8 aliquots, and grown for 14 days. Cell samples were collected at day one (24h), day two (48h), and subsequently in 48h intervals over the following 12 days. The quantities of r-proteins in the proteome were determined using SILAC based LC-MS/MS and normalized to the corresponding values of day one.

INSTRUMENT(S): LTQ Orbitrap XL

ORGANISM(S): Escherichia Coli

SUBMITTER: Kaspar Reier

LAB HEAD: Tiina Tamm

PROVIDER: PXD035927 | Pride | 2023-10-10

REPOSITORIES: Pride

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			Action	DRS
	200519_09_Orbi2_KN_SER_G240_TYMRI_Reier_G24_2ug.raw	Raw
	200519_11_Orbi2_KN_SER_G240_TYMRI_Reier_G11_2ug.raw	Raw
	200525_03_Orbi2_KN_SER_G240_TYMRI_Reier_G23_2ug.raw	Raw
	200525_05_Orbi2_KN_SER_G240_TYMRI_Reier_G2_2ug.raw	Raw
	200525_07_Orbi2_KN_SER_G240_TYMRI_Reier_G6_2ug.raw	Raw

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Project description:In this project, we employed a dynamic mass spectrometry-based proteomic approach to obtain global maps of basal autophagic flux in human primary fibroblasts (HCA2-hTert). The data provide a comparison of protein degradation rates between dividing and quiescent HCA2-hTert cells. To further investigate the role of autophagy in up-regulated protein degradation, protein degradation rates were also measured in autophagy-deficient (Atg5-/-) in both dividing and quiescent cells. Steady-state protein level changes in dividing and quiescent cells were measured by standard SILAC. To investigate the role of PSME1 in proteasome activity regulation, protein degradation rates in PSME1 knockdown cell line were measured by SILAC. Stable isotope labeling The media utilized for isotopic labeling was Eagle’s minimum essential medium (ATCC) supplemented with 15% dialyzed fetal bovine serum (Thermo Scientific), 100 U/ml penicillin, and 100 U/ml streptomycin. Both wildtype and Atg5-/- Cells were gradually adapted to this media and prepared for labeling experiments. Cells were then plated at a density of 500,000 cells per 10 cm plate. One day after plating, the dividing cultures were switched to MEM labeling media for SILAC (Thermo Scientific) supplemented with L-arginine:HCl (13C6, 99%) and L-lysine:2HCl (13C6, 99%, Cambridge Isotope Laboratories) at concentrations of 0.1264 g/l and 0,087 g/l and 15% dialyzed fetal bovine serum (Thermo Scientific). Cells were collected after 0, 1, 2, and 3 days of labeling, washed with PBS and cell pellets were frozen prior to further analysis. 8 days after plating, the confluent quiescent cultures were switched to MEM labeling media for SILAC (Thermo Scientific) supplemented with L-arginine:HCl (13C6, 99%) and L-lysine:2HCl (13C6, 99%; Cambridge Isotope Laboratories) at concentrations of 0.1264 g/l and 0.087 g/l and 15% dialyzed fetal bovine serum (Thermo Scientific). Cells were collected after 0, 2, 4, and 6 days of labeling, washed with PBS and cell pellets were frozen prior to further analysis. In order to measure changes in steady-state protein levels by standard SILAC, WT cells were gradually adapted to Eagle’s minimum essential medium (ATCC) supplemented with 15% dialyzed fetal bovine serum (Thermo Scientific), 100 U/ml penicillin, and 100 U/ml streptomycin. Then, WT cells were passaged in MEM labeling media for SILAC (Thermo Scientific) supplemented with L-arginine:HCl (13 C6, 99%) and L-lysine:2HCl (13 C6, 99%; Cambridge Isotope Laboratories) at concentrations of 0.1264 g/l and 0.087 g/l and 15% dialyzed fetal bovine serum (Thermo scientific) for eight passages. Cells were then plated at a density of 500,000 cells per 10-cm plate. 2 days after plating, dividing cells were collected and washed with PBS, and cell pellets were frozen prior to further analysis. Following extraction (see below), equal protein amounts of dividing and quiescent WT cells were mixed before mass spectrometric analysis. To measure protein degradation in PSME1 knockdown cell line, 8 days after plating, the confluent quiescent cultures were switched to MEM labeling media for SILAC (Thermo Scientific) supplemented with L-arginine:HCl (13C6, 99%) and L-lysine:2HCl (13D4, 96%-98%; Cambridge Isotope Laboratories) at concentrations of 0.1264 g/l and 0.087 g/l and 15% dialyzed fetal bovine serum (Thermo Scientific). Cells were collected after 3 days of labeling, washed with PBS and cell pellets were frozen prior to further analysis.

Project description:Protein extraction and proteolytic digestion were performed using a Filter-Assisted Sample Preparation (FASP) protocol. In case of phosphopeptide enrichment immobilized Fe(III) affinity chromatography (Fe-IMAC) was performed. The peptides were fractionated using an HPLC system fitted with an SCX column. 10 sample datasets were used: 1. CRC_N: Normal tissue samples were obtained from 20 colorectal cancer patients, no quantification.Phosphopeptide enrichment was performed. 2. CRC_T: Human colorectal cancer tissue samples were obtained from 20 patients, no quantification. Phosphopeptide enrichment was performed. 3. CRC_iTRAQ: Human colorectal cancer tissue samples were obtained from 24 patients, iTRAQ quantification. 4. CRC_phospho_iTRAQ:Human colorectal cancer tissue samples were obtained from 24 patients, iTRAQ quantification.Phosphopeptide enrichment was performed. 5. HCT116_iTRAQ: Human colon cancer cell HCT116, iTRAQ quantification. 6. HCT116_phospho_iTRAQ: Human colon cancer cell HCT116, phosphopeptide enrichment, iTRAQ quantification. 7. SW_SILAC_HL: Human colon cancer cell SW480 and SW620, SW480 and SW620 were grown in SILAC media, containing l-arginine (Arg0) and l-lysine (Lys0) (SW480; light), or 13C615N4-l-arginine (Arg10) and 13C6-l-Lysine (Lys6) (SW620; heavy). 8. SW_SILAC_LH: SW480 and SW620 were grown in SILAC media, containing l-arginine (Arg0) and l-lysine (Lys0) (SW620; light), or 13C615N4-l-arginine (Arg10) and 13C6-l-Lysine (Lys6) (SW480; heavy). 9. SW_phospho_SILAC_HL: SW480 and SW620 were grown in SILAC media, containing l-arginine (Arg0) and l-lysine (Lys0) (SW480; light), or 13C615N4-l-arginine (Arg10) and 13C6-l-Lysine (Lys6) (SW620; heavy). Phosphopeptide enrichment was performed. 10. SW_phospho_SILAC_LH: SW480 and SW620 were grown in SILAC media, containing l-arginine (Arg0) and l-lysine (Lys0) (SW620; light), or 13C615N4-l-arginine (Arg10) and 13C6-l-Lysine (Lys6) (SW480; heavy). Phosphopeptide enrichment was performed. For creation of xml and mgf files, Proteome Discoverer 1.3 and Mascot v2.3 software were used.

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Escherichia coli proteome during stationary phase

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