Dataset Information

DIA-PASEF–based FLAG-IP proteomic analysis of nuclear or cytosolic p62 condensates in HeLa cells

ABSTRACT: Cells were cross-linked with 0.1% formaldehyde for 10 min at 25 °C and quenched with 100 mM glycine. After washing with HBS (20 mM HEPES-NaOH, pH 7.5, 150 mM NaCl), cells were lysed in HEPES-RIPA buffer (20 mM HEPES-NaOH, pH 7.5, 150 mM NaCl, 1 mM MgCl₂, 1 mM EGTA, 1% NP-40, 0.25% sodium deoxycholate, 0.05% SDS) supplemented with EDTA-free protease inhibitors, phosphatase inhibitors, and Benzonase. Lysates were clarified by centrifugation (4 ºC), and supernatants were incubated with anti-FLAG M2 magnetic beads (Sigma, M8823) for 2 h at 4 °C with rotation. Beads were washed three times with HEPES-RIPA buffer and twice with 50 mM ammonium bicarbonate. On-bead digestion was performed with 200 ng of trypsin/Lys-C mix overnight at 37 °C. Peptides were reduced, alkylated, acidified, desalted using GL-Tip SDB, dried, and reconstituted in 0.1% TFA and 3% ACN. LC-MS/MS analysis of the resultant peptides was performed on a nanoElute 2 coupled with a timsTOF HT mass spectrometer (Bruker). The peptides were separated on a 75-μm inner diameter × 150 mm C18 reversed-phase column (Nikkyo Technos) with a linear 5%–35% ACN gradient for 0–60 min followed by an increase to 95% ACN for 1 min and a final hold at 80% ACN for 4 min. Data acquisition was conducted in dia-PASEF mode with an MS1 m/z range of 100–1700 and an ion mobility (1/K0) range of 0.6–1.6. The ramp time was set to 100 ms with a 100% duty cycle. The MS2 polygon was manually defined based on the region where peptides were predominantly detected, specifically using the following four vertices: Point 1 (m/z = 300, 1/K0 = 0.64), Point 2 (m/z = 300, 1/K0 = 0.8), Point 3 (m/z = 770, 1/K0 = 0.97), and Point 4 (m/z = 770, 1/K0 = 1.15). Within the defined polygon, an 8 Da isolation window was employed with an overlap of 0.5 Da for each window, resulting in 62 windows per cycle and a cycle time of 3.36 s. Protein identification was carried out in DIA-NN (version 1.9) against a human in silico spectral library. Parameters for constructing the library included: trypsin as the digestion enzyme, one allowed missed cleavage, peptide lengths ranging from 7 to 45 amino acids, precursor charges of 2 to 4, and a fragment ion m/z range of 200–1800. Features such as library-free search, deep learning-based retention time and ion mobility predictions, N-terminal methionine cleavage, and cysteine carbamidomethylation were enabled. In the DIA-NN search settings, MS1 and MS2 mass accuracies were set to auto, neural network classifiers were configured for single-pass mode, and the quantification strategy was set to QuantUMS (high precision).

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Aaron Ciechanover

PROVIDER: PXD072519 | JPOST Repository | Tue Dec 30 00:00:00 GMT 2025

REPOSITORIES: jPOST

ACCESS DATA

Dataset's files

Source:

			Action	DRS
	PJ1647_Aaron_10_sh_p62_WT_FLAGIP_timsHT_DIA_Slot2-19_1_4826.d.zip	Other
	PJ1647_Aaron_11_sh_p62_dNES_FLAGIP_timsHT_DIA_Slot2-20_1_4828.d.zip	Other
	PJ1647_Aaron_12_sh_p62_dNLS_FLAGIP_timsHT_DIA_Slot2-21_1_4830.d.zip	Other
	PJ1647_Aaron_13_sh_p62_MG123_FLAGIP_timsHT_DIA_Slot2-22_1_4832.d.zip	Other
	PJ1647_Aaron_14_sh_p62_WT_MG123_FLAGIP_timsHT_DIA_Slot2-23_1_4834.d.zip	Other

Items per page:

1 - 5 of 20

Publications

Proteasomal proteolysis in p62 condensates directs tumor suppression or growth depending on their subcellular localization.

