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Human induced pluripotent stem cells (iPSCs) can be differentiated into motor neurons (MNs), offering valuable models for studying neurodegenerative diseases such as ALS and SMA. While single-cell RNA sequencing has revealed considerable cellular heterogeneity, it cannot capture the full complexity of the proteome – and bulk proteomic methods mask cell-to-cell variation. The relationship between mRNA and protein abundance in individual cells, particularly during neuronal differentiation, remains poorly understood.
To address this, Zhemkov et al. applied next-generation single-cell proteomics (MS-SCP – an exploratory, global protein identification approach) to characterise proteomic heterogeneity across iPSC states and throughout a 60-day MN differentiation protocol. Single cells were isolated by fluorescence-activated cell sorting and analysed using label-free MS-SCP with parallelised nanoflow dual-trap single-column liquid chromatography (nano-DTSC). Peptide separation was performed on an Aurora® Rapid™ 5 cm ×75 μm CSI C18 UHPLC column, interfaced with a timsTOF Ultra 2 via CaptiveSpray ion source, delivering approximately 20% greater sensitivity over the previous platform and a ~175% increase in detection of low-abundance unique proteins.
Leveraging this workflow, the researchers from the Svendsen lab demonstrated that iPSC proteomic heterogeneity is linked to cell cycle stage, with pluripotency markers such as Oct4 peaking in G1 phase. They further revealed that mRNA-protein discordance increases substantially as cells commit to a MN fate, with the most dramatic divergence occurring during the neurosphere-to-MN transition – a finding invisible to transcriptomics alone.
These results suggest that proteomic regulation during neuronal differentiation is far more complex than transcriptomic data implies, with important implications for disease modelling and the development of cell-based therapies for neurodegenerative conditions.
Publication
bioRxiv
Authors
Vladimir A. Zhemkov, Aleksandra Binek, Ali Haghani, Edo Israely, Shaughn Bell, Alba Sansa, George Lawless, Jennifer Van Eyk, & Clive N. Svendsen;
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