{"database":"JPOST Repository","file_versions":[{"headers":{"Content-Type":["application/json"]},"body":{"files":{"Raw":["https://storage.jpostdb.org/JPST002443/files/20210824_AC16_Thapsigargin_R4_TR1.raw","https://storage.jpostdb.org/JPST002443/files/20210824_AC16_Control_R1_TR1.raw","https://storage.jpostdb.org/JPST002443/files/20210824_AC16_Control_R2_TR1.raw","https://storage.jpostdb.org/JPST002443/files/20210824_AC16_Thapsigargin_R1_TR2.raw","https://storage.jpostdb.org/JPST002443/files/20210824_AC16_Thapsigargin_R2_TR1.raw","https://storage.jpostdb.org/JPST002443/files/20210824_AC16_Control_R3_TR1.raw","https://storage.jpostdb.org/JPST002443/files/20210824_AC16_Thapsigargin_R3_TR1.raw","https://storage.jpostdb.org/JPST002443/files/20210824_AC16_Control_R1_TR2.raw","https://storage.jpostdb.org/JPST002443/files/20210824_AC16_Control_R4_TR1.raw","https://storage.jpostdb.org/JPST002443/files/20210824_AC16_Thapsigargin_R4_TR2.raw","https://storage.jpostdb.org/JPST002443/files/20210824_AC16_Control_R2_TR2.raw","https://storage.jpostdb.org/JPST002443/files/20210824_AC16_Thapsigargin_R1_TR1.raw","https://storage.jpostdb.org/JPST002443/files/20210824_AC16_Control_R5_TR1.raw","https://storage.jpostdb.org/JPST002443/files/20210824_AC16_Control_R3_TR2.raw","https://storage.jpostdb.org/JPST002443/files/20210824_AC16_Thapsigargin_R5_TR1.raw","https://storage.jpostdb.org/JPST002443/files/20210824_AC16_Thapsigargin_R5_TR2.raw","https://storage.jpostdb.org/JPST002443/files/20210824_AC16_Thapsigargin_R2_TR2.raw","https://storage.jpostdb.org/JPST002443/files/20210824_AC16_Thapsigargin_R3_TR2.raw","https://storage.jpostdb.org/JPST002443/files/20210824_AC16_Control_R4_TR2.raw","https://storage.jpostdb.org/JPST002443/files/20210824_AC16_Control_R5_TR2.raw"]},"type":"primary"},"statusCodeValue":200,"statusCode":"OK"}],"scores":null,"additional":{"omics_type":["Proteomics"],"submitter":["Edward Lau"],"species":["Homo Sapiens (human)"],"full_dataset_link":["https://repository.jpostdb.org/entry/JPST002443"],"submitter_affiliation":["University of Colorado"],"sample_protocol":[""],"repository":["jPOST"],"data_protocol":[""],"pubmed_abstract":["Protein turnover is a critical component of gene expression regulation and cellular homeostasis, yet methods for measuring turnover rates that are scalable and applicable to different models are still needed. We introduce an improved D<sub>2</sub>O (heavy water) labeling strategy to investigate the landscape of protein turnover in cell culture, with accurate calibration of per-residue deuterium incorporation in multiple cell types. Applying this method, we mapped the proteome-wide turnover landscape of pluripotent and differentiating human induced pluripotent stem cells (hiPSCs). Our analysis highlights the role of APC/C (anaphase-promoting complex/cyclosome) and SPOP (speckle-type POZ protein) degrons in the fast turnover of cell-cycle-related and DNA-binding hiPSC proteins. Upon pluripotency exit, many short-lived hiPSC proteins are depleted, while RNA-binding and -splicing proteins become hyperdynamic. The ability to identify fast-turnover proteins also facilitates secretome profiling, as exemplified in hiPSC-cardiomyocyte and primary human cardiac fibroblast analysis. This method is broadly applicable to protein turnover studies in primary, pluripotent, and transformed cells."],"pubmed_title":["Deuterium labeling enables proteome-wide turnover kinetics analysis in cell culture."],"pubmed_authors":["Alamillo Lorena L, Ng Dominic C M DCM, Currie Jordan J, Black Alexander A, Pandi Boomathi B, Manda Vyshnavi V, Pavelka Jay J, Schaal Peyton P, Travers Joshua G JG, McKinsey Timothy A TA, Lam Maggie P Y MPY, Lau Edward E"],"additional_accession":[]},"is_claimable":false,"name":"Calibration