Lulu-Shimron Chen C Luo Zhiwen Z Brekhman Vera V Huang Lina L Livneh Ido I Kosako Hidetaka H Cohen-Kaplan Victoria V Ciechanover Aaron A

Proceedings of the National Academy of Sciences of the United States of America 20260116 3

p62/SQSTM1 generates liquid-liquid phase-separated condensates that participate in diverse processes, including protein quality control (PQC) and autophagy. Nuclear p62 condensates were shown to act as ubiquitin- and proteasome-mediated degradation hubs, whereas the involvement of cytoplasmic condensates in this pathway has remained unclear. Here, we show that cytoplasmic p62 condensates serve as a hub for proteasomal degradation that displays distinct substrate preferences compared with nuclear ...[more]

PMID: 41543905

Similar Datasets

Project description:Cells were lysed in 6 M guanidine-HCl, 100 mM HEPES-NaOH, pH7.5, 10 mM TCEP, and 40 mM CAA. The lysates were dissolved by heating and sonication, followed by centrifugation at 20,000 × g for 15 min at 4 °C. The supernatants were recovered, and proteins (20 µg each) were purified by the SP3 method and digested with 0.2 µg trypsin/Lys-C mix (Promega) at 37 °C overnight. The resulting peptide solutions were desalted using GL-Tip SDB (GL Sciences), evaporated in a SpeedVac concentrator, and re-dissolved in 0.1% TFA and 3% ACN. LC-MS/MS analysis of the resultant peptides was performed on a nanoElute 2 coupled with a timsTOF HT mass spectrometer (Bruker). The peptides were separated on a 75-μm inner diameter × 150 mm C18 reversed-phase column (Nikkyo Technos) with a linear 5%–35% ACN gradient for 0–60 min followed by an increase to 95% ACN for 1 min and a final hold at 80% ACN for 4 min. Data acquisition was conducted in dia-PASEF mode with an MS1 m/z range of 100–1700 and an ion mobility (1/K0) range of 0.6–1.6. The ramp time was set to 100 ms with a 100% duty cycle. The MS2 polygon was manually defined based on the region where peptides were predominantly detected, specifically using the following four vertices: Point 1 (m/z = 300, 1/K0 = 0.64), Point 2 (m/z = 300, 1/K0 = 0.8), Point 3 (m/z = 770, 1/K0 = 0.97), and Point 4 (m/z = 770, 1/K0 = 1.15). Within the defined polygon, a 8 Da isolation window was employed with an overlap of 0.5 Da for each window, resulting in 62 windows per cycle and a cycle time of 3.36 s. Protein identification was carried out in DIA-NN (version 1.9) against a human in silico spectral library. Parameters for constructing the library included: trypsin as the digestion enzyme, one allowed missed cleavage, peptide lengths ranging from 7 to 45 amino acids, precursor charges of 2 to 4, and a fragment ion m/z range of 200–1800. Features such as library-free search, deep learning-based retention time and ion mobility predictions, N-terminal methionine cleavage, and cysteine carbamidomethylation were enabled. In the DIA-NN search settings, MS1 and MS2 mass accuracies were set to auto, neural network classifiers were configured for single-pass mode, and the quantification strategy was set to QuantUMS (high precision).

