OHSU Proteomics Shared Resource - Fazio Lab

OHSU Proteomics Shared Resource - Fazio Lab
Clustergrammer Heatmap
 
Download
NV0001_Mouse-Skin_mProphet_Panorama_2024-03-09_19-20-18.sky.zip2024-03-10 20:30:291,6595,7905,79028,90434
XW0008_Cas9Myc_DIAassayLIB_OmBcells_17Nov2023_2024-02-24_08-51-18.sky.zip2024-02-24 12:56:485,20383,67483,675605,04024
XW0009_DIAassayLIB_OmBcells_17Nov2023_2024-02-23_18-35-50.sky.zip2024-02-23 22:06:575,20383,64583,647604,72019
AutoQC-lumos-SysS-MouAD-PFC-C2-B5-B7.sky.zip2024-02-20 07:53:561889414
Lumos-Jax-Cortex-DIA-ind-8mz-ovlp-400to1000-C2_B07.sky.zip2024-02-18 11:31:099,778127,624127,624966,34712
Lumos-Jax-Cortex-DIA-ind-8mz-ovlp-400to1000-C2_B06.sky.zip2024-02-18 10:45:259,778127,624127,624966,34716
Lumos-Jax-Cortex-DIA-ind-8mz-ovlp-400to1000-C2_B05.sky.zip2024-02-18 09:51:569,778127,624127,624966,34716
Lumos-Jax-Cortex-DIA-ind-8mz-ovlp-400to1000-C2_B04.sky.zip2024-02-18 01:14:219,778127,624127,624966,34716
Lumos-Jax-Cortex-DIA-ind-8mz-ovlp-400to1000-C2_B03.sky.zip2024-02-18 00:22:039,778127,624127,624966,34716
Lumos-Jax-Cortex-DIA-ind-8mz-ovlp-400to1000-C2_B02.sky.zip2024-02-17 23:29:529,778127,624127,624966,34716
Lumos-Jax-Cortex-DIA-ind-8mz-ovlp-400to1000-C2_B01.sky.zip2024-02-17 18:20:009,778127,624127,624966,34716
Lumos-Jax-Cortex-DIA-ind-8mz-ovlp-400to1000-C1_B28.sky.zip2024-02-17 17:30:039,778127,624127,624966,3476
Lumos-Jax-Cortex-DIA-ind-8mz-ovlp-400to1000-C1_B27.sky.zip2024-02-17 16:57:559,778127,624127,624966,34716
Lumos-Jax-Cortex-DIA-ind-8mz-ovlp-400to1000-C1_B26.sky.zip2024-02-17 15:06:069,778127,624127,624966,34716
Lumos-Jax-Cortex-DIA-ind-8mz-ovlp-400to1000-C1_B25.sky.zip2024-02-17 14:11:069,778127,624127,624966,34716
Lumos-Jax-Cortex-DIA-ind-8mz-ovlp-400to1000-C1_B24.sky.zip2024-02-17 13:17:049,778127,624127,624966,34716
Lumos-Jax-Cortex-DIA-ind-8mz-ovlp-400to1000-C1_B23.sky.zip2024-02-17 10:45:369,778127,624127,624966,34716
Lumos-Jax-Cortex-DIA-ind-8mz-ovlp-400to1000-C1_B22.sky.zip2024-02-17 09:52:589,778127,624127,624966,34716
Lumos-Jax-Cortex-DIA-ind-8mz-ovlp-400to1000-C1_B21.sky.zip2024-02-17 09:01:129,778127,624127,624966,34716
Lumos-Jax-Cortex-DIA-ind-8mz-ovlp-400to1000-C1_B20.sky.zip2024-02-17 01:24:329,778127,624127,624966,34716
Lumos-Jax-Cortex-DIA-ind-8mz-ovlp-400to1000-C1_B19.sky.zip2024-02-17 00:31:539,778127,624127,624966,34716
Lumos-Jax-Cortex-DIA-ind-8mz-ovlp-400to1000-C1_B18.sky.zip2024-02-16 23:42:139,778127,624127,624966,34716
Lumos-Jax-Cortex-DIA-ind-8mz-ovlp-400to1000-C1_B17.sky.zip2024-02-16 21:59:109,778127,624127,624966,34716
Lumos-Jax-Cortex-DIA-ind-8mz-ovlp-400to1000-C1_B16.sky.zip2024-02-16 21:08:449,778127,624127,624966,34716
Lumos-Jax-Cortex-DIA-ind-8mz-ovlp-400to1000-C1_B15.sky.zip2024-02-16 19:45:379,778127,624127,624966,34716
