Ehime HBFP - BACDROP

Ehime HBFP - BACDROP
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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
BAC-DROP: Rapid digestion of proteome fractionated via dissolvable polyacrylamide gel electrophoresis and its application to bottom-up proteomics workflow
Data License: CC BY 4.0 | ProteomeXchange: PXD021489
  • Organism: Homo sapiens, Bos taurus, Hepatitis B virus
  • Instrument: QTRAP 5500
  • SpikeIn: No
  • Keywords: BAC-PAGE, Bottom-up proteomics, Dissolvable polyacrylamide gel, GeLC-MS, Rapid enzymatic digestion
  • Lab head: Nobuaki Takemori Submitter: Nobuaki Takemori
Abstract
The GeLC-MS workflow, which combines low-cost, easy-to-use SDS-polyacrylamide gel electrophoresis (SDS-PAGE) with liquid chromatography-mass spectrometry (LC-MS), is very popular in current bottom-up proteomics. However, GeLC-MS requires that PAGE-separated proteins undergo overnight enzymatic digestion in a gel, resulting in more than 20 hours of sample preparation for LC-MS. In this study, we overcame the limitations of GeLC-MS by developing a rapid digestion workflow for PAGE separation proteins using N,N'-bis(acryloyl)cystamine (BAC) cross-linked gels that can be solubilized by reductive treatment. Making use of an established workflow called BAC-DROP (BAC-gel dissolution to digest PAGE-resolved objective proteins), crude proteome samples were fractionated based on molecular weight by BAC cross-linked PAGE. After fractionation, the gel fragments were reductively dissolved in under 5 minutes, and in-solution trypsin digestion of the protein released from the gel was completed in less than 1 hour at 70 °C, equivalent to a 90–95 % reduction in time compared to conventional in-gel trypsin digestion. The introduction of the BAC-DROP workflow to the MS assays for inflammatory biomarker CRP and viral marker HBsAg allowed for serum sample preparation to be completed in as little as 5 hours, demonstrating successful marker quantification from a 0.5 μL sample of human serum. Combining reduced proteome complexity via PAGE fractionation with rapid trypsin digestion provides a unique opportunity for conducting high-throughput analysis of in-depth proteome with a focus on specific molecular weight ranges.
Experiment Description
The LC-SRM assay was performed using an Eksigent nanoLC 400 system (SCIEX) connected to a QTRAP 5500 mass spectrometer (SCIEX). The LC mobile phases consisted of 0.1% formic acid (solvent A) and 0.1 % (v/v) formic acid/80 % (v/v) acetonitrile (solvent B). Each peptide sample was injected onto a 200 µm i.d. × 0.5 mm cHiPLC trap column (SCIEX) and desalted at 5 μl/min for 10 minutes using 0.1 % (v/v) TFA. Concentrated peptides were then separated on a fused-silica capillary column packed with C18 resin (12.5 cm × 75 µm i.d.; Nikkyo Technos) at a flow rate of 300 nL/min according to the following gradient schedule: 0–15 minutes, 2–50 % B; 15–18 minutes, 50–90 % B; hold at 90 % B for 6 minutes; and re-equilibrate at 2% B for 20 minutes. Eluate was ionized using a NanoSpray III Ion Source (SCIEX). The QTRAP 5500 was operated in positive ion mode with the following parameters: ion spray voltage = 2300 V; curtain gas = 20; ion source gas1 = 20; collision gas = 12; interface heater temperature = 150; entrance potential, 10; collision cell exit potential = 9; and Q1/Q3 = low resolution. The SRM transitions for each target peptide are shown in Supplementary Table S4. The obtained SRM data was analyzed by Skyline software (University of Washington, Seattle, WA, USA).
Sample Description
For CRP quantification, human serum samples were collected from three healthy donors, one patient with rheumatoid arthritis (RA), four patients with antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV), and two patients with bacterial pneumonia (BP), all treated at Ehime University Hospital, following the procedures approved by the human ethics committee of Ehime University. For HBsAg quantification, human serum samples were collected from five HBV infected patients and one uninfected healthy donor following the procedures approved by the human ethics committee of Hamamatsu University School of Medicine. For HBV inactivation, 180 μL of the collected serum sample were mixed with 20 μL of 10 % (w/v) SDS solution and subjected to UV irradiation. The samples were then mixed with 160 μL of 150 mM Tris-HCl (pH 8.8) and stored at -80 °C until use. BSA was purchased from Wako (Osaka, Japan).
Created on 9/15/20, 5:46 AM