PXD048321

PXD048321 is an original dataset announced via ProteomeXchange.

Title	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. 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 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.
HostingRepository	jPOST
AnnounceDate	2025-07-23
AnnouncementXML	Submission_2025-07-23_14:24:00.945.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; alpha-amino acetylated residue
Instrument	Q Exactive HF

Revision	Datetime	Status	ChangeLog Entry
0	2024-01-08 08:25:50	ID requested
⏵ 1	2025-07-23 14:24:01	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: heavy water, protein turnover, ER stress, calibration

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

jPOST dataset URI

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