Dataset Information

TMTpro-18plex analysis of MM1.S cells treated with PROTACs

ABSTRACT: MM1.S cells were treated with DMSO, 0.3 µM Po-P, 0.3 µM Le-P, 0.3 µM F-P, 0.3 µM Cl-P, or 0.3 µM F3C-P for 16 h in three biological replicates. The cells were lysed in 100 µL guanidine buffer (6 M guanidine-HCl, 100 mM HEPES-NaOH, pH 7.5, 10 mM TCEP, 40 mM chloroacetamide). After heating and sonication, proteins (100 µg each) were purified by methanol–chloroform precipitation and resuspended in 20 µL 0.1% RapiGest SF (Waters) in 50 mM triethylammonium bicarbonate. After sonication and heating at 95 °C for 10 min, the proteins were digested with 2 µg trypsin/Lys-C mix (Promega) at 37 °C overnight. The digested peptides (40 µg each) were labeled with 0.5 mg TMTpro-18plex reagents (Thermo Fisher Scientific) for 1 h at 25 °C. After the reaction was quenched with hydroxylamine, all the TMT-labeled samples were pooled, acidified with TFA, and fractionated by offline high-pH reversed-phase chromatography on a Vanquish DUO UHPLC (Thermo Fisher Scientific) as described previously. Briefly, the peptides were loaded onto a 4.6 × 250 mm Xbridge BEH130 C18 column with 3.5 mm particles (Waters) and separated using a 30 min multistep gradient of solvents A (10 mM ammonium formate at pH 9.0 in 2% ACN) and B (10 mM ammonium formate pH 9.0 in 80% ACN), at a flow rate of 1 mL/min. Peptides were separated into 48 fractions, which were consolidated into 16 fractions. Each fraction was evaporated in a SpeedVac concentrator and dissolved in 0.1% TFA and 3% ACN. LC-MS/MS analysis of the resultant peptides (500 ng each) was performed on an EASY-nLC 1200 UHPLC connected to a Q Exactive Plus mass spectrometer through a nanoelectrospray ion source (Thermo Fisher Scientific). The peptides were separated on the analytical column (75 μm × 15 cm, 3 μm; Nikkyo Technos) with a linear gradient of 4–20% for 0–115 min and 20–32% for 115–160 min, followed by an increase to 80% ACN for 10 min and finally held at 80% ACN for 10 min. The mass spectrometer was operated in data-dependent acquisition mode with a top 10 MS/MS method. MS1 spectra were measured with a resolution of 70,000, an AGC target of 3e6, and a mass range from 375 to 1,400 m/z. MS/MS spectra were triggered at a resolution of 35,000, an AGC target of 1e5, an isolation window of 0.4 m/z, a maximum injection time of 100 ms, and a normalized collision energy of 31. Dynamic exclusion was set to 20 s. Raw data were directly analyzed against the SwissProt database restricted to Homo sapiens using Proteome Discoverer version 2.4 with the Sequest HT search engine for identification and TMT quantification. The search parameters were as follows: (a) trypsin as an enzyme with up to two missed cleavages; (b) precursor mass tolerance of 10 ppm; (c) fragment mass tolerance of 0.02 Da; (d) TMT of lysine and peptide N-terminus and carbamidomethylation of cysteine as fixed modifications; (e) oxidation of methionine as a variable modification. Peptides were filtered at an FDR of 1% using the Percolator node. TMT quantification was performed using the Reporter Ions Quantifier node. Normalization was performed such that the total sum of the abundance values for each TMT channel over all peptides was the same.

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Hidetaka Kosako

PROVIDER: PXD037178 | JPOST Repository | Sat Jul 22 00:00:00 BST 2023

REPOSITORIES: jPOST

ACCESS DATA

Dataset's files

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			Action	DRS
	20_Yamanaka_MM1S_TMTpro18_Vanquish16_1_500ng_QEp.raw	Raw
	211220_Yamanaka_MM1S_TMTpro18_Vanquish16_QEp.pdResult	Other
	211220_Yamanaka_MM1S_TMTpro18_Vanquish16_QEp.xlsx	Xlsx
	22_Yamanaka_MM1S_TMTpro18_Vanquish16_2_500ng_QEp.raw	Raw
	24_Yamanaka_MM1S_TMTpro18_Vanquish16_3_500ng_QEp.raw	Raw

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Publications

Lenalidomide derivatives and proteolysis-targeting chimeras for controlling neosubstrate degradation.