Lumos-Jax-Cortex-DIA-ind-8mz-ovlp-400to1000-C1_B14.sky.zip2024-02-16 18:50:509,778127,624127,624966,34716
Lumos-Jax-Cortex-DIA-ind-8mz-ovlp-400to1000-C1_B13.sky.zip2024-02-16 17:05:369,778127,624127,624966,34716
Lumos-Jax-Cortex-DIA-ind-8mz-ovlp-400to1000-C1_B12.sky.zip2024-02-16 16:13:309,778127,624127,624966,34716
XW0008-Myc248_DIAassayLIB_OmBcells_17Nov2023_2024-02-16_10-02-13.sky.zip2024-02-16 15:02:065,20383,67483,675605,04024
Lumos-Jax-Cortex-DIA-ind-8mz-ovlp-400to1000-C1_B11.sky.zip2024-02-16 11:03:589,778127,624127,624966,34716
Lumos-Jax-Cortex-DIA-ind-8mz-ovlp-400to1000-C1_B10.sky.zip2024-02-16 10:07:519,778127,624127,624966,34716
Lumos-Jax-Cortex-DIA-ind-8mz-ovlp-400to1000-C1_B09.sky.zip2024-02-16 09:14:539,778127,624127,624966,34716
Lumos-Jax-Cortex-DIA-ind-8mz-ovlp-400to1000-C1_B08.sky.zip2024-02-16 08:20:059,778127,624127,624966,34716
Lumos-Jax-Cortex-DIA-ind-8mz-ovlp-400to1000-C1_B07.sky.zip2024-02-16 01:08:409,778127,624127,624966,34716
Lumos-Jax-Cortex-DIA-ind-8mz-ovlp-400to1000-C1_B06.sky.zip2024-02-16 00:17:379,778127,624127,624966,34716
Lumos-Jax-Cortex-DIA-ind-8mz-ovlp-400to1000-C1_B05.sky.zip2024-02-15 23:29:389,778127,624127,624966,34716
XW0008_nanos3_DIAassayLIB_OmBcells_17Nov2023_2024-02-15_17-02-46.sky.zip2024-02-15 21:13:165,20383,67483,675605,04024
Lumos-Jax-Cortex-DIA-ind-8mz-ovlp-400to1000-C1_B04.sky.zip2024-02-15 16:37:369,778127,624127,624966,34716
Lumos-Jax-Cortex-DIA-ind-8mz-ovlp-400to1000-C1_B03.sky.zip2024-02-15 14:42:299,778127,624127,624966,34716
Lumos-Jax-Cortex-DIA-ind-8mz-ovlp-400to1000-C1_B02.sky.zip2024-02-15 13:44:359,778127,624127,624966,34716
Lumos-Jax-Cortex-DIA-ind-8mz-ovlp-400to1000-C1_B01.sky.zip2024-02-15 12:45:409,778127,624127,624966,34716
AutoQC-lumos-PCs-MouAD-PFC-C2-B5-B7.sky.zip2024-02-14 16:42:502141417344
AutoQC-lumos-PCs-MouAD-PFC-C2-B1-B4.sky.zip2024-02-14 16:42:332141417364
AutoQC-lumos-PCs-MouAD-PFC-C1-B9-B12.sky.zip2024-02-14 16:42:152141417364
AutoQC-lumos-PCs-MouAD-PFC-C1-B4-B8.sky.zip2024-02-14 16:42:002141417380
AutoQC-lumos-PCs-MouAD-PFC-C1-B25-B28.sky.zip2024-02-14 16:41:372141417354
AutoQC-lumos-PCs-MouAD-PFC-C1-B21-B24.sky.zip2024-02-14 16:41:002141417364
AutoQC-lumos-PCs-MouAD-PFC-C1-B17-B20.sky.zip2024-02-14 16:40:442141417365
AutoQC-lumos-PCs-MouAD-PFC-C1-B13-B16.sky.zip2024-02-14 16:40:282141417364
AutoQC-lumos-PCs-MouAD-PFC-C1-B1-B3.sky.zip2024-02-14 16:40:082141417347
AutoQC-lumos-SysS-MouAD-PFC-C2-B1-B4.sky.zip2024-02-14 16:10:161889417
AutoQC-lumos-SysS-MouAD-PFC-C1-B9-B12.sky.zip2024-02-14 16:06:251889416
AutoQC-lumos-SysS-MouAD-PFC-C1-B4-B8.sky.zip2024-02-14 16:02:231889422
AutoQC-lumos-SysS-MouAD-PFC-C1-B1-B3.sky.zip2024-02-14 15:59:501889418
AutoQC-lumos-SysS-MouAD-PFC-C1-B17-B20.sky.zip2024-02-14 14:48:381889410
