Temporal Proteomic Profiling of SH-SY5Y Differentiation with Retinoic Acid Using FAIMS and Real-Time Searching

Abstract

The SH-SY5Y cell line is often used as a surrogate for neurons in cell-based studies. This cell line is frequently differentiated with all-trans retinoic acid (ATRA) over a 7-day period, which confers neuron-like properties to the cells. However, no analysis of proteome remodeling has followed the progress of this transition. Here, we quantitatively profiled over 9400 proteins across a 7-day treatment with retinoic acid using state-of-the-art mass spectrometry-based proteomics technologies, including FAIMS, real-time database searching, and TMTpro16 sample multiplexing. Gene ontology analysis revealed that categories with the highest increases in protein abundance were related to the plasma membrane/extracellular space. To showcase our data set, we surveyed the protein abundance profiles linked to neurofilament bundle assembly, neuron projections, and neuronal cell body formation. These proteins exhibited increases in abundance level, yet we observed multiple patterns among the queried proteins. The data presented represent a rich resource for investigating temporal protein abundance changes in SH-SY5Y cells differentiated with retinoic acid. Moreover, the sample preparation and data acquisition strategies used here can be readily applied to any analogous cell line differentiation analysis.

Quantitative proteomics and in-cell cross-linking reveal cellular reorganisation during early neuronal differentiation of SH-SY5Y cells

“…Although many studies commonly employ undifferentiated or differentiated SH-SY5Y cells as neuronal model system, the comparative proteome analysis of the various sub-types is still incomplete 3 , 13 . A full proteome analysis, however, enables identification and relative quantification of neuronal marker proteins including changes in protein abundance associated with neuronal differentiation.…”

Neuronal Dynamics and miRNA Signaling Differ between SH-SY5Y APPSwe and PSEN1 Mutant iPSC-Derived AD Models upon Modulation with miR-124 Mimic and Inhibitor

“…In all ways, the expression of a variety of markers of mature neurons makes SH-SY5Y cells useful to investigate several diseases, including AD, and to obtain accurate results that may be translated into in vivo models [ 18 ]. In fact, the evaluation of RA-maturated SH-SY5Y cells by proteomic analysis revealed upregulated proteins that were involved in neuritogenesis [ 19 ]. Transfection with the APP695 Swedish mutant gene did not affect cell morphology and resulted in neurotoxicity [ 20 ].…”

Tris (2,3-Dibromopropyl) Isocyanurate (TDBP-TAZTO or TBC) Shows Different Toxicity Depending on the Degree of Differentiation of the Human Neuroblastoma (SH-SY5Y) Cell Line

“…The cells were seeded in 96-well culture plates at a density of 6 × 10 3 cells per well for 24-h treatment and initially cultured before the experiment for 24 h. Subsequently, the medium was replaced with a fresh one by increasing the concentrations (1, 10, 50, and 100 nM; and 1, 10, 50, and 100 µM) of TDBP-TAZTO for 24 h. The process of SH-SY5Y differentiation was induced by maintaining the cells in DMEM/F12 without phenol red supplemented with 1% FBS and 10 µM all-trans retinoic acid (RA). According to the literature, the minimal time of differentiation of SH-SY5Y is 7 days (Zhang et al 2021). However, the overwhelming majority of papers report that SH-SY5Y cells are not fully differentiated until day 14 (Schneider et al 2011;Shipley et al 2016).…”