Proteomic overview:

(a) Western blots showing the expression of the pluripotency factors NANOG, OCT4 and SOX2 across all hESC and hiSPC lines (b) Diagram showing the SPS-MS3 TMT proteomic workflow used for the experiment. (c) Venn diagram showing the overlap of proteins identified within the hiPSC and hESC populations. (d) Average copy number histogram for the hESCs. (e) Average copy number histogram for the hiPSCs. (f) Bubble plot showing proteins coloured by specific categories where the size is represented by the average hESC estimated protein copy numbers. (g) Bubble plot showing proteins coloured by specific categories where the size is represented by the average hiPSC estimated protein copy numbers. (h) PCA plot based on the log10 copy numbers for all 8 replicates. hESCs are shown in purple and hiPSCs in orange.

Normalisation and protein content:

(a) Concentration based volcano plot showing the -log10 p-value and the log2 fold change comparing hiPSCs to hESCs. Elements shaded in red are considered significantly changed. All dots above the red line have a q-value lower than 0.001 (b) Copy number-based volcano plot showing the -log10 p-value and the log2 fold change comparing hiPSCs to hESCs. Elements shaded in red are considered significantly changed. All dots above the red line have a q-value lower than 0.001 (c) Box plot showing the MS based estimated protein content for hESCs and hiPSCs. (d) Box plot showing the protein amount per million cells derived from the EZQ Protein Quantification Kit for all hESCs and hiPSCs (e) Boxplot showing the median forward scatter of hESCs and hiPSCs. (f) Boxplot showing the median side scatter of hESCs and hiPSCs. (g) Boxplot showing the median number of cells across cell cycle stages for hESCs and hiPSCs. For all boxplots, the bottom and top hinges represent the 1st and 3rd quartiles. The top whisker extends from the hinge to the largest value no further than 1.5 3 IQR from the hinge; the bottom whisker extends from the hinge to the smallest value at most 1.5 3 IQR of the hinge.

Fuelling growth:

(a) Boxplots showing the estimated copy numbers for the lactate (SLC16A1 and SLC16A3) and glucose transporters (SLC2A1 and SLC2A3) across hESCs and hiPSCs. (b) Schematic showing the glycolytic proteins and their fold change in hESC vs hiPSCs. (c) Chord diagram showing the 15 most upregulated solute carrier proteins along with their classification based on transport activities/localisation. (d) Boxplots showing the estimated copy numbers of the main amino acid transporters in hESCs and hiPSCs. (e) Boxplot showing the net glutamine uptake (see methods) in hESCs and hiPSCs. (f) Schematic showing the glutaminolysis proteins and their fold change in hESC vs hiPSCs. (g) Radar plot showing the median fold change (iPSC/ESC) for protein categories which are related to the pre-ribosomes. Boxplots showing the estimated copy numbers for (h) SREBF1, (i) FASN, (j) SCD, (k) PLIN3. in hESC vs hiPSCs. (l) Transmission electron microscopy images for hiPSCs. Lipid droplets are marked with red arrows. (m) Transmission electron microscopy images for hESCs. For all boxplots, the bottom and top hinges represent the 1st and 3rd quartiles. The top whisker extends from the hinge to the largest value no further than 1.5 3 IQR from the hinge; the bottom whisker extends from the hinge to the smallest value at most 1.5 3 IQR of the hinge.

Mitochondrial differences:

(a) Schematic showing the mitochondrial genome encoded proteins and their fold change in hESCs and hiPSCs. (b) Boxplot showing the estimated copy numbers of all mitochondrial ribosomal proteins. (c) Schematic showing proteins involved in mitochondrial translation and their fold change (hiPSCs/hESCs). (d) Treeplot showing all mitochondrial transporters, size is proportional to the estimated copy numbers in hiPSCs. Boxplot showing the estimated copy numbers for (e) SLC25A20. (f) CPT1A. (g) MCAT, (h) MECR, (i) OSXM, (j) Boxplot showing the log2 fold change (hiPSC/hESCs) of all subunits of the different complexes of the electron transport chain. The median fold change across all detected proteins is shown as a dotted line. (k) Schematic showing the fold change of citic acid cycle and glutaminolysis proteins in hESCs and hiPSCs. (l) Boxplot showing the P/E control ratio. All boxplots show the data for hESCs and hiPSCs. For all boxplots, the bottom and top hinges represent the 1st and 3rd quartiles. The top whisker extends from the hinge to the largest value no further than 1.5 3 IQR from the hinge; the bottom whisker extends from the hinge to the smallest value at most 1.5 3 IQR of the hinge.

Secreted proteins:

(a) Sankey diagram showing secreted proteins that are significantly increase in hiPSCs and belong to the the ECM matrix, Growth factor, Protease Inhibitor or Protease categories. (b) Schematic showing ECM proteins that are significantly increased in abundance in hiPSCs. (c) Boxplot showing the estimated copy numbers of TF in hESCs and hiSPCs. (d) Boxplot showing the estimated copy numbers of TFRC in hESCs and hiSPCs. (e) Boxplot showing the estimated copy numbers of FTH1. (f) Boxplot showing the estimated copy numbers of FTL. (g)Schematic showing the changes in abundance in vital primed pluripotency growth factors. (h) Boxplot showing the estimated protein copy numbers for VGF. (i) Boxplot showing the estimated protein copy numbers for MDK. (j) Boxplot showing the estimated protein copy numbers for TGFB1. (k) Boxplot showing the estimated protein copy numbers for ARG1. (l) Boxplot showing the estimated protein copy numbers for CD276. (m) Boxplot showing the estimated protein copy numbers for HLA-E. (n) Boxplot showing the estimated protein copy numbers for CD200. (o) Boxplot showing the estimated protein copy numbers for CD47. All boxplots show the data for hESCs and hiPSCs. For all boxplots, the bottom and top hinges represent the 1st and 3rd quartiles. The top whisker extends from the hinge to the largest value no further than 1.5 3 IQR from the hinge; the bottom whisker extends from the hinge to the smallest value at most 1.5 3 IQR of the hinge.

Changes within histones:

(a) Barplot showing the GO term enrichment results for proteins significantly decreased in abundance (see methods) in hiPSCs. (b) Boxplots showing estimated copy numbers for histones HIST1H1E in hESCs and hiPSCs. (c) Boxplots showing estimated copy numbers for histones HIST1H1D in hESCs and hiPSCs. (d) Boxplots showing estimated copy numbers for histones HIST1H1C in hESCs and hiPSCs. (e) Boxplots showing estimated copy numbers for histones H1FX in hESCs and hiPSCs. (f) Barplot showing the estimated copy numbers for all histones in hESCs and hiPSCs. (g) Boxplots showing estimated copy numbers for histones HIST1H3A in hESCs and hiPSCs. (h) Boxplots showing estimated copy numbers for histones HIST1H4A in hESCs and hiPSCs. Western blot showing the abundance of (i) H3 and (j) H4 histones in hESCs and hiPSCS. (k) Boxplots showing estimated copy numbers for histones H2AFV in hESCs and hiPSCs. (l) Boxplots showing estimated copy numbers for histones H2AFY in hESCs and hiPSCs. (m) Boxplots showing estimated copy numbers for histones H2AFY2 in hESCs and hiPSCs. For all boxplots, the bottom and top hinges represent the 1st and 3rd quartiles. The top whisker extends from the hinge to the largest value no further than 1.5 3 IQR from the hinge; the bottom whisker extends from the hinge to the smallest value at most 1.5 3 IQR of the hinge.