ZipChip_HR_Metabolomics_2024Protocol_2024-02-05_17-24-05.sky.zip2024-02-05 14:24:28100821594
22AminoAcids_Fully13CLabeled_2024-01-29_14-30-52.sky.zip2024-01-29 11:32:1410444936
RBD_M_Glyco_2024-01-25_15-29-41.sky.zip2024-01-26 17:23:2672923972,3829
20240104_Neg_FMT_MCBAs_isoRemove_Cleaned_Final_2024-01-25_21-40-19.sky.zip2024-01-26 16:43:471010030056
20231220_Neg_FMT_BA_Full_reduce_Res50_High_final_2024-01-04_15-44-59.sky.zip2024-01-26 16:43:47405112176
P179_UNCSet1_ACE_v0p3_2024-01-24_22-42-18.sky.zip2024-01-24 19:51:4423034963724
P179_UNCSet2_ACE_v0p3_2024-01-24_22-37-25.sky.zip2024-01-24 19:40:1117021336726
New_iRBD2024-01-15 23:30:5233474794292
Paired_CSF_Plasma_Serum2024-01-15 23:30:523347479460
Initial_Targeted_Proteomics2024-01-15 23:30:5233474794441
TPAD_VL_CSF_PRTC_APOA1_2024-01-07_23-01-46.sky.zip2024-01-07 23:08:493464642412
TPAD-CSF-SP3_1-5.sky.zip2024-01-05 06:03:432,90823,74323,743189,895396
173_peptides_iRTs_chromatogram_library_2023-12-22_00-47-19.sky.zip2023-12-22 01:06:36311833561,0822
Figure_8B_Freiburg_ALG1-CDG-Patients_Comparison_2023-12-22_02-34-55.sky.zip2023-12-22 01:06:2022691284006
Figures_4_5_6_7_8A_Heidelberg_CDG-Patients_2023-12-22_02-32-43.sky.zip2023-12-22 01:06:20206712439014
Figure_S5_Freiburg_ALG11_I-CDG_Natural_Variant_2023-12-22_01-59-41.sky.zip2023-12-22 01:06:2021112146
Figure_9_Freiburg_ALG11_I-CDG_Natural_Variant_2023-12-22_01-53-52.sky.zip2023-12-22 01:06:2021418404
Figures_3_and_S3_HEK_293T_Fibroblasts_HeLa_2023-12-22_01-03-03.sky.zip2023-12-22 01:06:2023701303989
20210301 Calibration Dev_DilutionOil_2023-12-11_10-57-35.sky.zip2023-12-20 00:34:263482654
20210607 Calibration Curve_DilutionDigest_2023-12-11_10-50-40.sky.zip2023-12-20 00:34:2634824108
20210212 Low range exploration 140K-fragmod_Pub_2023-12-08_16-04-13.sky.zip2023-12-20 00:34:263482456
HeatedOilSpike-LowTemp_HighTemp_Combined_Final_2022-05-26_12-00-47.sky.zip2023-12-20 00:34:26591548204
20200715_PeptideSpecificity_SignalRatio_2022-05-25_16-33-02.sky.zip2023-12-20 00:34:2611202212044
20200622_PeptideSpecificityTest_2022-05-25_16-30-20.sky.zip2023-12-20 00:34:2614252713545
20191112_Diff-TempConc_Oil-Spike_24pep_2022-05-25_14-24-35.sky.zip2023-12-20 00:34:2611242715040
20191007_HeatedOilSpike_Extraction_method_24pep_2022-05-25_14-16-21.sky.zip2023-12-20 00:34:2611242715032
20190904_Organic_Aqueous_Extraction_Oil_Spike_24pep_2022-05-25_14-12-26.sky.zip2023-12-20 00:34:2611242715636
September 21 Import V1 (Samples with IS) w Cal Curve_Blanks Deleted_2023-12-01_11-40-59.sky.zip2023-12-02 23:51:3320262687
September 21 Kaylie New Molecule Import v1 (Filtered)_2023-12-01_11-40-01.sky.zip2023-12-02 23:51:3310161698
September 21 Import V1 all samples (Neg mode only)_2023-12-01_11-35-34.sky.zip2023-12-02 23:51:338014514598