Project description:Three independent biological replicates of HCT116 cells transiently transfected with FLAG-tagged RPS19 expression vectors or empty vectors were fixed with 0.1% formaldehyde methanol-free (Pierce) and then lysed on ice in 1 ml of HEPES-RIPA2 buffer (20 mM HEPES-NaOH pH7.5, 1 mM EGTA, 1 mM MgCl2, 150 mM NaCl, 0.25% Na-deoxycholate, 0.05% SDS, 1% NP-40) containing multiple protease inhibitors and Benzonase (70664, Novagen). The lysates were then immunoprecipitated with anti-FLAG M2 Magnetic beads (M8823, Sigma). The beads were washed three times with 1 ml of HEPES-RIPA2 buffer and twice with 50 mM ammonium bicarbonate. Proteins on beads were digested by addition of 200 ng trypsin/Lys-C mix (Promega) for 16 h at 37°C. The digests were reduced, alkylated, acidified, and desalted using GL-Tip SDB (GL Sciences). The eluates were evaporated in a SpeedVac concentrator and dissolved in 0.1% trifluoroacetic acid and 3% acetonitrile (ACN). LC-MS/MS analysis of the resultant peptides was performed on a timsTOF HT mass spectrometer coupled to a nanoElute2 UHPLC system (Bruker). Peptides were separated on a 75 μm X 150 mm C18 reversed-phase column (Nikkyo Technos) using a segmented linear gradient: 5%–20% ACN from 0 to 40 min, 20%–35% ACN from 40 to 60 min, followed by a rapid increase to 95% ACN from 60 to 61 min, and held at 95% ACN from 61 to 75 min. Data acquisition was conducted in DIA-PASEF mode with an MS1 m/z range of 100–1700 and an ion mobility (1/K0) range of 0.6–1.6. The ramp time was set to 100 ms with a 100% duty cycle. The MS2 polygon was manually defined based on the region where peptides were predominantly detected, specifically using the following four vertices: Point 1 (m/z = 300, 1/K0 = 0.64), Point 2 (m/z = 300, 1/K0 = 0.8), Point 3 (m/z = 770, 1/K0 = 0.97), and Point 4 (m/z = 770, 1/K0= 1.15). Within the defined polygon, an 8 Da isolation window was employed with an overlap of 0.5 Da for each window, resulting in 62 windows per cycle and a cycle time of 3.36 s. DIA-MS data were analyzed using DIA-NN (version 1.9) against a human in silico spectral library. Library generation parameters included trypsin digestion with one missed cleavage allowed, peptide lengths ranging from 7 to 45 amino acids, precursor charges of 2 to 4, and a fragment ion m/z range of 200–1800. Features such as library-free search, deep learning-based retention time and ion mobility predictions, N-terminal methionine cleavage, and cysteine carbamidomethylation were enabled. For DIA-NN search settings, MS1 and MS2 mass accuracies were set to auto, both neural network classifiers were configured for single-pass mode, and QuantUMS algorithm was used for quantification.

Project description:CD4 SP thymocytes were sorted and unstimulated or stimulated with α-CD3 mAb and α-CD28 mAb for 16 hours. Cells were washed with PBS and lysed in 150 μl of 6 M guanidine-HCl, 100 mM HEPES-NaOH, pH7.5, 10 mM TCEP, and 40 mM CAA. The lysates were dissolved by heating and sonication, followed by centrifugation at 20,000 × g for 15 min at 4 °C. The supernatants were recovered, and proteins were purified by methanol–chloroform precipitation and solubilized in 20 µl of 8 M urea, 50 mM Tris-HCl, pH8.0. After sonication, the protein solutions were diluted 8-fold with 50 mM Tris-HCl, pH8.0 and digested with 1 µg trypsin/Lys-C mix (Promega) at 37 °C overnight. The resulting peptide solutions were desalted using GL-Tip SDB (GL Sciences), evaporated in a SpeedVac concentrator, and re-dissolved in 0.1% TFA and 3% ACN. LC-MS/MS analysis of the resultant peptides was performed on a nanoElute 2 coupled with a timsTOF HT mass spectrometer (Bruker). The peptides were separated on a 75-μm inner diameter × 150 mm C18 reversed-phase column (Nikkyo Technos) with a linear 5%–35% ACN gradient for 0–60 min followed by an increase to 95% ACN for 1 min and a final hold at 80% ACN for 4 min. Data acquisition was conducted in dia-PASEF mode with an MS1 m/z range of 100–1700 and an ion mobility (1/K0) range of 0.6–1.6. The ramp time was set to 100 ms with a 100% duty cycle. The MS2 polygon was manually defined based on the region where peptides were predominantly detected, specifically using the following four vertices: Point 1 (m/z = 300, 1/K0 = 0.64), Point 2 (m/z = 300, 1/K0 = 0.8), Point 3 (m/z = 770, 1/K0 = 0.97), and Point 4 (m/z = 770, 1/K0 = 1.15). Within the defined polygon, a 8 Da isolation window was employed with an overlap of 0.5 Da for each window, resulting in 62 windows per cycle and a cycle time of 3.36 s. Protein identification was carried out in DIA-NN (version 1.9) against a mouse in silico spectral library. Parameters for constructing the library included: trypsin as the digestion enzyme, one allowed missed cleavage, peptide lengths ranging from 7 to 45 amino acids, precursor charges of 2 to 4, and a fragment ion m/z range of 200–1800. Features such as library-free search, deep learning-based retention time and ion mobility predictions, N-terminal methionine cleavage, and cysteine carbamidomethylation were enabled. In the DIA-NN search settings, MS1 and MS2 mass accuracies were set to auto, neural network classifiers were configured for single-pass mode, and the quantification strategy was set to QuantUMS (high precision).