Yamanaka Satoshi S Furihata Hirotake H Yanagihara Yuta Y Taya Akihito A Nagasaka Takato T Usui Mai M Nagaoka Koya K Shoya Yuki Y Nishino Kohei K Yoshida Shuhei S Kosako Hidetaka H Tanokura Masaru M Miyakawa Takuya T Imai Yuuki Y Shibata Norio N Sawasaki Tatsuya T

Nature communications 20230818 1

Lenalidomide, an immunomodulatory drug (IMiD), is commonly used as a first-line therapy in many haematological cancers, such as multiple myeloma (MM) and 5q myelodysplastic syndromes (5q MDS), and it functions as a molecular glue for the protein degradation of neosubstrates by CRL4<sup>CRBN</sup>. Proteolysis-targeting chimeras (PROTACs) using IMiDs with a target protein binder also induce the degradation of target proteins. The targeted protein degradation (TPD) of neosubstrates is crucial for ...[more]

PMID: 37596276

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Project description:NTERA-2 cells were treated with DMSO, 0.3 µM Po-P, 0.3 µM Le-P, 0.3 µM F-P, 0.3 µM Cl-P, or 0.3 µM F3C-P for 16 h in three biological replicates. The cells were lysed in 100 µL guanidine buffer (6 M guanidine-HCl, 100 mM HEPES-NaOH, pH 7.5, 10 mM TCEP, 40 mM chloroacetamide). After heating and sonication, proteins (100 µg each) were purified by methanol–chloroform precipitation and resuspended in 20 µL 0.1% RapiGest SF (Waters) in 50 mM triethylammonium bicarbonate. After sonication and heating at 95 °C for 10 min, the proteins were digested with 2 µg trypsin/Lys-C mix (Promega) at 37 °C overnight. The digested peptides (40 µg each) were labeled with 0.5 mg TMTpro-18plex reagents (Thermo Fisher Scientific) for 1 h at 25 °C. After the reaction was quenched with hydroxylamine, all the TMT-labeled samples were pooled, acidified with TFA, and fractionated by offline high-pH reversed-phase chromatography on a Vanquish DUO UHPLC (Thermo Fisher Scientific) as described previously. Briefly, the peptides were loaded onto a 4.6 × 250 mm Xbridge BEH130 C18 column with 3.5 mm particles (Waters) and separated using a 30 min multistep gradient of solvents A (10 mM ammonium formate at pH 9.0 in 2% ACN) and B (10 mM ammonium formate pH 9.0 in 80% ACN), at a flow rate of 1 mL/min. Peptides were separated into 48 fractions, which were consolidated into 16 fractions. Each fraction was evaporated in a SpeedVac concentrator and dissolved in 0.1% TFA and 3% ACN. LC-MS/MS analysis of the resultant peptides (500 ng each) was performed on an EASY-nLC 1200 UHPLC connected to a Q Exactive Plus mass spectrometer through a nanoelectrospray ion source (Thermo Fisher Scientific). The peptides were separated on the analytical column (75 μm × 15 cm, 3 μm; Nikkyo Technos) with a linear gradient of 4–20% for 0–115 min and 20–32% for 115–160 min, followed by an increase to 80% ACN for 10 min and finally held at 80% ACN for 10 min. The mass spectrometer was operated in data-dependent acquisition mode with a top 10 MS/MS method. MS1 spectra were measured with a resolution of 70,000, an AGC target of 3e6, and a mass range from 375 to 1,400 m/z. MS/MS spectra were triggered at a resolution of 35,000, an AGC target of 1e5, an isolation window of 0.7 m/z, a maximum injection time of 150 ms, and a normalized collision energy of 33. Dynamic exclusion was set to 20 s. Raw data were directly analyzed against the SwissProt database restricted to Homo sapiens using Proteome Discoverer version 2.4 with the Sequest HT search engine for identification and TMT quantification. The search parameters were as follows: (a) trypsin as an enzyme with up to two missed cleavages; (b) precursor mass tolerance of 10 ppm; (c) fragment mass tolerance of 0.02 Da; (d) TMT of lysine and peptide N-terminus and carbamidomethylation of cysteine as fixed modifications; (e) oxidation of methionine as a variable modification. Peptides were filtered at an FDR of 1% using the Percolator node. TMT quantification was performed using the Reporter Ions Quantifier node. Normalization was performed such that the total sum of the abundance values for each TMT channel over all peptides was the same.