and heavy water labeling in normal and stressed human AC16 cells","description":"For calibration curve samples, human AC16 cells (Millipore) were cultured in DMEM/F12 supplemented with 10% FBS and either 6% D2O (heavy labeled population) or 6% H2O (control population) at 37°C, 5% CO2. The cells were maintained in this medium for 3 passages, each passage with a split ratio of 1:8. This growth was estimated to constitute approximately 9 doublings of the cell populations. The cells were harvested by trypsinization, pelleted, washed once with phosphate buffered saline, and pelleted again before snap freezing in liquid nitrogen and storing at –80°C. At the time of processing each pellet was resuspended in 1 mL of RIPA buffer (Thermo Scientific) supplemented with Halt Protease and Phosphatase Inhibitor Cocktail (Thermo Scientific). Proteins were extracted with sonication in a Biorupter Pico (Diagenode) with settings 10x 30 sec on 30 sec off at 4°C. Insoluble debris was pelleted and removed from all samples by centrifugation at 14,000 ×g, 5 minutes. For the baseline and stressed samples, AC16 cells were cultured in 6% D2O with or without 1 µM thapsigargin for 16 to 24 hours, then processed as above. Protein concentration of all samples was measured with Rapid Gold BCA (Pierce). Cell lysates from the D2O and H2O media populations were then combined in a labelling series expressed as the proportion of protein that was labeled with heavy water: 0, 0.125, 0.25, 0.375, 0.5, 0.625, 0.75, 0.875 and 1. The samples were trypsin digested using a modified version of the filter-aided sample preparation approach as previously described 18. A total of 50 µg protein per sample in 250 µL 8M urea was loaded onto Pierce Protein Concentrators PES, 10K MWCO (Thermo Scientific) pre-washed with 100 mM ammonium bicarbonate (Ambic). The samples were again washed with 8 M urea to denature proteins and remove SDS. The samples were washed with 300 uL 100 mM ABC twice. The samples were then reduced and alkylated with final concentrations 5 mM dithiothreitol (DTT) and 18 mM iodoacetamide (IAA) for 30 minutes at 37°C in the dark. DTT and IAA were removed with centrifugation and the samples were washed 3× with 100 mM ABC. Samples were digested atop the filters overnight at 37°C with mass spectrometry grade trypsin (Promega) at a ratio of 1:50 enzyme:protein. The following day samples were cleaned with Pierce C18 spin columns (Thermo Scientific) according to the manufacturer’s protocol. Eluted peptides were dried under vacuum and redissolved resuspended in 0.1% (v/v) formic acid.\n\nThe samples were analyzed on a Thermo Q-Exactive HF quadrupole-Orbitrap mass spectrometer coupled to a nanoflow Easy-nLC UPLC with the Thermo EasySpray electrospray ionization source. Peptides were separated with a PepMap RSLC C18 column 75 μm × 15 cm, 3 μm particle size (Thermo Scientific) with a 90 minute gradient from 0 to 100% pH 2 solvent B (0.1% formic acid in 80% v/v LC-MS grade acetonitrile). The mass spectrometer was operated in data-dependent acquisition mode with scans between m/z 200 and 1650 acquired at a mass resolution of 60,000. The maximum injection time was 20 ms, and the automatic gain control was set to 3e6. MS2 scans of the 15 most intense precursor ions with charge states of 2+ to 5+ were acquired with an isolation window of 2 m/z units, maximum injection time 110 ms, and automatic gain control of 2e5. Fragmentation of the peptides was by stepped normalized collision-induced dissociation energy (NCE) of 25 to 27. Dynamic exclusion of m/z values was used with an exclusion time of 30 s.\n","dates":{"publication":"Wed Jul 23 00:00:00 BST 2025"},"accession":"PXD048321","cross_references":{"TAXONOMY":["9606"],"pubmed":["40645189"]}}