Project description:After control or Shu knockdown (n = 3 each), OSCs were lysed in binding buffer [50 mM Tris-HCl (pH 8.0), 150 mM KOAc, 5 mM Mg(OAc)2, 5 mM DTT, 0.1% NP-40, 2 µg/mL leupeptin, 2 µg/mL pepstatin A, 0.5% aprotinin, 10% glycerol] and centrifuged to remove insoluble debris. The lysates were then incubated with anti-Shu antibody bound to Dynabeads Protein G (Thermo Fisher Scientific) at 4°C for 2 h. The beads were washed five times with binding buffer and twice with 50 mM ammonium bicarbonate. Proteins on the beads were digested by adding 400 ng of trypsin/Lys-C mix (Promega) at 37 °C overnight. The digests were reduced, alkylated, acidified, and desalted using GL-Tip SDB (GL Sciences). The eluates were evaporated in a SpeedVac concentrator and dissolved in 0.1% trifluoroacetic acid and 3% acetonitrile (ACN). LC-MS/MS analysis of the resultant peptides was performed on a nanoElute 2 coupled with a timsTOF HT mass spectrometer (Bruker). The peptides were separated on a 75-μm inner diameter × 150 mm C18 reversed-phase column (Nikkyo Technos) using a segmented linear gradient: 5%–20% ACN from 0 to 40 min, 20%–35% ACN from 40 to 60 min, followed by a rapid increase to 95% ACN from 60 to 61 min, and held at 95% ACN from 61 to 75 min. Data acquisition was conducted in dia-PASEF mode with an MS1 m/z range of 100–1700 and an ion mobility (1/K0) range of 0.6–1.6. The ramp time was set to 100 ms with a 100% duty cycle. The MS2 polygon was manually defined based on the region where peptides were predominantly detected, specifically using the following four vertices: Point 1 (m/z = 300, 1/K0 = 0.64), Point 2 (m/z = 300, 1/K0 = 0.8), Point 3 (m/z = 770, 1/K0 = 0.97), and Point 4 (m/z = 770, 1/K0 = 1.15). Within the defined polygon, an 8 Da isolation window was employed with an overlap of 0.5 Da for each window, resulting in 62 windows per cycle and a cycle time of 3.36 s. Protein identification was carried out in DIA-NN (version 1.9)81 against a Drosophila melanogaster (UP000000803) in silico spectral library. Parameters for constructing the library included: trypsin as the digestion enzyme, one allowed missed cleavage, peptide lengths ranging from 7 to 45 amino acids, precursor charges of 2 to 4, and a fragment ion m/z range of 200–1800. Features such as library-free search, deep learning-based retention time and ion mobility predictions, N-terminal methionine cleavage, and cysteine carbamidomethylation were enabled. In the DIA-NN search settings, MS1 and MS2 mass accuracies were set to auto, neural network classifiers were configured for single-pass mode, and the quantification strategy was set to QuantUMS (high precision).

