Data for whole and mitochondrial proteome of human embryonic stem cells

The data presented here pertain to the research article entitled “Proteome Analysis of Human Embryonic Stem Cell Organelles” (Shekariet al., 2017 [1]). In the present article we endeavour to locate new proteins and pathways in human embryonic stem cells (hESCs) by mass spectrometry and bioinformatics analysis. We have analyzed total and mitochondrial proteins extracted from three biological replicates of the hESC H9 cell line according to mass spectrometry proteomics and bioinformatics investigations.


Value of the data
Data present the whole proteome of the hESC H9 cell line. This data is the first report of the mitochondria proteome of hESCs. Western blot analysis and a mitochondrial proteome database search have confirmed the efficiency of the reported protocol for mitochondria isolation from hESCs. This data could enable other researchers to use the reported protocol for isolation of mitochondria.
This data may be used to develop and enrich our knowledge about localization of proteins in mitochondria.

Mass spectrometry based whole proteome profiling and bioinformatics analysis
A total of 470 out of 1516 proteins (Supplementary Table S1) identified in 3 replicates of hESCs (H9 cell line) were observed in only one replicate ( Supplementary Fig. S2). Although most lacked transmembrane helices, approximately 60 proteins had at least two transmembranes comprised of up to 14 helices (Supplementary Table S2).
We mapped the proteins to KEGG biochemical pathways using KOBAS 3.0. The signaling components of 46 KEGG signaling pathways were found in the hESCs (Supplementary Table S3). The significantly enriched pathways included HIF-1, cGMP-dependent protein kinase (cGMP-PKG), Glucagon, Rap1, and Hippo, which highlighted the importance of these pathways in hESCs.

Mass spectrometry based mitochondrial proteome profiling and bioinformatics analysis
We identified approximately 1500 proteins in three mitochondrial replicates (Supplementary  Table S5). Approximately 200 proteins had at least two transmembranes of up to 15 helices. We analyzed the list of the 958 proteins found in at least two replicates with the MitoMiner 4.0 v 2016 APR database of the mitochondrial proteome (MRC Mitochondrial Biology Unit, University of Cambridge, UK; Table 1) [4]. A comparison of the identified mitochondrial proteomes with other subcellular fractions has been published in the article entitled "Proteome Analysis of Human Embryonic Stem Cell Organelles" [1].

Experimental design, materials and methods
All materials were purchased from Sigma unless otherwise noted.  Table 1 Localization of identified proteins in the mitochondrial fraction of human embryonic stem cells (hESCs) according to MitoMiner 4.0 v2016 APR. MitoMiner reported mitochondrial localization of proteins based on subcellular immunofluorescent staining results from the Human Protein Atlas, large-scale mass-spectrometry, and GFP tagging data sets as well as computational predictions of mitochondrial targeting sequences from three popular mitochondrial target sequence prediction programs: iPSORT [5], TargetP [6], and MitoProt [7].

Western blot analysis
A total of 10 mg of extracted protein from isolated mitochondria (based on the BCA assay) were resolved by 12% SDS-PAGE using a Mini-PROTEAN 3 electrophoresis cell (Bio-Rad), then transferred onto PVDF membrane by wet transfer in Towbin electroblotting transfer buffer (Fig. 1).

Protein identification
Peptide and protein identifications were performed using the Mascot search engine (version 2.3.02, Matrix Science). Database searching was restricted to human tryptic peptides (IPI_hu-man_3.87; 91464 sequences) and variable modifications of human deamidated (NQ), methylthio (C), and oxidation (M). We allowed a maximum of two missed cleavages. The peptide mass tolerance was set at 10 ppm.
The Mascot engine searched the decoy database. Both the decoy score and false discovery rates were considered for identification.

Transparency document. Supplementary material
Transparency data associated with this article can be found in the online version at http://dx.doi. org/10.1016/j.dib.2017.05.036.