The neuroprotective E3-ubiquitin ligase CHIP is linked to healthy aging. Here, we present a protocol using a patient-derived iPSC line with a triplication of the α-synuclein gene to produce gene-edited cells isogenic for CHIP. We describe iPSC differentiation into cortical neurons and their identity validation. We then detail mass spectrometry-based approaches (SWATH-MS) to identify dominant changes in the steady state proteome generated by loss of CHIP function. This protocol can be adapted to other proteins that impact proteostasis in neurons. For complete details on the use and execution of this protocol, please refer to Dias et al. (2021).

Institute of Genetics and Cancer University of Edinburgh EH4 2XU Edinburgh UK

International Centre for Cancer Vaccine Science University of Gdańsk ul Kładki 24 80 822 Gdańsk Poland

MRC Centre for Regenerative Medicine Institute for Stem Cell Research School of Biological Sciences The University of Edinburgh EH16 4UU Edinburgh UK

Research Centre for Applied Molecular Oncology Masaryk Memorial Cancer Institute 656 53 Brno Czech Republic

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