Project description:After expression of Shu-FLAG or luciferase-FLAG (n = 3 each), OSCs were lysed in binding buffer [50 mM Tris-HCl (pH 8.0), 150 mM KOAc, 5 mM Mg(OAc)2, 5 mM DTT, 0.1% NP-40, 2 µg/mL leupeptin, 2 µg/mL pepstatin A, 0.5% aprotinin, 10% glycerol] and centrifuged to remove insoluble debris. The lysates were then incubated with anti-FLAG antibody (Merck) bound to Dynabeads Protein G (Thermo Fisher Scientific) at 4°C for 2 h. The beads were washed five times with binding buffer and twice with 50 mM ammonium bicarbonate. Proteins on the beads were digested by adding 400 ng of trypsin/Lys-C mix (Promega) at 37 °C overnight. The digests were reduced, alkylated, acidified, and desalted using GL-Tip SDB (GL Sciences). The eluates were evaporated in a SpeedVac concentrator and dissolved in 0.1% trifluoroacetic acid and 3% acetonitrile (ACN). LC-MS/MS analysis of the resultant peptides was performed on a nanoElute 2 coupled with a timsTOF HT mass spectrometer (Bruker). The peptides were separated on a 75-μm inner diameter × 150 mm C18 reversed-phase column (Nikkyo Technos) using a segmented linear gradient: 5%–20% ACN from 0 to 40 min, 20%–35% ACN from 40 to 60 min, followed by a rapid increase to 95% ACN from 60 to 61 min, and held at 95% ACN from 61 to 75 min. Data acquisition was conducted in dia-PASEF mode with an MS1 m/z range of 100–1700 and an ion mobility (1/K0) range of 0.6–1.6. The ramp time was set to 100 ms with a 100% duty cycle. The MS2 polygon was manually defined based on the region where peptides were predominantly detected, specifically using the following four vertices: Point 1 (m/z = 300, 1/K0 = 0.64), Point 2 (m/z = 300, 1/K0 = 0.8), Point 3 (m/z = 770, 1/K0 = 0.97), and Point 4 (m/z = 770, 1/K0 = 1.15). Within the defined polygon, an 8 Da isolation window was employed with an overlap of 0.5 Da for each window, resulting in 62 windows per cycle and a cycle time of 3.36 s. Protein identification was carried out in DIA-NN (version 1.9)81 against a Drosophila melanogaster (UP000000803) in silico spectral library. Parameters for constructing the library included: trypsin as the digestion enzyme, one allowed missed cleavage, peptide lengths ranging from 7 to 45 amino acids, precursor charges of 2 to 4, and a fragment ion m/z range of 200–1800. Features such as library-free search, deep learning-based retention time and ion mobility predictions, N-terminal methionine cleavage, and cysteine carbamidomethylation were enabled. In the DIA-NN search settings, MS1 and MS2 mass accuracies were set to auto, neural network classifiers were configured for single-pass mode, and the quantification strategy was set to QuantUMS (high precision).

Project description:HEK293T cells were cultured in 6-well plates and treated with DMSO or 2 µM MLN4924. After 6 h (MLN4924) or 24 h (DMSO or MLN4924), the cells (n=3) were collected using 1 mL of PBS by pipetting. All samples were centrifuged at 500 × g for 3 min at 4°C, and cell pellets were lysed in 160 µL of guanidine buffer. After heating and sonication, the lysates were centrifuged at 20,000 × g for 15 min at 4°C. The supernatants were recovered, and proteins (50 µg each) were purified by methanol–chloroform precipitation and resuspended in 20 µl of 8 M urea, 50 mM Tris-HCl, pH 8.0. After sonication, the protein solutions were diluted 8-fold with 50 mM Tris-HCl, pH 8.0, and digested with 1 µg of trypsin/Lys-C mix at 37 °C overnight. The digests were acidified, centrifuged, and desalted using GL-Tip SDB. The eluates were evaporated and dissolved in 0.1% TFA and 3% ACN. LC-MS/MS analysis of the resultant peptides was performed using a timsTOF HT system (equipped with a CaptiveSpray2 ion source) coupled to a nanoElute 2 (Bruker). A 150-mm C18 reversed-phase column with an inner diameter of 75 µm was employed. Mobile phase A consisted of ultrapure water containing 0.1% formic acid, and mobile phase B consisted of ACN containing 0.1% formic acid. The gradient was initiated at 5% solvent B, increased to 20% at 40 min, then to 35% at 60 min, followed by a rapid increase to 95% at 61 min, and finally hold at 95% until 75 min. Data acquisition was conducted in DIA-PASEF mode with an MS1 m/z range of 100–1700 and an ion mobility (1/K0) range of 0.6–1.6. The MS2 polygon was manually defined based on the region where peptides were predominantly detected, specifically using the following four vertices: Point 1 (m/z = 300, 1/K0 = 0.64), Point 2 (m/z = 300, 1/K0 = 0.8), Point 3 (m/z = 770, 1/K0 = 0.97), and Point 4 (m/z = 770, 1/K0 = 1.15). Within the defined polygon, a 8 Da isolation window was employed with an overlap of 0.5 Da for each window, resulting in 62 windows per cycle and a cycle time of 3.36 s. DIA-MS data were searched using DIA-NN (version 1.9) against a human in silico spectral library. To construct the library from the UniProt human protein sequence database, the following parameters were applied: trypsin as the digestion enzyme, one allowed missed cleavage, peptide lengths ranging from 7 to 45 amino acids, precursor charges of 2 to 4, and a fragment ion m/z range of 200–1800. Features such as library-free search, deep learning-based retention time and ion mobility predictions, N-terminal methionine cleavage, and cysteine carbamidomethylation were enabled. In the DIA-NN search settings, MS1 and MS2 mass accuracies were set to auto, both neural network classifiers were configured for single-pass mode, and the quantification strategy was set to QuantUMS (high precision).

