Cedars-Sinai Parker Lab - ACE10 Bernstein Study

Cedars-Sinai Parker Lab - ACE10 Bernstein Study
ACE over expression in myeloid cells increases oxidative metabolism and cellular ATP
Data License: CC BY 4.0
  • Organism: Mus musculus
  • Instrument: QTRAP 6500+
  • SpikeIn: Yes
  • Keywords: ACE overexpression, Mouse Monocyte Metabolism, Mitoplex
  • Lab head: Sarah Parker Submitter: Sarah Parker
Abstract
Angiotensin converting enzyme (ACE) affects blood pressure. In addition, ACE over expression in myeloid cells increases their immune response. We identified marked changes of intermediate metabolites in ACE over expressing macrophages and neutrophils, with increased cellular ATP and Krebs cycle intermediates including citrate, isocitrate, succinate, and malate. Increased ATP is due to ACE activity; it is reversed by an ACE inhibitor but not by an angiotensin II AT1 receptor antagonist. ACE over expression does not change cell or mitochondrial size or number. However, expression levels of the electron transport chain proteins NDUFB8 (complex I), ATP5A, and ATP5β (complex V) are increased in macrophages and neutrophils, and MTCO1 and COX2 (complex IV) in macrophages, over expressing ACE. Macrophages over expressing ACE have increased mitochondrial ATP production rates and maximal respiratory rates. Increased cellular ATP underpins increased myeloid cell superoxide production and phagocytosis associated with increased ACE expression. Myeloid cells over expressing ACE indicate the existence of a novel pathway in which myeloid cell function can be enhanced, with a key feature being increased cellular ATP.
Experiment Description
Comparison of mitochondrial protein expression between ACE10-overexpressing and wild-type mouse monocytes
Sample Description
Monocyte pellets (N=4 per mouse genotype, ACE10 vs WT) were lysed in 8M Urea dissolved in 50mM TRIS-HCl buffer, pH 8.0. Lysis was facilitated by high pressure treatment on a Pressure Biosciences (location) barocycler (Model number), with 60 one-minute cycles consisting of 50 seconds of 45 kPSI followed by 10 seconds at Atmospheric Pressure. Following extraction, protein concentration was assayed using the BCA (Pierce, Company Location). Fifty micrograms of protein from each sample were aliquoted for digestion, and each sample was reduced using 10mM Dithiothreitol (10mM) and subsequently alkylated with iodoacetamide (100mM). Samples were diluted with 200mM Ammonium Bicarbonate buffer and supplemented with 10% Acetonitrile, to a final Urea concentration < 2M. Trypsin was added at a ratio of 1 ug to 50ug total protein and subjected to an additional series of 60 cycles (45kPSI for 50s, ATM for 10s) at 37 degrees C, after which they were left to incubate at ATM overnight at 37C. Digestion was quenched with 1% Trifluoroacetic acid and samples were desalted on Nest C18 tips (Nest Group, Location). Peptides were dried to completion and re-suspended in a solution of 0.1% Formic Acid (FA) in ddH20 into which a 1:250 dilution of stable isotope labeled reference peptides derived for targeted mitochondrial protein analysis as described in detail in Stotland et al., 2019 (https://www.biorxiv.org/content/10.1101/820167v1). A total of 8ug of digested peptides, injected twice as duplicate technical replicates, were separated on a Prominence UFLCXR HPLC system (Shimadzu, Japan) with a Waters Xbridge BEH30 C18 2.1mm x 100mm, 3.5µm column (Waters) flowing at 0.25 mL/min and 36 °C coupled to a QTRAP® 6500 (SCIEX, Framingham, MA). Mobile phase A consisted of 2% ACN, 98% water, and 0.1% formic acid and mobile phase B of 95% ACN, 5% water, and 0.1% formic acid. After loading, the column was equilibrated with 5% B for 5 minutes. Peptides were then eluted over 30 minutes with a linear 5% to 35% gradient of buffer B. The column was washed with 98% B for 10 minutes and then returned to 5% B for 5 minutes before loading the next sample. A scheduled, targeted acquisition method optimized for each peptide was used to monitor fragments within a 2-minute window of the optimized retention time.
