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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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Shotgun proteomic analysis of isolated hair cells and non-sensory cells.(A) False discovery rates (FDR) at both protein and peptide levels were applied to filter the data to a representative number of PSMs, which result in the number of proteins identified. The majority of proteins were represented by more than one PSM. (B) Demonstration of a MSMS peptide spectral match (PSM) of the peptide ETLYGQEIDQASFLTILK from the protein otolin-1 (OTOL1). The peptide was identified with better than 2 parts-per-million (ppm) mass accuracy, where the experimental mass/charge (mz) was 1035.0452 Da and the theoretical 1035.0437 Da. The annotated b and y ions are indicative of peptide backbone bond cleavage between carbonyl carbon and nitrogen, whereas in the case of the amino-terminus, b-ions result, and for the carboxyl-terminus, y-ions. OTOL1 was only identified in the AM1-43-low cell fraction (see Fig. 3C), and suggests that this protein is present in non-sensory cells, presumably supporting cells, and not in hair cells. (C) Venn diagram displaying the number of proteins unique to hair cells (64), non-sensory cells (104) and the number of proteins shared between the two cell types (467) at the stringent filter setting of requiring at least 2 different peptides per protein. This corresponds to the FDR settings shown in the third line of the table shown in (A).
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pone-0066026-g004: Shotgun proteomic analysis of isolated hair cells and non-sensory cells.(A) False discovery rates (FDR) at both protein and peptide levels were applied to filter the data to a representative number of PSMs, which result in the number of proteins identified. The majority of proteins were represented by more than one PSM. (B) Demonstration of a MSMS peptide spectral match (PSM) of the peptide ETLYGQEIDQASFLTILK from the protein otolin-1 (OTOL1). The peptide was identified with better than 2 parts-per-million (ppm) mass accuracy, where the experimental mass/charge (mz) was 1035.0452 Da and the theoretical 1035.0437 Da. The annotated b and y ions are indicative of peptide backbone bond cleavage between carbonyl carbon and nitrogen, whereas in the case of the amino-terminus, b-ions result, and for the carboxyl-terminus, y-ions. OTOL1 was only identified in the AM1-43-low cell fraction (see Fig. 3C), and suggests that this protein is present in non-sensory cells, presumably supporting cells, and not in hair cells. (C) Venn diagram displaying the number of proteins unique to hair cells (64), non-sensory cells (104) and the number of proteins shared between the two cell types (467) at the stringent filter setting of requiring at least 2 different peptides per protein. This corresponds to the FDR settings shown in the third line of the table shown in (A).

Mentions: For the interpretation of the resulting mass spectrometry based datasets, refined statistical search strategies that mitigate and control incorrect peptide identifications were used. Specifically, we utilized concatenated target-decoy database searches, which employ a strategy using composite protein target sequence databases and decoy sequences that comprise the reversed target sequences to estimate false positive identification rates for individual peptide-spectral matches (PSMs) [18]. With this method, it is possible to distinguish between correctly identified spectra, which should be derived solely or mainly from target sequences, and incorrectly identified PSMs, which should be traceable more or less in equal proportions to target and decoy sequences. Based on the resulting hits, a false discovery rate (FDR) was generated for each PSM dataset, which we used to filter matching PSMs (Fig. 4A).


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)

Shotgun proteomic analysis of isolated hair cells and non-sensory cells.(A) False discovery rates (FDR) at both protein and peptide levels were applied to filter the data to a representative number of PSMs, which result in the number of proteins identified. The majority of proteins were represented by more than one PSM. (B) Demonstration of a MSMS peptide spectral match (PSM) of the peptide ETLYGQEIDQASFLTILK from the protein otolin-1 (OTOL1). The peptide was identified with better than 2 parts-per-million (ppm) mass accuracy, where the experimental mass/charge (mz) was 1035.0452 Da and the theoretical 1035.0437 Da. The annotated b and y ions are indicative of peptide backbone bond cleavage between carbonyl carbon and nitrogen, whereas in the case of the amino-terminus, b-ions result, and for the carboxyl-terminus, y-ions. OTOL1 was only identified in the AM1-43-low cell fraction (see Fig. 3C), and suggests that this protein is present in non-sensory cells, presumably supporting cells, and not in hair cells. (C) Venn diagram displaying the number of proteins unique to hair cells (64), non-sensory cells (104) and the number of proteins shared between the two cell types (467) at the stringent filter setting of requiring at least 2 different peptides per protein. This corresponds to the FDR settings shown in the third line of the table shown in (A).
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Related In: Results  -  Collection

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pone-0066026-g004: Shotgun proteomic analysis of isolated hair cells and non-sensory cells.(A) False discovery rates (FDR) at both protein and peptide levels were applied to filter the data to a representative number of PSMs, which result in the number of proteins identified. The majority of proteins were represented by more than one PSM. (B) Demonstration of a MSMS peptide spectral match (PSM) of the peptide ETLYGQEIDQASFLTILK from the protein otolin-1 (OTOL1). The peptide was identified with better than 2 parts-per-million (ppm) mass accuracy, where the experimental mass/charge (mz) was 1035.0452 Da and the theoretical 1035.0437 Da. The annotated b and y ions are indicative of peptide backbone bond cleavage between carbonyl carbon and nitrogen, whereas in the case of the amino-terminus, b-ions result, and for the carboxyl-terminus, y-ions. OTOL1 was only identified in the AM1-43-low cell fraction (see Fig. 3C), and suggests that this protein is present in non-sensory cells, presumably supporting cells, and not in hair cells. (C) Venn diagram displaying the number of proteins unique to hair cells (64), non-sensory cells (104) and the number of proteins shared between the two cell types (467) at the stringent filter setting of requiring at least 2 different peptides per protein. This corresponds to the FDR settings shown in the third line of the table shown in (A).
Mentions: For the interpretation of the resulting mass spectrometry based datasets, refined statistical search strategies that mitigate and control incorrect peptide identifications were used. Specifically, we utilized concatenated target-decoy database searches, which employ a strategy using composite protein target sequence databases and decoy sequences that comprise the reversed target sequences to estimate false positive identification rates for individual peptide-spectral matches (PSMs) [18]. With this method, it is possible to distinguish between correctly identified spectra, which should be derived solely or mainly from target sequences, and incorrectly identified PSMs, which should be traceable more or less in equal proportions to target and decoy sequences. Based on the resulting hits, a false discovery rate (FDR) was generated for each PSM dataset, which we used to filter matching PSMs (Fig. 4A).

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