Dataset Information

DIA-PASEF–based total proteome profiling of HeLa cells expressing nuclear or cytosolic p62 condensates

ABSTRACT: 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).

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Aaron Ciechanover

PROVIDER: PXD072518 | JPOST Repository | Tue Dec 30 00:00:00 GMT 2025

REPOSITORIES: jPOST

ACCESS DATA

Dataset's files

Source:

			Action	DRS
	PJ1646_Aaron_10_sh_p62_WT_timsHT_DIA_Slot2-43_1_4697.d.zip	Other
	PJ1646_Aaron_11_sh_control_timsHT_DIA_Slot2-44_1_4699.d.zip	Other
	PJ1646_Aaron_12_sh_p62_timsHT_DIA_Slot2-45_1_4701.d.zip	Other
	PJ1646_Aaron_13_sh_p62_dNES_timsHT_DIA_Slot2-46_1_4703.d.zip	Other
	PJ1646_Aaron_14_sh_p62_dNLS_timsHT_DIA_Slot2-47_1_4705.d.zip	Other

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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

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Project description: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).

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: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: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: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:Cyanophora paradoxa muroplasts were isolated on a Percoll gradient. Muroplasts were fractionated into soluble (stroma), envelope, and thylakoid proteins. Protein samples from envelope membranes, thylakoids, stroma and total muroplasts were separated by 12% SDS-PAGE. Each Protein lanewas cut into 10 slices of variable size to match similar protein amounts. The gel slices were washed and diced to 1mm3 cubes. Gel pieces were destained by two consecutive washes with a 50:50 mixture of 200 mM ammonium bicarbonate and acetonitrile (ACN) for 20 min at 37°C. Proteins were reduced with 10mM DTT for 45 min at 56°C, followed by alkylation with 55mM iodoacetamide for 45 min at room temperature in the dark. After reduction and alkylation, gel pieces were washed twice with 100 mM ammonium bicarbonate, dehydrated with 100% ACN for 10 min, and dried in a SpeedVac. Gel pieces were fully covered in 50 mM ammonium bicarbonate containing 10ng/ml trypsin and rehydrated at 4°C. The samples were incubated overnight at 37°C and peptides in the supernatant were pooled after successive extraction steps using 50 mM ammonium bicarbonate, 100% ACN and 2.5% formic acid (FA), 50% ACN. The samples were dried in a SpeedVac and reconstituted in 5% ACN, 0.1% FA prior to MS analysis. Each of the samples were analysed in duplicates. Tryptic peptides of the thylakoid, envelope and stroma fractions were loaded onto a fritless 100 μm capillary packed in-house with 10cm of ProntoSIL C18 ace-EPS, 3µm. A gradient of 5-80% (v/v) ACN in 0.1% (v/v) FA was passed over the column with a duration of 115 min. The eluted peptides were directly electrosprayed into the LTQ orbitrap XL MS at a flow rate of 250 nl min-1 and a spray voltage of 1.3 kV. The instrument was operated in a 4-step data-dependent positive mode to identify the top three most abundant ions in a high-resolution MS scan (400 to 2000 m/z), which were then selected for fragmentation. MudPIT analyses were performed on the muroplast samples using an in-house packed analytical column consisting of 10 cm ProntoSIL C18 ace-EPS, 3µm (Bischoff) and 2 cm of a strong cationic exchange (SCX) material, 3µm in combination with an 6-step salt pulse gradient (0, 50, 100, 150, 200 and 500 mM ammonium acetate). Each salt-step peptides were eluted from the SCX to the C18-phase of the analytical column and eluated with a 130- min reverse-phase solvent gradient (5–80% ACN containing 0.1% FA). The eluate was directly electrosprayed into the LTQ orbitrap XL MS which was operated as described before. All MS/MS samples were analyzed using Sequest and X! Tandem. Sequest was set up to search the C. paradoxa nuclear and muroplast protein database with a total of 32,286 entries, assuming the digestion enzyme trypsin. X! Tandem was set up to search a subset of the C. paradoxa database also assuming trypsin. Sequest and X! Tandem were searched with a fragment ion mass tolerance of 0,80 Da and a parent ion tolerance of 10,0 ppm. Oxidation of methionine and iodoacetamide derivatives of cysteine were specified in Sequest and X! Tandem as variable modifications. Scaffold (version 3.5.1, Proteome Software Inc., Portland, OR) was used to validate MS/MS based peptide and protein identifications. Peptide identifications were accepted if they could be established at greater than 95,0% probability as specified by the Peptide Prophet algorithm. Protein identifications were accepted if they could be established at greater than 99,0% probability and contained at least 2 identified peptides.

