Cargando…

Identification of Kv4.2 protein complex and modifications by tandem affinity purification-mass spectrometry in primary neurons

Proteins usually form complexes to fulfill variable physiological functions. In neurons, communication relies on synapses where receptors, channels, and anchoring proteins form complexes to precisely control signal transduction, synaptic integration, and action potential firing. Although there are m...

Descripción completa

Detalles Bibliográficos
Autores principales: Hu, Jia-Hua, Liu, Ying, Hoffman, Dax A.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9788671/
https://www.ncbi.nlm.nih.gov/pubmed/36568885
http://dx.doi.org/10.3389/fncel.2022.1070305
_version_ 1784858807920230400
author Hu, Jia-Hua
Liu, Ying
Hoffman, Dax A.
author_facet Hu, Jia-Hua
Liu, Ying
Hoffman, Dax A.
author_sort Hu, Jia-Hua
collection PubMed
description Proteins usually form complexes to fulfill variable physiological functions. In neurons, communication relies on synapses where receptors, channels, and anchoring proteins form complexes to precisely control signal transduction, synaptic integration, and action potential firing. Although there are many published protocols to isolate protein complexes in cell lines, isolation in neurons has not been well established. Here we introduce a method that combines lentiviral protein expression with tandem affinity purification followed by mass-spectrometry (TAP-MS) to identify protein complexes in neurons. This protocol can also be used to identify post-translational modifications (PTMs) of synaptic proteins. We used the A-type voltage-gated K(+) channel subunit Kv4.2 as the target protein. Kv4.2 is highly expressed in the hippocampus where it contributes to learning and memory through its regulation of neuronal excitability and synaptic plasticity. We tagged Kv4.2 with the calmodulin-binding-peptide (CBP) and streptavidin-binding-peptide (SBP) at its C-terminus and expressed it in neurons via lentivirus. Kv4.2 was purified by two-step TAP and samples were analyzed by MS. MS identified two prominently known Kv4.2 interacting proteins [dipeptidyl peptidase like (DPPs) and Kv channel-interacting proteins (KChIPs)] in addition to novel synaptic proteins including glutamate receptors, a calcium channel, and anchoring proteins. Co-immunoprecipitation and colocalization experiments validated the association of Kv4.2 with glutamate receptors. In addition to protein complex identification, we used TAP-MS to identify Kv4.2 phosphorylation sites. Several known and unknown phosphorylation sites were identified. These findings provide a novel path to identify protein-protein interactions and PTMs in neurons and shed light on mechanisms of neuronal signaling potentially involved in the pathology of neurological diseases.
format Online
Article
Text
id pubmed-9788671
institution National Center for Biotechnology Information
language English
publishDate 2022
publisher Frontiers Media S.A.
record_format MEDLINE/PubMed
spelling pubmed-97886712022-12-24 Identification of Kv4.2 protein complex and modifications by tandem affinity purification-mass spectrometry in primary neurons Hu, Jia-Hua Liu, Ying Hoffman, Dax A. Front Cell Neurosci Cellular Neuroscience Proteins usually form complexes to fulfill variable physiological functions. In neurons, communication relies on synapses where receptors, channels, and anchoring proteins form complexes to precisely control signal transduction, synaptic integration, and action potential firing. Although there are many published protocols to isolate protein complexes in cell lines, isolation in neurons has not been well established. Here we introduce a method that combines lentiviral protein expression with tandem affinity purification followed by mass-spectrometry (TAP-MS) to identify protein complexes in neurons. This protocol can also be used to identify post-translational modifications (PTMs) of synaptic proteins. We used the A-type voltage-gated K(+) channel subunit Kv4.2 as the target protein. Kv4.2 is highly expressed in the hippocampus where it contributes to learning and memory through its regulation of neuronal excitability and synaptic plasticity. We tagged Kv4.2 with the calmodulin-binding-peptide (CBP) and streptavidin-binding-peptide (SBP) at its C-terminus and expressed it in neurons via lentivirus. Kv4.2 was purified by two-step TAP and samples were analyzed by MS. MS identified two prominently known Kv4.2 interacting proteins [dipeptidyl peptidase like (DPPs) and Kv channel-interacting proteins (KChIPs)] in addition to novel synaptic proteins including glutamate receptors, a calcium channel, and anchoring proteins. Co-immunoprecipitation and colocalization experiments validated the association of Kv4.2 with glutamate receptors. In addition to protein complex identification, we used TAP-MS to identify Kv4.2 phosphorylation sites. Several known and unknown phosphorylation sites were identified. These findings provide a novel path to identify protein-protein interactions and PTMs in neurons and shed light on mechanisms of neuronal signaling potentially involved in the pathology of neurological diseases. Frontiers Media S.A. 2022-12-09 /pmc/articles/PMC9788671/ /pubmed/36568885 http://dx.doi.org/10.3389/fncel.2022.1070305 Text en Copyright © 2022 Hu, Liu and Hoffman. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Cellular Neuroscience
Hu, Jia-Hua
Liu, Ying
Hoffman, Dax A.
Identification of Kv4.2 protein complex and modifications by tandem affinity purification-mass spectrometry in primary neurons
title Identification of Kv4.2 protein complex and modifications by tandem affinity purification-mass spectrometry in primary neurons
title_full Identification of Kv4.2 protein complex and modifications by tandem affinity purification-mass spectrometry in primary neurons
title_fullStr Identification of Kv4.2 protein complex and modifications by tandem affinity purification-mass spectrometry in primary neurons
title_full_unstemmed Identification of Kv4.2 protein complex and modifications by tandem affinity purification-mass spectrometry in primary neurons
title_short Identification of Kv4.2 protein complex and modifications by tandem affinity purification-mass spectrometry in primary neurons
title_sort identification of kv4.2 protein complex and modifications by tandem affinity purification-mass spectrometry in primary neurons
topic Cellular Neuroscience
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9788671/
https://www.ncbi.nlm.nih.gov/pubmed/36568885
http://dx.doi.org/10.3389/fncel.2022.1070305
work_keys_str_mv AT hujiahua identificationofkv42proteincomplexandmodificationsbytandemaffinitypurificationmassspectrometryinprimaryneurons
AT liuying identificationofkv42proteincomplexandmodificationsbytandemaffinitypurificationmassspectrometryinprimaryneurons
AT hoffmandaxa identificationofkv42proteincomplexandmodificationsbytandemaffinitypurificationmassspectrometryinprimaryneurons