Project description:Single-cell RNA-seq data from the mouse Dorsomedial Hypothalamic Nucleus (DMH) was generated following a modified protocol from https://www.nature.com/articles/s41586-020-2821-8. Briefly, six 7.5 week old C57B6 mice (3 male, 3 female) were anaesthetized with isoflurane. Brains were rapidly extracted and dropped into ice-cold carbogenated NMDG-HEPES-ACSF. Brain sections (2 mm) containing DMH were cut with a razor blade on a stainless steel brain matrix (51392, Stoelting) and transferred to a dissection dish on ice containing NMDG-HEPES-ACSF. DMH was bilaterally microdissected in ~700·700·350 micron tissue block. Microdissected tissue was aggregated in a collection tube on ice containing NMDG-HEPES-ACSF and 30 μM actinomycin D. For enzymatic tissue digestion, NMDG-HEPES-ACSF was replaced by trehalose-HEPES-ACSF containing papain (50 U/ml; P3125, Sigma Aldrich, pre-activated with 2.5 mM cysteine and a 30-min incubation at 34 °C and supplemented with 0.5 mM EDTA) and 15 μM actinomycin D. Extracted DMH tissue was incubated at room temperature with gentle carbogenation for 55 min. During enzymatic digestion the tissue was pipetted periodically every 10 min. At the end of enzymatic digestion, the medium was replaced with 200μl of room temperature trehalose-HEPES-ACSF containing 3 mg/ml ovomucoid inhibitor (OI-BSA,Worthington)25 U/ml DNase I (90083, Thermo Scientific) and 15 μM actinomycin D and tissue was gently triturated into a uniform single-cell suspension with fire-polished glass Pasteur pipettes. The resulting cell suspension volume was brought up to 1 ml with trehalose-HEPES-ACSF with 3 mg/ml ovomucoid inhibitor and pipetted through a 40μm cell strainer. Single-cell suspension was centrifuged down at 300g for 5 min at 4 °C and the supernatant was replaced with 1 ml fresh ice-cold trehalose-HEPES-ACSF and the cell pellet was resuspended. Cells were pelleted again and resuspended in 100 μl of ice-cold resuspension-ACSF. Cell suspensions were kept on ice while cell densities were quantified with a haemocytometer and the final cell densities were verified to be in the range of 300–1,000 cells/μl. Cell suspension volumes estimated to retrieve ~10,000 single-cell transcriptomes were added to the 10x Genomics RT reaction mix and loaded to the 10X Next GEM Chip G (2000177, 10x Genomics) per the manufacturer’s protocol. We used the Chromium Next GEM Single Cell 3’ Reagents Kit v3.1 (1000128, 10x Genomics) and the Single Index Kit Set A (100213) to prepare Illumina sequencing libraries downstream of reverse transcription following the manufacturer’s protocol. Resulting scRNA-seq sequencing library was sequenced on an Illumina NovaSeq 6000 sequencers (paired-end 150). Sequencing data were aligned to optimized pre-mRNA reference transcriptome52 and digital gene-cell matrices were generated with the 10x Genomics Cell Ranger v.6.0.0 count pipeline. The resulting scRNA-seq data were analyzed in R (4.1.2) using Seurat (v.4.1.0.9007) as previously described51. Briefly, expression data were filtered to exclude cells with fewer than 1,000 unique transcripts as well as cells exhibiting more than 15% of mitochondrial transcripts. We followed the standard Seurat workflow to identify transcriptomic cell-types and assigned cel class identities based on canonical cell-type markers: Ndrg4 for neurons, Ntsr2 for astrocytes, Slco1c1 for endothelial cells, Fcer1g for microglia and Mag for oligodendrocytes. We digitally extracted neuronal clusters from our dataset resulting in expression data for 4587 DMH neurons that we used to establish an unbiased neuronal nomenclature for DMH.

