Benchmarking In-Cell Proteomics For Profiling Neuroblastoma Cell Differentiation And The Ubiquitin-Proteasome System

ABSTRACT: The human neuroblastoma SH-SY5Y cell line is a widely utilized model for studying neurodegenerative diseases, owing to its ability to differentiate into cells with a neuron-like phenotype. However, a comprehensive understanding of the cellular and molecular mechanisms underpinning the SH-SY5Y cell differentiation and maturation is still lacking from a deep proteomics perspective. We systematically benchmarked an “in-cell proteomics” strategy against the SDS lysate-based processing method and showed superior performance in terms of simplicity, sensitivity and quantitation accuracy while requiring minimal inputs. Next, we employed the in-cell proteomics strategy to characterize SH-SY5Y cells at undifferentiated, partially and terminally differentiated states, respectively. Among over 9,000 proteins quantified in total, we were able to detect marker proteins in neuronal development and integrity, and observed increases in glutamatergic synapse-related proteins and proteins previously reported in mature neurons as well as in differentiated neuroblastoma cells. Lastly, we examined proteins involved in the ubiquitin-proteasome pathway and found stage-specific expression of E3 ubiquitin ligases, deubiquitinases and proteasome subunits, revealing an important role of protein homeostasis in neuroblastoma cell differentiation. In summary, our study presented the first benchmark dataset of neuroblast cells using an in-cell proteomics strategy, and demonstrated its great potential in cataloging neuronal function and disease.

INSTRUMENT(S): Orbitrap Eclipse

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Zhihao Zhuang  

PROVIDER: MSV000097787 | MassIVE | Fri May 02 19:29:00 BST 2025

REPOSITORIES: MassIVE