Project description:hiPSC-CM heavy water labeling calibration standard
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). To produce calibration cells for hiPSC-CM, fully labeled or unlabeled hiPSC were replated into Geltrex coated 12 well plates at a density of 4.5 × 10^5 cells/well and daily media changes continued until the cells reached 80% confluency, day 0 of cardiac differentiation. The hiPSC were differentiated into hiPSC-CM using a small molecule-based GSK-3β inhibition/Wnt inhibition protocol. Briefly, on day 0, cell media was replaced with 2 mL/well RPMI supplemented with B-27 minus insulin (Gibco) and 6 μM CHIR99021 (STEMCELL); on day 2, the media was changed to 2 mL/well RPMI+B-27 minus insulin. On day 3, the media was changed to 2 mL/well RPMI+B27 without insulin supplemented with 5 μM IWR-1-Endo (STEMCELL). On day 7, the media was changed to 2 mL RPMI+B27 with insulin. Differentiation was confirmed via visualization of morphology, spontaneous contraction of cells, and imaging of the GFP tagged MYL7/MLC-2a. Media was refreshed every other day with RPMI+B27 with insulin until day 14. Attached hiPSC-CMs were washed twice with PBS before incubating with 0.5 mL per well of TrypLE Express (1x, Thermo Fisher) for 11 minutes. An additional 1.5 mL PBS was added to dilute the TrypLE and the cells were triturated until fully detached. Cells were pelleted by centrifugation at 300 ×g for 3 minutes, washed with PBS before repelleting, and stored at -80°C until protein extraction.
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
Secretome analysis in D2O labeled hiPSC-CM
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% vol/vol D2O. The collected conditioned media was centrifuged for 5 minutes at 300 ×g and 5 minutes at 14,000 ×g. Spun media was stored at –80°C. 24 hours later (48 hours of labeling total), the media was collected again and centrifuged as described. At the same time, the intracellular samples were collected and reduced, alkylated, and digested using an on-filter digestion protocol as described above.
To analyze secretome content, 300 µL of spun conditioned media per sample was depleted using the Seer Proteograph XT workflow (Seer, Inc.) utilizing two distinct nanoparticle (NP) mixtures (NPA, NPB). NP protein coronas were reduced, alkylated, and digested with Trypsin/Lys-C to generate tryptic peptides for LC-MS analysis. Digested peptides were desalted, eluted in a high-organic buffer into a deep-well collection plate, and quantified. In hiPSC-CM experiments, cleaned peptides were reconstituted in 0.1% formic acid to a final concentration of 0.0325 µg/µL and 0.116 µg/µL from NPA and NPB, respectively. 3 µL of peptides per sample were separated with online low-pH reversed-phase LC (PepMap C18 column, 3-μm particle, 100-Å pore; 75 µm × 15 cm; Thermo Fisher Scientific) via the EASYnLC 1200 system coupled to the Easy-Spray ion source (Thermo Fisher Scientific) at 300 nL/min with a 90-min gradient: 0–75 min: 0 to 30% B; 75-80 min: 30 to 70% B; 80-85 min: 70% to 100% B; 85-90 min: 100% B (solvent A: 0.1% v/v formic acid; solvent B: 0.1% formic acid in 80% v/v acetonitrile). Mass spectra were acquired on a Thermo Scientific Q-Exactive HF Orbitrap mass spectrometer with the following settings: polarity, positive; data independent acquisition (DIA); MS resolution, 120,000; maximum ion injection time, 230 ms; MS automatic gain control (AGC) target, 3e6; normalized collision energy (NCE), 30; MS2 resolution, 30,000; MS2 maximum ion injection time, 45 ms; isolation window, 11 m/z; and MS2 AGC target, 1e6.
File naming
Calibration standards:
20240619_[xxx]_D2O.raw where xxx is the mixture proportion of fully labeled hiPSC-CM (e.g., 250 = 25.0%)
Intracellular samples:
202312xx_CM_Intracellular_48h_[Treatment]_[Sample_name].raw
Treatment: DOX for doxorubicin, or DMSO
Sample_name: 01 - 12
Secretome samples:
202308xx_Secretome_[Sample_name]_0_[Hour]_[Bead]_DIA_TR1.raw
Sample_name: 01 - 12, corresponding to intracellular samples
Hour: 24 or 48 - hours of enrichment/collection
Bead: Nanoparticle A or B
2025-07-24 | PXD060295 | JPOST Repository