Project description:Female Sprague-Dawley rats were randomly separated into two groups: i) trained (Ex group) and ii) sedentary (Cont group). Animals from the Ex group were adapted to treadmill exercise for two consecutive weeks, involving a gradual increase in the running time till 60 min/day at 20 m/min, 0% grade, 5 days/week. This exercise program remained constant until the end of the 54 weeks. Mitochondria isolation from 10 animals (5 Ex group and 5 Cont group) was performed using the conventional methods of differential centrifugation. Mitochondrial protein extracts were reduced with dithiothreitol, alkylated with iodoacetamide and digested with trypsin using the FASP procedure (18). Briefly, each sample was solubilized in 4 % SDS, 0.1 M DTT, 0.1 M HEPES and incubated for 30 min at 60 C. Then, samples were mixed with 0.2 mL of a solution of 8 M Urea, 0.1 M HEPES, pH 8.5 (UH solution), loaded into the filtration devices (3k Microcon, Millipore), centrifuged at 14,000 g for 20 min, and washed twice with UH solution. After centrifugation, the concentrates were alkylated with 50 mM iodoacetamide in UH solution (dark, 25 C, 20 min), and washed twice with UH solution. The concentrate was diluted with 0.1 mL of 50 mM ammonium bicarbonate (ABC) and digested overnight (37 C) with the addition of 2 ug of trypsin diluted in 30 uL of 50 mM ABC. The resulting tryptic peptides were collected by centrifugation and the filter were washed twice with 50 uL of 50 mM ABC. Eluted peptides were acidified with 10 uL of formic acid and cleaned up on a homemade Empore C18 column (3M, St. Paul, MN, USA) (ref) for subsequent phospho-enrichment and/or analysis by LC-MS/MS. The dried protein digest was dissolved with 10 uL (3 % ACN, 0.1 % TFA), diluted 1:5 with loading buffer (1 M glycolic acid, 5 % TFA, 80 % ACN) and phosphopeptides were enriched on TiO2-beads as previously described (19). Briefly 5 mg of "Titansphere TiO2 5 um" were suspended in 100 uL of 100 % ACN, immobilized in a pipette-column and washed using 5 uL of loading buffer. Each sample was loaded in each pipette-column and then washed with 30 uL of washing buffer (1 % TFA, 80 % ACN). Phosphopeptides were eluted from the beads with 25 uL of 25 % ACN (v/v) containing 25 % NH4OH (m/v), acidified with 10 uL of 10 % TFA and vacuum- concentrated to approximately 4 uL. Samples were finally diluted to 10 uL with H2O + 0.1% formic acid prior to injection. The peptides mixtures were analyzed by online nanoflow liquid chromatography tandem mass spectrometry (nanoLC-MS/MS) on an EASY-nLC system (Proxeon Biosystems, Odense, Denmark) connected to the LTQ Orbitrap Velos instrument (Thermo Fisher Scientific, Bremen, Germany) through a nanoelectrospray ion source. Six microliters of the peptide mixture were auto-sampled directly onto the analytical HPLC column (120 mm x75 um I.D., 3 um particle size (Nikkyo Technos Co., Ltd.) with a 120-min gradient from 5 % to 50 % ACN in 0.1 % FA. The effluent from the HPLC column was directly electrosprayed into the mass spectrometer. The LTQ Orbitrap Velos instrument was operated in data-dependent acquisition mode to automatically switch between full scan MS and MS/MS acquisition. The MS survey scan was performed in the FT cell recording a window between 350 and 2000 m/z. The resolution was set to 60 000, and the automatic gain control was set to 1,000,000 ions with a maximal acquisition time of 400 ms. The 20 most intense peptide ions with charge states great than or equal to 2 were sequentially isolated to a target value of 5,000 and fragmented in the high-pressure linear ion trap by low-energy CID with normalized collision energy of 35% Q value of 0.25, and an activation time of 10 ms. Raw files were processed using Proteome Discoverer version 1.3 (Thermo Fisher Scientific, Bremen). Peak lists were searched using Mascot software version 2.3 (Matrix Science, UK) against a SwissProt database containing entries corresponding to Rattus norvegicus (version of July 2012), a list of common contaminants, and all the corresponding decoy entries. Trypsin was chosen as enzyme and a maximum of two miscleavages were allowed. Carbamidomethylation (C) was set as a fixed modification, whereas oxidation (M) and phosphorylation (STY) were used as variable modifications. Searches were performed using a peptide tolerance of 7 ppm, a product ion tolerance of 0.5 Da. Resulting data files were filtered for FDR less than 1 % at peptide level.