PXD060287

PXD060287 is an original dataset announced via ProteomeXchange.

Title	Human induced pluripotent stem cells under dynamic heavy water labeling
Description	Fractional synthesis calibration cells for hiPSC AICS-0052-003 hiPSC (mono-allelic C-terminus mEGFP-tagged MYL7 WTC-11; Allen Institute Cell Collection) line was acquired from Coriell Institute and seeded onto Geltrex (Gibco) coated 6 well plates and maintained in mTesR Plus basal media with Supplement (STEMCELL) media. The basal medium but not supplement was diluted with either 6% D2O (heavy labeled population) or 6% H2O (control population), and the cells were cultured at 37 °C, 5% CO2 with daily media changes. At 80% confluency, cells were passaged at 1:6 using EDTA before resuspension in media supplemented with 10 µM Y-27632 (Selleck), for a total of 4 passages (equivalent to ≥10 doublings). Proteins were extracted with sonication in a Bioruptor Pico (Diagenode) with settings 10× 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. 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. A total of 50 µg protein per sample in 250 µL 8 M urea was loaded onto Pierce Protein Concentrators PES, 10K MWCO (Thermo Scientific) pre-washed with 100 mM ammonium bicarbonate (ABC). The samples were again washed with 8 M urea to denature proteins and remove SDS. The samples were washed with 300 µL 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. The 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 (DDA) 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 seconds. Dynamic labeling time course For hiPSC-CM single-point labeling, AICS-0052-003 hiPSC (mono-allelic C-terminus mEGFP-tagged MYL7 WTC-11; Allen Institute Cell Collection) were expanded and differentiated into hiPSC-CM as above. Media was refreshed every other day with RPMI+B27 with insulin until day 41, at which time the media was supplemented with 6% v/v D2O (Cambridge Isotope), and 1 µM doxorubicin or vehicle. 24 hours later, the media was collected and replaced with media supplemented with 6% v/v D2O. At 48 hours of total labeling, the intracellular samples were collected and reduced, alkylated, and digested using an on-filter digestion protocol as described above, and analyzed with DDA mass spectrometry. Directed differentiation of hiPSC for dynamic D2O labeling experiments For hiPSC differentiation toward mesoderm/mesendoderm specification, ACIS-52 hiPSC were plated onto a 6-well plate coated with geltrex in mTesR Plus basal media with Supplement (STEMCELL) media and 10 µM Y-27632 (Selleck). Media changes were performed every day with mTesR Plus basal media with Supplement (STEMCELL) media. At 80% confluency, cells were split with TrypLE Select Enzyme (10X, Thermo Fisher) onto geltrex coated 12-well plates at 0.2 × 106 cells/well in mTesR Plus basal media with Supplement (STEMCELL) media and 10 µM Y-27632 (Selleck). Media changes were performed every day with mTesR Plus basal media with Supplement (STEMCELL) media. At 80% confluency, the medium was removed and replaced with 2 mL/well RPMI1640 supplemented with B-27 minus insulin (Gibco), 6 μM CHIR99021 (STEMCELL), and 6% v/v D2O (Cambridge Isotope), except for time point 0. At each timepoint of 0, 1, 2, 3, 4, 6, 8, 12 and 24 hours, media was aspirated from cells and cells were collected with 100 µL TrypLE Select Enzyme (10X, Thermo Fisher) for 2 minutes, then quenched with 500 µL RPMI1640. For each replicate in each condition, 2 wells were combined, pelleted at 200 ×g for 5 minutes, rinsed 1× with 1 mL PBS, pelleted once more at 200 ×g for 5 minutes and immediately flash frozen. Protein extraction, digestion and peptide desalting were performed using the same protocol described above. The digested peptides were resuspended in 0.1% formic acid (v/v) and separated using a Vanquish Neo UHPLC system (Thermo Fisher Scientific) on an Easy-Spray™ analytical column (ES900 C18, 75µm x 150 mm, 3 µm) with a 60-minute gradient. Solvent A was 0.1% formic acid in LCMS-grade water and solvent B was 0.1% Formic acid in 80% LCMS-grade acetonitrile. The LC-system was coupled to an Orbitrap Exploris 480 (Thermo Fisher Scientific) operating in positive mode with the spray voltage at 2 kV and ion transfer tube temperature of 275 °C. Full-mass spectra were acquired from 350–1650 m/z at 60,000 resolution with an AGC target at 300% and maximum ion injection time set to auto. The mass spectrometer was operated in DDA mode, selecting precursors within a charge state of 2–6 and minimum intensity of 3 x 102. The top 20 precursors were subsequently selected with a 2 m/z isolation window for HCD fragmentation with NCE set at 30%. The resolution was set to 15,000 with an AGC target at 200%, maximum ion injection time of 26 ms and dynamic exclusion of 45 seconds.
HostingRepository	jPOST
AnnounceDate	2025-07-23
AnnouncementXML	Submission_2025-07-23_14:22:42.154.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Edward Lau
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationList	S-carboxamidomethyl-L-cysteine; L-methionine sulfoxide
Instrument	Q Exactive HF; instrument

Revision	Datetime	Status	ChangeLog Entry
0	2025-01-28 09:39:17	ID requested
⏵ 1	2025-07-23 14:22:43	announced

Alamillo L, Ng DCM, Currie J, Black A, Pandi B, Manda V, Pavelka J, Schaal P, Travers JG, McKinsey TA, Lam MPY, Lau E, Deuterium labeling enables proteome-wide turnover kinetics analysis in cell culture. Cell Rep Methods, 5(7):101104(2025) [pubmed]

submitter keyword: iPSC, heavy water

Edward Lau
lab head
Edward Lau
contact affiliation	University of Colorado
dataset submitter

jPOST dataset URI

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