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A simple method for purification of vestibular hair cells and non-sensory cells, and application for proteomic analysis.

Herget M, Scheibinger M, Guo Z, Jan TA, Adams CM, Cheng AG, Heller S - PLoS ONE (2013)

Bottom Line: Our conservative analysis identified more than 600 proteins with a false discovery rate of <3% at the protein level and <1% at the peptide level.Analysis of proteins exclusively detected in either population revealed 64 proteins that were specific to hair cells and 103 proteins that were only detectable in non-sensory cells.Statistical analyses extended these groups by 53 proteins that are strongly upregulated in hair cells versus non-sensory cells and vice versa by 68 proteins.

View Article: PubMed Central - PubMed

Affiliation: Department of Otolaryngology - HNS, Stanford University, Stanford, California, USA.

ABSTRACT
Mechanosensitive hair cells and supporting cells comprise the sensory epithelia of the inner ear. The paucity of both cell types has hampered molecular and cell biological studies, which often require large quantities of purified cells. Here, we report a strategy allowing the enrichment of relatively pure populations of vestibular hair cells and non-sensory cells including supporting cells. We utilized specific uptake of fluorescent styryl dyes for labeling of hair cells. Enzymatic isolation and flow cytometry was used to generate pure populations of sensory hair cells and non-sensory cells. We applied mass spectrometry to perform a qualitative high-resolution analysis of the proteomic makeup of both the hair cell and non-sensory cell populations. Our conservative analysis identified more than 600 proteins with a false discovery rate of <3% at the protein level and <1% at the peptide level. Analysis of proteins exclusively detected in either population revealed 64 proteins that were specific to hair cells and 103 proteins that were only detectable in non-sensory cells. Statistical analyses extended these groups by 53 proteins that are strongly upregulated in hair cells versus non-sensory cells and vice versa by 68 proteins. Our results demonstrate that enzymatic dissociation of styryl dye-labeled sensory hair cells and non-sensory cells is a valid method to generate pure enough cell populations for flow cytometry and subsequent molecular analyses.

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Related in: MedlinePlus

Categorization of subcellular localization and predicted cellular function of identified hair cell and non-sensory cell proteins.(A) All 634 identified hair cell and non-sensory cell proteins were classified according to gene ontology annotations and classified according to their subcellular localizations (upper bar graphs, before quantification). Taking into account the spectral counts of each identified protein resulted in slight changes of the distribution (lower bar graphs, after quantification). (B) Display of the gene ontology classifications of predicted cellular functions (upper bar graphs, before quantification) and after taking into account the spectral counts of each identified protein (lower bar graphs, after quantification).
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pone-0066026-g005: Categorization of subcellular localization and predicted cellular function of identified hair cell and non-sensory cell proteins.(A) All 634 identified hair cell and non-sensory cell proteins were classified according to gene ontology annotations and classified according to their subcellular localizations (upper bar graphs, before quantification). Taking into account the spectral counts of each identified protein resulted in slight changes of the distribution (lower bar graphs, after quantification). (B) Display of the gene ontology classifications of predicted cellular functions (upper bar graphs, before quantification) and after taking into account the spectral counts of each identified protein (lower bar graphs, after quantification).

Mentions: To characterize the actual proteins that we identified with our proteomic approach in more detail, we subcategorized all identified proteins according to their annotated subcellular localization (Fig. 5A) and function (Fig. 5B). With respect to all proteins that we detected in hair cells and all proteins detected in non-sensory cells, we found no significant differences in proteome composition (Fig. 5A, before quantification). This was not very surprising because the majority of identified proteins (467) were observed in both cell types (see Fig. 4C). Nearly 50% of all identified proteins were cytoplasmic, 16% were nuclear and 13% were of mitochondrial origin. The residual 21% localized to vesicles, plasma membrane, Golgi apparatus, lysozymes or are secreted proteins. A small portion of proteins was not annotated and could not be assigned to a subcellular localization. Regarding function, the largest fraction (16–18%) of proteins identified in both cell types was found to be involved in energy metabolism, followed by trafficking, signal transduction, protein synthesis and degradation (Fig. 5B, before quantification). 1% of all identified proteins of each cell type function as extracellular matrix proteins.


