Fig 1.
Overview of the three-pronged approach.
A three-pronged approach was used to investigate the two hypotheses addressing the identity of the target antigen of GALD antibodies. Not all samples were investigated in all 3 experimental approaches. No convincing leads were identified from the 3 different experimental approaches.
Fig 2.
The pedigrees of the three families included in this study for the exome analysis are shown. DNA from the fathers and mothers was used in the exome analysis as well as DNA from the child II.8. For children II.6 and II.7 the mother was treated with IVIG during pregnancy. The diagnosis of GALD was clinically verified for the affected and deceased children.
Fig 3.
Immunoprecipitaion from Huh-7.
Normalized log2 intensities of Primary Biliary Cirrosis (PBC) antigens in IPs with Huh-7 cell line.
Fig 4.
Immunoprecipitation from human fetal liver.
Normalized log2 Label Free Quantification (LFQ) intensities of PBC antigens in IPs with fetal liver material.
Fig 5.
Principle components 1 and 2 of PCA of MS results of IPs with Huh-7 cell line.
The PBC sample and the GALD samples were prepared with EDTA while the healthy samples were prepared with citrate.
Fig 6.
Differential protein abundance in GALD plasma vs healthy controls in Huh-7 cell line.
A) Volcano plot depicting differential abundance of proteins in IPs with GALD plasma vs healthy plasma with Huh-7 cell line. Proteins with Benjamin-Hochberg adjusted p-value (FDR) < 0.05 in orange: Complement C3 (C3), Complement C4-A (C4A), Complement C4-B (C4B), Monocarboxylate transporter 1 (SLC16A1), Tropomyosin beta chain (TPM2), Complement C1q subcomponent subunit B (C1QB). B) Normalized log2 LFQ intensities of significantly differentially abundant proteins in 3 conditions GALD, healthy and PBC plasma. Number of unique peptides in brackets. LogFC = log Fold Change.
Fig 7.
Differential protein abundance in GALD plasma vs healthy controls in fetal liver.
A, B, C) Volcano plots depicting differential abundance of proteins in IPs with GALD plasma vs healthy plasma in IP1, IP2 and IP3 respectively. Proteins with Benjamin-Hochberg adjusted p-value (FDR) < 0.05 in orange: Ig lambda chain V-II region TOG (P01704) and Carbamoyl-phosphate synthase 1, mitochondrial (CPS1). Proteins with log2 Fold Change > 2 are labeled with gene name or UniProt ID. Plotted below are average log2 Fold Change of proteins present in either GALD plasma or healthy controls and in at least 2 samples and the lower 0.1 percentile of normalized LFQ intensities in the respective IP. Top 5 proteins (Fold Change) are labeled. D) Normalized log2 LFQ intensities of CPS1 in 3 conditions GALD, healthy and PBC plasma in 3 IPs. LogFC = log Fold Change. https://github.com/HenriettaHolze/GALD_proteomics/blob/main/scripts/fetal_liver_tissue_analysis.Rmd.