Created on 11/14/19, 3:58 PM
Clustergrammer Heatmap
 
Download
DDR2_chromatogram5.sky.zip2021-04-08 06:02:52112141
DDR2_chromatogram1.sky.zip2021-04-07 18:04:172738765261
DDR2_chromatogram2.sky.zip2021-04-07 18:03:1078161101
DDR2_chromatogram4.sky.zip2021-04-07 18:02:2011281
DDR2_chromatogram3.sky.zip2021-04-07 18:01:31112141
ARP_Plasma_0.2_vs_2.0_mg_2021-02-24_18-55-48.sky.zip2021-04-02 14:46:32404124891,40511
ARP-OxHSA-PlasmaMatrix_DilSeries_2021-02-25_11-31-34.sky.zip2021-04-02 14:46:32224129686218
Plasma-OxHSA_2021-02-25_11-37-12.sky.zip2021-04-02 14:46:32626330991811
3cm_preliminary_data_2021-03-26_18-17-25.sky.zip2021-03-31 10:24:111371312
LINCS_P100_DIA_Plate15_annotated_minimized_2018-05-02_19-32-22.sky.zip2021-03-19 20:17:4890961921,20296
LINCS_P100_DIA_Plate16_annotated_minimized_2018-05-02_19-43-10.sky.zip2021-03-19 20:17:4890961921,12295
LINCS_P100_DIA_Plate17_annotated_minimized_2018-05-02_17-35-29.sky.zip2021-03-19 20:17:4890961921,05495
LINCS_P100_DIA_Plate19_annotated_minimized_2018-05-02_17-18-22.sky.zip2021-03-19 20:17:4890961921,16096
LINCS_P100_DIA_Plate20_annotated_minimized_2018-05-02_21-04-33.sky.zip2021-03-19 20:17:4890961921,07496
LINCS_P100_DIA_Plate22_annotated_minimized_2018-05-02_22-33-32.sky.zip2021-03-19 20:17:4890961921,09695
LINCS_P100_DIA_Plate23_annotated_minimized_2018-05-02_18-13-55.sky.zip2021-03-19 20:17:48909619296096
LINCS_P100_DIA_Plate24_annotated_minimized_2018-05-02_19-07-44.sky.zip2021-03-19 20:17:48909619273296
LINCS_P100_DIA_Plate27_annotated_minimized_2018-05-02_19-16-47.sky.zip2021-03-19 20:17:4890961921,06496
LINCS_P100_DIA_Plate30_annotated_minimized_2018-05-02_18-33-32.sky.zip2021-03-19 20:17:48909619278596
LINCS_P100_DIA_Plate33_annotated_minimized_2018-05-02_17-47-07.sky.zip2021-03-19 20:17:48909619279696
LINCS_P100_DIA_Plate34_annotated_minimized_2019-08-30_17-25-21.sky.zip2021-03-19 20:17:48909619275296
LINCS_P100_DIA_Plate58_annotated_minimized_2017-11-28_11-50-08.sky.zip2021-03-19 20:17:48909619283495
LINCS_P100_DIA_Plate59a_annotated_minimized_2018-11-30_14-13-58.sky.zip2021-03-19 20:17:4890961922,76494
LINCS_P100_DIA_Plate60_annotated_minimized_2017-11-29_16-22-26.sky.zip2021-03-19 20:17:48909619297895
LINCS_P100_DIA_Plate61_annotated_minimized_2018-02-06_17-09-06.sky.zip2021-03-19 20:17:48909619299095
LINCS_P100_DIA_Plate62_annotated_minimized_2018-04-20_15-55-05.sky.zip2021-03-19 20:17:48909619298696
LINCS_P100_DIA_Plate63a_annotated_minimized_2019-11-14_15-28-26.sky.zip2021-03-19 20:17:4890961922,76494
LINCS_P100_DIA_Plate64_annotated_minimized_2018-05-02_20-34-19.sky.zip2021-03-19 20:17:48909619298295
LINCS_P100_DIA_Plate66_annotated_minimized_2018-08-31_10-36-13.sky.zip2021-03-19 20:17:4790961922,76496
LINCS_P100_PRM_Plate25_annotated_minimized_2018-05-02_18-24-10.sky.zip2021-03-19 20:17:4790961921,24296
LINCS_P100_DIA_Plate67_annotated_minimized_2018-08-02_13-56-24.sky.zip2021-03-19 20:17:4790961922,75496
LINCS_P100_PRM_Plate18_annotated_minimized_2020-01-08_17-11-19.sky.zip2021-03-19 20:17:4790961921,53896
LINCS_P100_PRM_Plate28_annotated_minimized_2018-05-02_18-51-02.sky.zip2021-03-19 20:17:4790961921,15896