Project description:Human Umbilical Vein Endothelial Cells pooled from several donors, were purchased from Lonza and cultured in FBS reduced Endopan 3 media. HUVEC cells of passages 5-10 were used for experiments. MPO treatment was performed with protein purified from human blood (Planta) and at 1 μg/ml final concentration. Immunoprecipitation was performed from isolated nuclei (after 8 hours MPO treatment), non-treated cells were used as negative control. For immunoprecipitation antibody against MPO (Calbiochem, 475915) was used. Cell nuclei were isolated by incubating cells for 15 min on ice in NIB buffer (15 mM Tris-HCL pH 7.5, 60 mM KCl, 15 mM NaCl, 5 mM MgCl2, 1 mM CaCl2, 250 mM sucrose) containing 0.3% NP-40. Nuclei were pelleted for 5 min 800 × g at 4 °C, washed twice in the same buffer, lysed for 10 min on ice in IP buffer (150 mM LiCl, 50 mM Tris-HCl pH 7.5, 1 mM EDTA, 0.5% Empigen) freshly supplemented with 2 mM sodium vanadate, 1× protease inhibitor cocktail (Roche), PMSF (10 µl), 0,5 mM DTT, and 50 units Benzonase per ml of IP buffer, before preclearing cell debris by centrifugation at >15,000 × g at 4 °C. Finally, 1 mg of the lysate was incubated with anti-MPO antisera overnight at 4 °C. Magnetic beads (Active Motif) were then washed once with 1× PBS-Tween and combined with the antibody-lysate mixture. Following a 2-h incubation at 4 °C, beads were separated on a magnetic rack and washed 5×, 5 min each in wash buffer (150 mM KCl, 5 mM MgCl2, 50 mM Tris-HCl pH 7.5, 0.5% NP-40) and another two times in wash buffer without NP-40. Captured proteins were predigested and eluted from the beads using digestion buffer (2 M Urea, 50 mM Tris-HCl pH 7.5, 1 mM DTT) supplemented with trypsin and eluted from the beads with elution buffer (2 M Urea, 50 mM Tris-HCl pH 7.5, 5 mM chloroacetamide) supplemented with trypsin and LysC, before subjected to mass-spectrometry on a Q-Exactive Plus Orbitrap platform coupled to an EASY nLC (Thermo Scientific). Peptides were loaded in solvent A (0.1% formic acid in water) onto an in-house packed analytical column (50 cm length, 75 µm I.D., filled with 2.7 µm Poroshell EC120 C1; Agilent)

Project description:Bacteria was grown at 30C in 3 different conditions, i.e. SYN (syngas and minimal medium- ATCC no 1789), AC (0.3% acetate in minimal medium- ATCC no 1789) and TSB (tryptic soy broth). After harvesting by centrifugation, O. carboxidovorans pellets (1g) were lysed in 100 mM Tris-Cl pH 8.0, 2% Triton X-100, 2.6 mg/ml sodium azide, 8 mM PMSF by sonication on ice (4 pulses of 15 s duration each). For each condition of growth 4 samples (1g pellets) were separately treated (i.e. lysed and processed further). The supernatants were treated with 50% cold TCA, and the precipitated protein washed with acetone. The pellets were resuspended in solubilization buffer (7M urea, 20 mM tris-Cl, pH 8.0, 5 mM EDTA, 5 mM MgCl2, 4% CHAPS), and protein concentration was determined using the Plus One 2-D Quant Kit (Amersham) following the manufacturers instructions. Protein samples from each treatment were stored at -80 C. One hundred micrograms of each protein sample was resuspended in 0.1 M ammonium bicarbonate, 5% HPLC grade ACN, reduced in 5 mM DTT (65 C, 5 min), alkylated in 10 mM iodoacetamide (30 C, 30 min), and then trypsin digested until there was no visible pellet (1:50 w/w 37 C, 16 h). Peptides were desalted using a peptide microtrap (Michrom BioResources, Auburn, CA) and eluted using a 0.1% TFA, 95% ACN solution. Desalted peptides were dried in a vacuum centrifuge and resuspended in 20 ?l of 0.1% formic acid. Peptides were separated by strong cation exchange (SCX) liquid chromatography (LC) followed by reverse phase (RP) LC coupled directly in line with electrospray ionization (ESI) tandem mass spectrometry (MS/MS). 2DLC ESI MS/MS was done exactly as described (1). All searches were done using TurboSEQUEST (Bioworks Browser 3.2; Thermo Electron). Mass spectra and tandem mass spectra were searched against all annotated proteins from the strain OM5 including all the annotated plasmid-encoded proteins. Cysteine carbamidomethylation and methionine oxidation (single and double) were included in the search strategy. We used the reverse database functionality in Bioworks 3.2 and searched MS2 data against a reversed OM5 database using identical search criteria.