Project description:Prefrontal cortex tissue slices from a cognitively and neurpathologically normal human subject were obtained from the University of Pennsylvania (UPenn) Brain Bank. 300-350 mg grey matter was homogenized in 1.5 ml solution A (0.32 M sucrose, 1mM MgCl2 and 0.1mM CaCl2) with a Teflon pestle. ~100 ?l of the homogenate was saved, solubilized with 1% SDS and clarified by centrifugation. To prepare the [13C6]brain ISTD, 400 mg labeled MouseExpress brain tissue (Cambridge Isotopes, MA) was homogenized in 4 ml buffer A as described above, centrifuged at 1,000 x g for 10 min to clarify, agitated on a rocker at 4�C for 30 min, and centrifuged at 10,000 x g for 20 min. The pellet, a crude membrane fraction, was dissolved in 2 ml 20 mM Tris pH 7.4 with 1% SDS. Total protein in all preparations was quantified with the micro BCA assay (Pierce) and all solutions were supplemented with protease and phosphate inhibitor cocktails (Sigma) as well as 1 mM sodium fluoride and sodium orthovanadate (Sigma). Human cortex homogenate was mixed with the [13C6]brain ISTD (1?g/?l) at a ratio of 2:1(?g/?g) and processed for LC-MS/MS: 40 ?l preparations were heated with lithium dodecyl sulfate (LDS) (Invitrogen) buffer at 95�C for 20 min, separated on a 1.5 mm 4-12% Bis-Tris Gel (Invitrogen), cut into five fractions, chopped into ?2 mm cubes, washed in 200 ?l 50% acetonitrile (ACN) containing 25 mM NH4HCO3, reduced in 300 ?l 10 mM DTT, alkylated in 300 ?l 55 mM iodoacetamide (Sigma), and digested with 80-120 ?l trypsin (.025 ?g/?l) (Promega) overnight at 37�C, so that the amount of trypsin was 1:5 of the total protein to be digested by mass. Peptides were recovered from gel cubes into 200 ?l 50/50 H2O/ACN with 3% formic acid by vortex-mixing and sonication for 20 min each, twice. Samples were then evaporated to a 100 ?l volume, brought to 1ml in H20 with 0.1% formic acid, desalted with Oasis� HLB cartridges (Waters), evaporated almost to completion and suspended in ~ 45?l H2O with 0.1% formic acid, and filtered with 0.22 �m Ultra free-MC filter cartridges (Millipore). LC-data dependant -MS/MS analyses were conducted on a Q Exactive (ThermoFisher Scientific) quadrupole orbi-trap hybrid instrument with an Easy nLC-II (ThermoFisher Scientific) nano-pump/autosampler. 3?l peptide sample (~ 1 ug) was loaded and resolved on a 20 cm x 75 �m proteopepII C18 packed tip column over a 90 min gradient at a flow rate of 350 nL/min, 0-35% mobile phase B (ACN, 0.1 formic acid). A data-dependent top 10 method was used to acquire a 70,000 resolution full scan to trigger ten 17,500 resolution HCD scans. Ions were isolated for MS/MS analysis with a 2.0 Da window on the quadrupole. Average cycle times were 1.2 sec. Each sample was analyzed in triplicate. Raw files from triplicate injections were searched together within Proteome Discoverer 1.3 (ThermoFisher Scientific) using SEQUEST and the human refseq. database (release 47, ftp://ftp.ncbi.nih.gov/refseq/H_sapiens/mRNA_Prot/). Search parameters allowed trypsin to cleave after Lysine and Arginine and have 2 missed cleavages. Precursor ion mass tolerance was set to 15 ppm and fragment ion mass tolerance was 20 mmu. The dynamic modification of methionine (oxidation = 15.995 Da) and lysine (13C6 = 6.020 Da), in addition to the static modification of cysteine (carbamidomethylation = 57.021 Da) was accepted on up to 4 residues per peptide. Within the Proteome Discoverer Software, SILAC pairs were identified using the �2 plex� workflow node, and all peptides were rescored using the Percolator algorithm node. Finally, peptides were filtered at 1% false discovery rate (FDR).

Project description:Lung tissues from uninfected and infected mice (n = 5 per group) were homogenized in 0.5 mL of guanidine buffer. After heating and sonication, the lysates were centrifuged at 20,000 g for 15 min at 4° C. The resulting supernatants were collected, and the proteins (20 µg each) were purified using the SP3 method and digested with 200 ng trypsin/Lys-C mixture at 37° C overnight. The digests were acidified, centrifuged, and desalted using a GL-Tip SDB. The eluates were evaporated and dissolved in 0.1% TFA and 3% ACN. LC-MS/MS analysis of the resulting peptides was performed using a timsTOF HT system (equipped with a CaptiveSpray 2 ion source) coupled to a nanoElute 2 (Bruker, Billerica, MA). A 150-mm C18 reversed-phase column with an inner diameter of 75 µm was used. 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 and increased to 20% at 40 min. Then, it increased to 35% at 60 min, followed by a rapid increase to 95% at 61 min. Finally, it was held at 95% until 75 min. Data were acquired 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, it was defined 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). An 8 Da isolation window with an overlap of 0.5 Da was employed within the defined polygon, 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 mouse in silico spectral library. The following parameters were applied to construct the library from the UniProt mouse protein sequence database: trypsin as the digestion enzyme; one missed cleavage; peptide lengths ranging from seven to 45 amino acids; precursor charges of two to four; and a fragment ion m/z range of 200–1,800. The following features were enabled: library-free search, deep learning–based retention time and ion mobility predictions, N-terminal methionine cleavage, and cysteine carbamidomethylation. MS1 and MS2 mass accuracies were automatically optimized; both neural network classifiers were run in single-pass mode; and quantification was performed using the high-precision QuantUMS strategy.