LINCS_P100_PRM_Plate29_03H_annotated_minimized_2018-05-02_21-16-28.sky.zip2021-03-19 20:17:4790961921,53696
LINCS_P100_DIA_Plate65_annotated_minimized_2019-10-21_16-54-32.sky.zip2021-03-19 20:17:4790961921,58694
LINCS_P100_PRM_Plate31_annotated_minimized_2018-05-02_18-00-17.sky.zip2021-03-19 20:17:47909619299684
LINCS_P100_PRM_Plate32_annotated_minimized_2018-05-02_20-45-26.sky.zip2021-03-19 20:17:4790961921,14696
ZaharaN_MM_Final_SkylineReport_2020-07-30_12-54-33.sky.zip2021-03-18 16:36:239212321832
Martinez et al_PRM Replicates 5-7_MCP_2021-01-28_10-13-34.sky.zip2021-03-18 16:36:23919192378
DDR2_repeatability2.sky.zip2021-03-18 07:29:152365057
DDR2_repeatability1.sky.zip2021-03-18 07:29:0931499868857
DDR2_response curve2.sky.zip2021-03-18 07:22:231242827
DDR2_response curve.sky.zip2021-03-18 07:22:2027469264634
Diab_tear_SRM_2021-03-18_13-32-33.sky.zip2021-03-18 05:33:45182649102112
BSA_method_dev.sky.zip2021-03-14 13:29:58143545443
BSA_and_PRTC.sky.zip2021-03-14 13:29:5361021171,1683
Skin AMP_2021-03-09_15-18-28.sky.zip2021-03-09 06:19:5716244882460
Giannaki_et_al_P171_01_NBCe1_phosphorylation_2021-02-25_17-22-02.sky.zip2021-03-04 09:45:02120206016
T11X_R2021-02-23 07:17:23177564
T11X_QS2021-02-23 07:17:23118181444
T6X_ACDE2021-02-23 07:17:23136362884
T6X_FGHIL2021-02-23 07:17:23136362884
A7X_KMNP2021-02-23 07:17:23136362884
A7X_QS2021-02-23 07:17:23118181444
R8X_ACDEFGH2021-02-23 07:17:23121211684
K9X_ACDE2021-02-23 07:17:23136362884
K9X_KMNP2021-02-23 07:17:23136362884
K9X_QS2021-02-23 07:17:22118181444
A1X_QS2021-02-23 07:17:22118181444
A1X_R2021-02-23 07:17:22177564
A1X_ACDE2021-02-23 07:17:22136362884
T3X_FGHIL2021-02-23 07:17:22136362884
T3X_R2021-02-23 07:17:22177564
R2X_RSTVWY2021-02-23 07:17:22124241924
R2X_ILKMNPQ2021-02-23 07:17:22118181444
K4X_TVWY2021-02-23 07:17:22136362884
A1X_KMNP2021-02-23 07:17:22136362884
A1X_1-10_KMNP2021-02-23 07:17:22136362864
K4X_KMNP2021-02-23 07:17:22136362884
K4X_QS2021-02-23 07:17:22127272164
K4X_ACDE2021-02-23 07:17:22136362884
A1X_FGHIL2021-02-23 07:17:22136362884
T3X_QS2021-02-23 07:17:22118181444
Q5X_FGHIL2021-02-23 07:17:21137372964
K9X_R2021-02-23 07:17:21177564
S10X_R2021-02-23 07:17:21177564
T11X_TVWY2021-02-23 07:17:21136362884
G12X_ACDE2021-02-23 07:17:21136362884
G12X_FGHIL2021-02-23 07:17:21136362884
G12X_QS2021-02-23 07:17:21118181444
R2X_ACDEFGH2021-02-23 07:17:21121211684
A1X_TVWY2021-02-23 07:17:21236362884
T3X_TVWY2021-02-23 07:17:21136362884
T3X_KMNP2021-02-23 07:17:21136362884
K4X_FGHIL2021-02-23 07:17:21136362884
K4X_R2021-02-23 07:17:21177564
Q5X_ACDE2021-02-23 07:17:21136362884
T6X_TVWY2021-02-23 07:17:20136362854
Q5X_R2021-02-23 07:17:20177564
Q5X_TVWY2021-02-23 07:17:20136362884
T6X_KMNP2021-02-23 07:17:20136362884
Q5X_QS2021-02-23 07:17:20127272164
R8X_ILKMNPQ2021-02-23 07:17:20118181444
R8X_RSTVWY2021-02-23 07:17:20124241924
Q5X_KMNP2021-02-23 07:17:20136362884
A7X_TVWY2021-02-23 07:17:20136362884
K9X_FGHIL2021-02-23 07:17:20136362864
S10X_ACDE2021-02-23 07:17:20136362884
S10X_KMNP2021-02-23 07:17:20136362864
T11X_ACDE2021-02-23 07:17:20136362884

This data is available under the CC BY 4.0 license.