THP1_IFN_PRM_Skyline_2023-11-14_14-22-42.sky.zip2023-11-16 13:26:09711771771,5148
CCS_library_v2.sky.zip2023-11-15 14:36:301061,86361,8630
IdentExpression_2023-09-25_14-39-47.sky.zip2023-10-02 20:45:1381581941,16419
2DGel_II_2023-09-25_14-37-39.sky.zip2023-10-02 20:45:13897408805,28034
2DGel_I_2023-09-25_14-36-13.sky.zip2023-10-02 20:45:13252142321,39232
InGelDigest_Der_p_SEA_II_2023-09-18_10-01-52.sky.zip2023-09-21 11:03:445004,0584,42026,52051
InGelDigest_Der_p_SEA_I_2023-09-15_16-14-11.sky.zip2023-09-21 11:03:443272,3822,52115,12652
FASP_Der_p_SEA_2023-09-15_14-24-26.sky.zip2023-09-21 11:03:445504,2964,77128,6261
230504_Myllys_231247ff_2023-09-20_13-22-59.sky.zip2023-09-21 10:31:2410434398
230512MyllysUrea_231247ff_2023-09-21_15-27-13.sky.zip2023-09-21 10:31:24102292
230511Myllys231247ff_AA_Crea_2023-09-20_13-36-24.sky.zip2023-09-21 10:31:242044166126
AutoQC-lumos-SysS-MouAD-C2-B8-10.sky.zip2023-09-17 11:22:321889414
AutoQC-lumos-SysS-MouAD-C2-B5-B7.sky.zip2023-09-17 11:22:301889415
AutoQC-lumos-SysS-MouAD-C2-B1-B4.sky.zip2023-09-17 11:22:281889419
AutoQC-lumos-SysS-MouAD-C1-B9-B12.sky.zip2023-09-17 11:22:261889413
Insights in the molecular kinetics and dynamics of the furin-cleaved form of PCSK9
Data License: CC BY 4.0 | ProteomeXchange: PXD018700
  • Organism: Homo sapiens
  • Instrument: LTQ Velos
  • SpikeIn: No
  • Keywords: cardiovascular disease, cholesterol, lipoprotein metabolism, LDL, LDLR, proteases, secretory pathway, posttranslational modifications, function
  • Lab head: Sergio Fazio Submitter: Ashok Reddy
Abstract
Abstract Proprotein convertase subtilisin/kexin type 9 (PCSK9) regulates cholesterol metabolism by inducing the degradation of hepatic low-density lipoprotein receptor (LDLR). Plasma PCSK9 has two main molecular forms: a 62-kDa mature form (PCSK9_62) and a 55-kDa, furin-cleaved form (PCSK9_55). PCSK9_55 is considered less active than PCSK9_62 in degrading LDLR. We aimed to identify the site of PCSK9_55 formation (intra- vs. extracellular) and to further characterize the function of PCSK9_55 relative to PCSK9_62. Co-expressing PCSK9_62 with furin in cell culture induced formation of PCSK9_55, most of which was found extracellularly (97-times more). Under the same conditions, i) adding a cell-permeable furin inhibitor preferentially affected the formation of PCSK9_55 out of the cell, and ii) substituting PCSK9_62 for a mutant that cannot get secreted completely abolished furin action on PCSK9, even though the majority of furin locates intracellularly. Further, the expression of a recombinant PCSK9_55 in cells demonstrated that the intracellular pool of PCSK9_55 fails to be secreted and is retained intracellularly. Nevertheless, this intracellular pool of PCSK9_55 induced degradation of LDLR, though with 50% lower efficiency when compared with PCSK9_62. Collectively, our data show that PCSK9_55 is 1) generated in the extracellular space, and that the small intracellular pool of PCSK9_55 is 2) not secreted but is 3) capable of inducing LDLR degradation through an intracellular pathway.
