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Proteogenomic analysis of hiPSC cultures from a healthy donor

ABSTRACT: Proteogenomic analysis of three stem cell samples from a healthy donor. The cell lines used here are commercially available as induced pluripotent stem cells (iPSC) via retroviral reprogramming of skin fibroblasts isolated from the PGP1 donor from the Personal Genome Project (PGP) (Coriell, GM23338). hiPSC cultures were maintained as previously described [doi.org/10.1387/ijdb.230220lg]. Cell lysis, protein extraction, trypsin digestion, and peptide desalting were performed using the AccelerOme automated system from Thermo Fisher Scientific according to the manufacturer's instructions. Peptides were separated on a 110 cm micro-PAC HPLC column (Thermo Fisher Scientific) with a Vanquish Neo HPLC system (Thermo Fisher Scientific) coupled through a nano-electrospray source to a Tribrid Ascend mass spectrometer (Thermo Fisher Scientific) with a non-linear gradient of 1 - 50 % buffer B (0.1 % formic acid, 80 % acetonitrile) at a flow rate of 300 nL/min over 160min. The column temperature was kept at 50 C. Samples were acquired using a DDA MS2 data acquisition, where the Tribrid mass spectrometer was switching between a full scan (120 K resolution, Auto max. injection time, AGC target 100%), to a data-dependent (Top-20) MS/MS scans in the Orbitrap analyzer (15K resolution, 27 ms max. injection time, AGC target 400%). Isolation window was set to 1.4 (m/z), and normalized collision energy to 25. Precursors were filtered by charge state of 2-6 and multiple sequencing of peptides was minimized by excluding the selected peptide candidates for 60 s. Protein sequence database was generated by ProHap [doi.org/10.1101/2023.12.24.572591] using the phased genotype of the healthy donor, available from [https://my.pgp-hms.org/profile_public?hex=hu43860C].

INSTRUMENT(S): Orbitrap Ascend

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Marc Vaudel

PROVIDER: MSV000095226 | MassIVE | Wed Jul 03 05:07:00 BST 2024

SECONDARY ACCESSION(S): PXD053601

REPOSITORIES: MassIVE

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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.

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Proteogenomic analysis of hiPSC cultures from a healthy donor

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