Summary

Title

HipSci project proteomics batch 1, comprising 6 IPS cell lines from 3 donors

Description

The Human Induced Pluripotent Stem Cells Initiative (HipSci) is generating a large, high-quality reference panel of human IPSC lines. This is a submission of mass-spectrometry analyses from 6 induced pluripotent stem cell lines generated by the HipSci project.

Sample Processing Protocol

The frozen iPS cell pellets were solubilized using lysis buffer (8M urea, 100 mM TEAB). The cell lysates were digested using a two-step digestion protocol (overnight Lys-C digestion followed by 4 hour-tryptic digestion). Peptides were labelled using TMT six plex. Basic Reverse phase LC (RP-LC) chromotography was used to fractionate the peptides for subsequent LC-MS/MS analyses.

Data Processing Protocol

Three seperate proteomics analyses were performed: 1) with Q-exactive mass spectrometer, 2) data-dependent acquisition (DDA) with Orbitrap Fusion mass spectrometer (SPS-MS3 method), 3) data-dependent acquisition (DDA) with Oribtrap Fusion mass spectrometer with inclusion list (SPS-MS3). Data processing was carried out using Maxquant version 1.5.3.30. Data were processed with the following settings: 2 missed cleavages allowed, enzyme used was LysC + trypsin/P, 20 ppm tolerance was used for fragment ions, FDR cut off of 0.05 was used. One fixed modification: carbamidomethylation (Cys) and TMT six plex, and few variable modifications were selected: Oxidation (M); Acetylation (N-term), deamidation (NQ), pyroglutamate conversion of glutamine, phospho(STY).

Contact

HipSci Project, Human Induced Pluripotent Stem Cells Initiative Human Induced Pluripotent Stem Cells Initiative Human Induced Pluripotent Stem Cells Initiative
Angus Lamond, College of Life Sciences, University of Dundee ( lab head )

Submission Date

06/12/2016

Publication Date

12/05/2017

Corresponding dataset(s) in other omics resources

SAMEA2536417 (ENA, EMBL-EBI)

Publication

    Kilpinen H, Goncalves A, Leha A, Afzal V, Alasoo K, Ashford S, Bala S, Bensaddek D, Casale FP, Culley OJ, Danecek P, Faulconbridge A, Harrison PW, Kathuria A, McCarthy D, McCarthy SA, Meleckyte R, Memari Y, Moens N, Soares F, Mann A, Streeter I, Agu CA, Alderton A, Nelson R, Harper S, Patel M, White A, Patel SR, Clarke L, Halai R, Kirton CM, Kolb-Kokocinski A, Beales P, Birney E, Danovi D, Lamond AI, Ouwehand WH, Vallier L, Watt FM, Durbin R, Stegle O, Gaffney DJ. Common genetic variation drives molecular heterogeneity in human iPSCs. Nature. 2017 May 10 PubMed: 28489815