A simple method for purification of vestibular hair cells and non-sensory cells, and application for proteomic analysis.

Herget M, Scheibinger M, Guo Z, Jan TA, Adams CM, Cheng AG, Heller S - PLoS ONE (2013)

Categorization of subcellular localization and predicted cellular function of identified hair cell and non-sensory cell proteins.(A) All 634 identified hair cell and non-sensory cell proteins were classified according to gene ontology annotations and classified according to their subcellular localizations (upper bar graphs, before quantification). Taking into account the spectral counts of each identified protein resulted in slight changes of the distribution (lower bar graphs, after quantification). (B) Display of the gene ontology classifications of predicted cellular functions (upper bar graphs, before quantification) and after taking into account the spectral counts of each identified protein (lower bar graphs, after quantification).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3672136&req=5

pone-0066026-g005: Categorization of subcellular localization and predicted cellular function of identified hair cell and non-sensory cell proteins.(A) All 634 identified hair cell and non-sensory cell proteins were classified according to gene ontology annotations and classified according to their subcellular localizations (upper bar graphs, before quantification). Taking into account the spectral counts of each identified protein resulted in slight changes of the distribution (lower bar graphs, after quantification). (B) Display of the gene ontology classifications of predicted cellular functions (upper bar graphs, before quantification) and after taking into account the spectral counts of each identified protein (lower bar graphs, after quantification).
Mentions: To characterize the actual proteins that we identified with our proteomic approach in more detail, we subcategorized all identified proteins according to their annotated subcellular localization (Fig. 5A) and function (Fig. 5B). With respect to all proteins that we detected in hair cells and all proteins detected in non-sensory cells, we found no significant differences in proteome composition (Fig. 5A, before quantification). This was not very surprising because the majority of identified proteins (467) were observed in both cell types (see Fig. 4C). Nearly 50% of all identified proteins were cytoplasmic, 16% were nuclear and 13% were of mitochondrial origin. The residual 21% localized to vesicles, plasma membrane, Golgi apparatus, lysozymes or are secreted proteins. A small portion of proteins was not annotated and could not be assigned to a subcellular localization. Regarding function, the largest fraction (16–18%) of proteins identified in both cell types was found to be involved in energy metabolism, followed by trafficking, signal transduction, protein synthesis and degradation (Fig. 5B, before quantification). 1% of all identified proteins of each cell type function as extracellular matrix proteins.

Bottom Line: Our conservative analysis identified more than 600 proteins with a false discovery rate of <3% at the protein level and <1% at the peptide level.Analysis of proteins exclusively detected in either population revealed 64 proteins that were specific to hair cells and 103 proteins that were only detectable in non-sensory cells.Statistical analyses extended these groups by 53 proteins that are strongly upregulated in hair cells versus non-sensory cells and vice versa by 68 proteins.

View Article: PubMed Central - PubMed

Affiliation: Department of Otolaryngology - HNS, Stanford University, Stanford, California, USA.

ABSTRACT
Mechanosensitive hair cells and supporting cells comprise the sensory epithelia of the inner ear. The paucity of both cell types has hampered molecular and cell biological studies, which often require large quantities of purified cells. Here, we report a strategy allowing the enrichment of relatively pure populations of vestibular hair cells and non-sensory cells including supporting cells. We utilized specific uptake of fluorescent styryl dyes for labeling of hair cells. Enzymatic isolation and flow cytometry was used to generate pure populations of sensory hair cells and non-sensory cells. We applied mass spectrometry to perform a qualitative high-resolution analysis of the proteomic makeup of both the hair cell and non-sensory cell populations. Our conservative analysis identified more than 600 proteins with a false discovery rate of <3% at the protein level and <1% at the peptide level. Analysis of proteins exclusively detected in either population revealed 64 proteins that were specific to hair cells and 103 proteins that were only detectable in non-sensory cells. Statistical analyses extended these groups by 53 proteins that are strongly upregulated in hair cells versus non-sensory cells and vice versa by 68 proteins. Our results demonstrate that enzymatic dissociation of styryl dye-labeled sensory hair cells and non-sensory cells is a valid method to generate pure enough cell populations for flow cytometry and subsequent molecular analyses.

Show MeSH
Related in: MedlinePlus