Experiment Description
Two biological replicates including the products of a negative control reaction (no furin; PCSK9 62-kDa) and the experimental reaction PCSK9_62 with furin (PCSK9 55-kDa) were evaluated separately in two independent LC-MS analyses to detect the expected furin cleavage product containing residues 153-218 (average mass = 7730.5). With no further digestion, aliquots of 2.2 µg of PCSK9 from a control sample (no furin) and a furin incubated sample were dried using a DNA120 SpeedVac (Thermo Scientific) and stored at -20 ̊C until analysis. Samples were solubilized in 22 µL of 8 M urea 24 h prior to analysis. A total of 1 µg of protein was analyzed using a LTQ Velos Pro linear ion trap (Thermo Scientific, San Jose, CA). Samples were injected onto a micro protein trap cartridge (Optimize Technologies, Oregon City, OR) at a flow rate of 20 µl/min in mobile phase containing 0.1% formic acid. After 5 min, the flow was diverted to a 1 x 250 mm C4 column (Vydac, SN 214MS51, Grace, Deerfield, IL). Protein was eluted by increasing the acetonitrile concentration from 2-7.5% over one minute, then 7.5-60% over 30 minutes and data collection on the mass spectrometer started 10 min into the separation. The instrument used a HESI-II probe fitted with a 34 gauge metal needle, 5.0 kV source voltage, 325 °C ion transfer tube temperature, sheath gas setting of 5, full MS scans in profile mode over a range of m/z = 400-2000, and averaging of 10 µscans. Spectra acquired during elution of the PCSK9 153-218 peaks was then averaged and deconvoluted using the Manual Respect module for isotopically unresolved data in Protein Deconvolution 4.0 software (Thermo Scientific). Single ion chromatographic traces were produced using Qual Browser software within the Xcalibur Suite (Thermo Scientific).
Sample Description
PCSK9 from different sources (cells overexpressing PCSK9 (3.4 µL of media or 3.4 µL of cell extract) or a purified recombinant form (1900 nM, Cat# 20631, Cayman Chemical) was set to react with furin (680 nM; Cat# 450-47, PeproTech) under optimized buffer, temperature and time conditions (4 mM CaCl2, 150 mM NaCl, 20 mM KCl and 50 mM Tris-HCl pH 7.4 in 10 µL of final volume at 37 ̊ C for 24 hour) to achieve the highest yield of PCSK9 proteolysis. Reactions were stopped by chelating calcium with 4 mM EDTA.
Created on 4/20/20, 3:39 PM