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

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.