MSACL 2023 Abstract
Self-Classified Topic Area(s): Proteomics > Emerging Technologies
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Podium Presentation in Steinbeck 2 on Thursday at 17:10 (Chair: Noortje de Haan / Xueheng Zhao)
 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
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John Yates, PhD (Presenter)
Scripps Research Institute |
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Presenter Bio: John R. Yates is the Ernest W. Hahn Professor in the Departments of Molecular Medicine and Neurobiology at The Scripps Research Institute. 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, and the 2019 John B. Fenn Distinguished Contribution to Mass Spectrometry award from the ASMS. 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 Thomson Reuters 2015 List of Highly Cited Scientists. He has published over 950 scientific articles with >125,000 citations, and an H index of 174 (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. |
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Abstract 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. |
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Financial Disclosure
| Description | Y/N | Source |
| Grants | no | |
| Salary | yes | Cambridge Isotope Labs, 908 Devices, OMASS |
| Board Member | yes | Partnership for Clean Competition |
| Stock | yes | Yatiri Bio |
| Expenses | no | |
| IP Royalty | yes | SEQUEST license U Washington |
| Planning to mention or discuss specific products or technology of the company(ies) listed above: |
no |
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= Expected to be clinically available in 1 to 4 years. (39.50%, 2023)
= Clinically available now. (36.13%, 2023)
