Fig 1.
Concept and result of surface protein enrichment in iPSC-derived.
Sensory-like neurons were processed as follows: (A) Matured sensory-like neurons were incubated with sulfo-NHS-SS-biotin solution in vitro. Biotin binds to extracellular portions of plasma membrane proteins, enabling the isolation of surface protein fractions through NeutrAvidin-mediated pulldown after cell lysis. Following this, biotin was cleaved by trypsin digestion, and surfaceome-enriched protein samples were subjected to mass spectrometry analysis. (B) Representative phase-contrast photomicrographs of FD and healthy control sensory-like neurons, taken after six weeks of maturation and before biotin labeling, show dense neuronal networks comprising both clusters and single cells for both genotypes. (C) An Euler diagram of CSPA-based surface protein annotation within the complete set of 1,508 detected proteins, as well as the top 100 most abundant proteins, highlights a distinct enrichment of cell surface proteins. Abbreviations: CSPA = Cell Surface Protein Atlas, FD = Fabry disease, iPSC = induced pluripotent stem cells, NanoLC-MS/MS = liquid chromatography-tandem mass spectrometry.
Fig 2.
Volcano plot of altered surfaceome in FD sensory-like neurons normalized to a healthy control line.
Samples from three independent differentiations, pairwise analyzed in independent proteomics experiments, were included for each group. Adjusted p-values were calculated using the R package “limma.” Dotted horizontal and vertical lines represent the p-value and LFQ intensity ratio thresholds, respectively. In FD sensory-like neurons, 86 proteins were upregulated, while 12 were downregulated compared to healthy controls. Abbreviations: FD = Fabry disease, LFQ = label-free quantitation.
Fig 3.
qPCR analysis of CACNA2D3, GPM6A, EGFR, and ABCA7 gene expression in iPSC-derived sensory-like neurons.
All four targets with potential relevance to FD pathology exhibited expression at the mRNA level, with no significant differences found between FD and control neurons (nFD = 4, nControl = 4 independent differentiations). Data is shown as normalized fold change with regard to one of the control line values that was chosen as the calibrator for each gene assessed, respectively. Abbreviations: ABCA7 = ATP-binding cassette subfamily A member 7; CACNA2D3 = calcium voltage-gated channel auxiliary subunit alpha2delta 3; EGFR = epidermal growth factor receptor; FD = Fabry disease; GPM6A = neuronal membrane glycoprotein M6-A; iPSC = induced pluripotent stem cells; qPCR = quantitative polymerase chain reaction.
Fig 4.
Protein-protein-interaction network generated from the deregulated surface proteins with STRING database.
The network revealed 38 connections, exceeding the expected number of 10 (average interaction degree: 1.58). K-means clustering was conducted with a pre-defined cluster number of five. A detailed description of these interactions is provided in S6 Table. Abbreviations: FD = Fabry disease, STRING = Search Tool for the Retrieval of Interacting Genes.