Barr-Gillespie - Pou4f3-Gfp FACs DIA

Barr-Gillespie - Pou4f3-Gfp FACs DIA
Mass spectrometry identification and quantitation of proteins from small pools of developing auditory and vestibular cells
  • Organism: Mus musculus
  • Instrument: Q Exactive HF
  • SpikeIn: No
Abstract
Hair cells undergo postnatal development that leads to formation of their sensory organelles, synaptic machinery, and in the case of cochlear outer hair cells, their electromotile mechanism. To examine the proteome changes over development, we isolated pools of 5000 Pou4f3-Gfp positive or negative cells from the cochlea or utricles at three different developmental time points; these cell pools were analysed by data-dependent and data-independent acquisition (DDA and DIA) mass spectrometry. DDA data were used to generate spectral libraries, which enabled identification and accurate quantitation of specific proteins using the DIA datasets. We also isolated and pooled individual inner and outer hair cells from adult cochlea and compared their proteomes to those of developing hair cells.
Experiment Description
Protein digests were separated using liquid chromatography with a NanoAcquity UPLC system (Waters); analytes were ionized using electrospray with a Nano Flex Ion Spray Source (Thermo Fisher) fitted with a 20 µm stainless steel nano-bore emitter spray tip and 1.0 kV source voltage, and were delivered to a Q Exactive HF (Thermo Fisher). Xcalibur version 4.0 was used to control the system. Samples were first bound to a trap cartridge (Symmetry C18 trap cartridge; Waters) at 15 µl/min to a for 10 min; the system then switched to a 75 µm x 250 mm NanoAcquity BEH 130 C18 column with 1.7 µm particles (Waters) using mobile phases water and acetonitrile containing 0.1% formic acid. A 7.5-30% acetonitrile gradient was delivered over 60 min at a flow rate of 300 nl/min. The DIA mass spectrometry method was constructed using 20 m/z quadrupole isolation windows. First, a full scan at 30,000 FWHM resolving power (at 200 m/z) was performed, followed by sequential HCD-MS/MS scans at normalized collision energy of 26 and 15,000 FWHM resolution. The range between 395 and 1005 m/z was surveyed by these experiments with maximum injection times of 55 ms and auto for MS and MS/MS, respectively. AGC values were set to 3 x 106 for MS and 1 x 106 for MS/MS. The MS/MS scan range was set to 200–2000 m/z. DIA data were analysed with Skyline23,24 version 3.7.
Sample Description
To selectively isolate hair cells, we used animals of either sex from the Tg(Pou4f3-Isl1-eGFP) transgenic mouse line, which expresses enhanced GFP under control of the Pou4f3 promoter. The high specificity of the Pou4f3 promoter ensures that the only labelled cells are hair cells. Utricles and cochleae were dissected in less than 1 hr using ice-cold PBS, then were transferred to ice-cold DMEM (Life Technologies) with 5% FBS. Cells were collected at three time points (postnatal day 0, 4 and 7) in triplicate. Cell aliquots were pooled and diluted if necessary to create samples of ~5000 GFP-positive or negative cells per condition. Cochlear hair cells are 0.5-1.0 pl in volume; if their total protein concentration was ~200 g/l, each cell would contain 0.1-0.2 ng of protein. Prior to sample preparation, the 5000 hair cells analysed in each sample thus together have 0.5-1.0 µg of total protein. The GFP-negative cells are likely somewhat larger and thus have larger amounts of total protein. In-solution tryptic digests of the samples were prepared using an enhanced filter-aided sample preparation (eFASP) method. Proteins were digested in the filter unit in 100 µl digestion buffer with 200 ng sequencing-grade modified trypsin (Promega) at 37°C for 12-16 hours. Peptides were isolated by centrifugation and were extracted with ethyl acetate to remove remaining deoxycholic acid.
Created on 1/26/18, 8:34 AM
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