Project description:Cells were treated with DMSO (biological triplicate) or degrader at indicated dose and time (Meta Treatment Table) and cells were harvested by centrifugation. Lysis buffer (8 M Urea, 50 mM NaCl, 50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (EPPS) pH 8.5, Protease and Phosphatase inhibitors from Roche) was added to the cell pellets and homogenized by 20 passes through a 21 gauge (1.25 in. long) needle to achieve a cell lysate with a protein concentration between 1 - 4 mg/mL. A micro-BCA assay (Pierce) was used to determine the final protein concentration in the cell lysate. 200 µg of protein for each sample were reduced and alkylated as previously described1. Proteins were precipitated using methanol/chloroform. In brief, four volumes of methanol were added to the cell lysate, followed by one volume of chloroform, and finally three volumes of water. The mixture was vortexed and centrifuged to separate the chloroform phase from the aqueous phase. The precipitated protein was washed with three volumes of methanol, centrifuged and the resulting washed precipitated protein was allowed to air dry. Precipitated protein was resuspended in 4 M Urea, 50 mM HEPES pH 7.4, followed by dilution to 1 M urea with the addition of 200 mM EPPS, pH 8. Proteins were first digested with LysC (1:50; enzyme:protein) for 12 hours at room temperature. The LysC digestion was diluted to 0.5 M Urea with 200 mM EPPS pH 8 followed by digestion with trypsin (1:50; enzyme:protein) for 6 hours at 37 °C. Tandem mass tag (TMT) reagents (Thermo Fisher Scientific) were dissolved in anhydrous acetonitrile (ACN) according to manufacturer’s instructions. Anhydrous ACN was added to each peptide sample to a final concentration of 30% v/v, and labeling was induced with the addition of TMT reagent to each sample at a ratio of 1:4 peptide:TMT label. The 11 or 16-plex labeling reactions were performed for 1.5 hours at room temperature and the reaction quenched by the addition of hydroxylamine to a final concentration of 0.3% for 15 minutes at room temperature. Each of the sample channels were combined in a 1:1 ratio, desalted using C18 solid phase extraction cartridges (Waters) and analyzed by LC-MS for channel ratio comparison. Samples were then combined using the adjusted volumes determined in the channel ratio analysis and dried down in a speed vacuum. The combined sample was then resuspended in 1% formic acid and acidified (pH 2 - 3) before being subjected to desalting with C18 SPE (Sep-Pak, Waters). Samples were then offline fractionated into 96 fractions by high pH reverse-phase HPLC (Agilent LC1260) through an aeris peptide xb-c18 column (phenomenex) with mobile phase A containing 5% acetonitrile and 10 mM NH4HCO3 in LC-MS grade H2O, and mobile phase B containing 90% acetonitrile and 10 mM NH4HCO3 in LC-MS grade H2O (both pH 8.0). The 96 resulting fractions were then pooled in a non-continuous manner into 24 fractions and these fractions were used for subsequent mass spectrometry analysis. Data were collected using an Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA) coupled with a Proxeon EASY-nLC 1200 LC pump (Thermo Fisher Scientific) or an Orbitrap Eclipse Tribrid mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA) coupled with an UltiMate 3000 RSLCnano System. Peptides were separated on an EasySpray ES803a/ES803a.rev2 75 μm inner diameter microcapillary column (Thermo Fisher Scientific) or a 100 µm inner diameter microcapillary column packed with ~ 50 cm of Accucore C18 resin (2.6 µM, 100 Å, Thermo Fisher Scientific). Peptides were separated using a 190 min gradient of 6 - 27% acetonitrile in 1.0% formic acid with a flow rate of 300 or 350 nL/min. Each analysis used an MS3-based TMT method as described previously. The data were acquired using a mass range of m/z 340 – 1350, resolution 120,000, AGC target 5 x 105, maximum injection time 100 ms, dynamic exclusion of 120 seconds for the peptide measurements in the Orbitrap. Data dependent MS2 spectra were acquired in the ion trap with a normalized collision energy (NCE) set at 35%, AGC target set to 1.8 x 104 and a maximum injection time of 120 ms. MS3 scans were acquired in the Orbitrap with a HCD collision energy set to 55%, AGC target set to 2 x 105, maximum injection time of 150 ms, resolution at 50,000 and with a maximum synchronous precursor selection (SPS) precursors set to 10.

