MSACL 2023 Abstract

Combined Single Neuron Patch-Clamp/Mass Spectrometry (PatchC-MS) Analyses

Jolene K. Diedrich, Matt Albertolle, Nima Dolatabadi, Swagata Ghatak, Maria Talantova, Stuart A. Lipton, Larry Rodriguez, Marisa Roberto, John R. Yates 3rd
The Scripps Research Institute, LaJolla, CA 92037

John Yates, PhD (Presenter) Scripps Research Institute

Presenter Bio: John R. Yates is the Ernest W. Hahn Professor in the Departments of Molecular Medicine and Neurobiology at The Scripps Research Institute. He received a B.A in Zoology and an M.S. in Chemistry from the University of Maine at Orono. He obtained his Ph.D. in Chemistry at the University of Virginia in the laboratory of Donald F. Hunt with a dissertation entitled Protein Sequencing by Tandem Mass Spectrometry. He performed postdoctoral research in the laboratory of Leroy E. Hood at California Institute of Technology. At the University of Washington, he obtained the rank of Associate Professor with tenure before moving to The Scripps Research Institute in LaJolla, CA. His research interests include development of integrated methods for tandem mass spectrometry analysis of protein mixtures, bioinformatics using mass spectrometry data, and biological studies involving proteomics. He is the lead inventor of the SEQUEST software for correlating tandem mass spectrometry data to sequences in the database and developer of the shotgun proteomics technique for the analysis of protein mixtures. His laboratory has developed the use of proteomic techniques to analyze protein complexes, posttranslational modifications, organelles and quantitative analysis of protein expression for the discovery of new biology. Many proteomic approaches developed by Yates have become a national and international resource to many investigators in the scientific community. He has received the American Society for Mass Spectrometry research award, the Pehr Edman Award in Protein Chemistry, the American Society for Mass Spectrometry Biemann Medal, the HUPO Distinguished Achievement Award in Proteomics, Herbert Sober Award from the ASBMB, and the Christian Anfinsen Award from The Protein Society, the 2015 ACS’s Analytical Chemistry award, 2015 The Ralph N. Adams Award in Bioanalytical Chemistry, the 2018 Thomson Medal from the International Mass Spectrometry Society, the 2019 John B. Fenn Distinguished Contribution to Mass Spectrometry award from the ASMS, the 2019 HUPO Award in Discovery, and the 2024 Pittsburgh Society Award in Analytical Chemistry. He was ranked by Citation Impact, Science Watch as one of the Top 100 Chemists for the decade, 2000-2010. He was #1 on a List of Most Influential in Analytical Chemistry compiled by The Analytical Scientist 10/30/2013 and is on the List of Most Highly Influential Biomedical Researchers, 1996-2011 (European J. Clinical Investigation 2013, 43, 1339-1365) and the Clarivate List of Highly Cited Scientists in 2015 and 2019-2024. He has published over 1000 scientific articles with >183,000 citations, and an H index of 211 (Google Scholar). Dr. Yates served as an Associate Editor at Analytical Chemistry for 15 years and is currently the Editor in Chief at the Journal of Proteome Research.

Relevant Financial Disclosures (within past 24 months, reported on Apr 21, 2026) Committee/Board/Advisory Board Partnership for Clean Competition Stock/Bonds Yatiri Bio Salary Cambridge Isotope Labs, 908 Devices, OMASS Royalty / IP / Other Income SEQUEST license U Washington Discussing Products? NO, presenter will NOT present educational content related to products/services of the ineligible company(ies) listed above. Conflict mitigation NOT REQUIRED.

As interest in single-cell analysis increases, performing single cell MS still remains a challenge. Herein we demonstrate patch-clamp electrophysiological recordings of single human iPSC-derived neurons followed by mass spectrometry analysis of the same cell. Human induced pluripotent stem cell (hiPSC)-derived cerebrocortical neurons are evaluated electrophysiologically by whole-cell recordings with a patch electrode capillary. The neuron is then aspirated into the capillary and expelled into a microtube. A simple digestion protocol is performed, and samples are analyzed by mass spectrometry. The single-cell digests are separated by nanoflow UPLC coupled to a Bruker timsTOF or a Thermo Eclipse, both operating in data dependent modes. Whole-cell recordings were performed on Alzheimer’s disease (AD) and isogenic, gene-corrected control (wild-type/WT) hiPSC-derived cerebrocortical neurons. WT neurons of interest were chosen based on their ability to fire action potentials, manifest voltage-gated sodium and potassium currents, and neurotransmitter-mediated postsynaptic currents. We have previously published that AD hiPSC neurons, like those in human AD brain, exhibit enhanced spontaneous action potential frequency, increased voltage gated sodium currents, and increased excitatory postsynaptic current frequency compared to WT neurons (Ghatak et al., eLIFE, 2019). We selected these AD neurons to compare to WT controls for further proteomic analysis. MS data analysis was performed with ProLuCID, Byonic and MSFragger. When injecting half of the contents of a single digested neuron, we were able to identify between 400-2000 proteins per sample. Advances in this methodology are used to perform patch clamping and proteomics analysis on neurons from brain tissue slices. We performed single-cell patch-clamp electrophysiology combined with mass spectrometry proteomic analysis.