Project description:MM1.S cells were treated with DMSO or 10 µM thalidomide or its derivatives for 5 h in two biological replicates. The cells were lysed in 200 µL guanidine buffer (6 M guanidine-HCl, 100 mM HEPES-NaOH, pH 7.5, 10 mM TCEP, 40 mM chloroacetamide). After heating and sonication, proteins (100 µg each) were purified by methanol–chloroform precipitation and resuspended in 20 µL 0.1% RapiGest SF (Waters) in 50 mM triethylammonium bicarbonate. After sonication and heating at 95 ºC for 10 min, the proteins were digested with 2 µg trypsin/Lys-C mix (Promega) at 37 ºC overnight. The digested peptides (40 µg each) were labeled with 0.5 mg TMTpro-18plex reagents (Thermo Fisher Scientific) for 1 h at 25 ºC. After the reaction was quenched with hydroxylamine, all the TMT-labeled samples were pooled, acidified with TFA, and fractionated by offline high-pH reversed-phase chromatography on a Vanquish DUO UHPLC (Thermo Fisher Scientific) as described previously. Briefly, the peptides were loaded onto a 4.6 × 250 mm Xbridge BEH130 C18 column with 3.5 µm particles (Waters) and separated using a 30 min multistep gradient of solvents A (10 mM ammonium formate at pH 9.0 in 2% ACN) and B (10 mM ammonium formate pH 9.0 in 80% ACN), at a flow rate of 1 mL/min. Peptides were separated into 48 fractions, which were consolidated into 16 fractions. Each fraction was evaporated in a SpeedVac concentrator and dissolved in 0.1% TFA and 3% ACN. LC-MS/MS analysis of the resultant peptides (500 ng each) was performed on an EASY-nLC 1200 UHPLC connected to a Q Exactive Plus mass spectrometer through a nanoelectrospray ion source (Thermo Fisher Scientific). The peptides were separated on the analytical column (75 μm × 15 cm, 3 μm; Nikkyo Technos) with a linear gradient of 4–20% for 0–115 min and 20–32% for 115–160 min, followed by an increase to 80% ACN for 10 min and finally held at 80% ACN for 10 min. The mass spectrometer was operated in data-dependent acquisition mode with a top 15 MS/MS method. MS1 spectra were measured with a resolution of 70,000, an AGC target of 3e6, and a mass range from 375 to 1,400 m/z. MS/MS spectra were triggered at a resolution of 35,000, an AGC target of 1e5, an isolation window of 0.7 m/z, a maximum injection time of 150 ms, and a normalized collision energy of 32. Dynamic exclusion was set to 20 s. Raw data were directly analyzed against the SwissProt database restricted to Homo sapiens using Proteome Discoverer version 2.5 with the Sequest HT search engine for identification and TMT quantification. The search parameters were as follows: (a) trypsin as an enzyme with up to two missed cleavages; (b) precursor mass tolerance of 10 ppm; (c) fragment mass tolerance of 0.02 Da; (d) TMT of lysine and peptide N-terminus and carbamidomethylation of cysteine as fixed modifications; (e) oxidation of methionine as a variable modification. Peptides were filtered at an FDR of 1% using the Percolator node. TMT quantification was performed using the Reporter Ions Quantifier node. Normalization was performed such that the total sum of the abundance values for each TMT channel over all peptides was the same.

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.