Limits...
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.

Show MeSH

Related in: MedlinePlus

Qualitative analysis of identified hair cell markers by immunocytochemistry.Shown are cross sections of E18 chicken utricles (A and C) and transverse projections of utricle whole mounts (B and D). (A, B) Co-immunolabeling of the identified hair cell markers otoferlin (green), myosin VIIA (red), and Sox2 (blue), which is detectable in hair cells and supporting cells. (C, D) Co-immunolabeling with antibodies to otoferlin (green), the identified hair cell marker AIFM1 (red), and Sox2 (blue).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3672136&req=5

pone-0066026-g007: Qualitative analysis of identified hair cell markers by immunocytochemistry.Shown are cross sections of E18 chicken utricles (A and C) and transverse projections of utricle whole mounts (B and D). (A, B) Co-immunolabeling of the identified hair cell markers otoferlin (green), myosin VIIA (red), and Sox2 (blue), which is detectable in hair cells and supporting cells. (C, D) Co-immunolabeling with antibodies to otoferlin (green), the identified hair cell marker AIFM1 (red), and Sox2 (blue).

Mentions: Not surprisingly, we identified a number of proteins in hair cells and non-sensory cells that previously were known markers for these cell types. Otoferlin for example, is a known hair cell protein [26], [35] that was identified in our analysis as highly enriched in hair cells after Fisher exact analysis. Monoclonal antibody staining confirmed that otoferlin is detectable in E18 by chicken utricular hair cells, co-labeled with antibodies to myosin VIIA, and that otoferlin immunolabeling is absent from non-sensory cells (Fig. 7A,B). We used Sox2 immunostaining to distinguish sensory epithelium cells from mesenchymal stromal cells. In the E18 chicken utricle, Sox2 protein is expressed by supporting cells and hair cells (Fig. 7A,B). We also confirmed hair cell expression of the mitochondrial protein apoptosis-inducing factor 1 (AIFM1), which was identified by our mass spectrometry analysis as hair cell only protein (Fig. 7C,D). The protein was, however, also detectable albeit with lower intensity in non-sensory cells. This result revealed, as previously discussed, a limitation of the comparative analyses that is a general lack of sensitivity for proteins that are not highly abundant. AIFM1, for example appears to be strongly enriched in hair cells and was identified via two independent peptides in one of the mass spectrometry experiments (Table 1). The protein was not detected by mass spectrometry in the non-hair cell fraction. Immunolabeling revealed a clear difference in staining intensity between hair cells and non-sensory cells, highlighting the differential expression of AIFM1 in these two cell types, but it also demonstrated expression of AIFM1 in non-sensory and supporting cells. This result shows that absence of detection of a protein in mass spectrometry does not mean that the protein is not present. Mass spectrometry has detection limits, which has been elegantly shown and discussed in a recent quantitative study of hair bundle proteins [21]. Overall, as reported in these recent results, we also observed that the detection limit for spectra is limited, leveling out at about 104 per mass spectrometry run in the best cases. Particularly for abundant and large proteins, such a detection limit is not a big problem because the statistical likelihood that these proteins are represented by multiple peptides in a single run is quite high. For smaller proteins that are less abundant, the limit of detection might not be reached in a single run. In addition, it is reasonable to presume that simple biochemical features also limit the representation of certain groups of proteins – for example globular cytoplasmic versus membrane-spanning proteins, or detergent solubility, charge, protein degradation sensitivity, etc. For better representation and less variability, a substantial increase of the detection limit and methods for exclusion of abundant proteins would probably be the most efficient means.


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)

Qualitative analysis of identified hair cell markers by immunocytochemistry.Shown are cross sections of E18 chicken utricles (A and C) and transverse projections of utricle whole mounts (B and D). (A, B) Co-immunolabeling of the identified hair cell markers otoferlin (green), myosin VIIA (red), and Sox2 (blue), which is detectable in hair cells and supporting cells. (C, D) Co-immunolabeling with antibodies to otoferlin (green), the identified hair cell marker AIFM1 (red), and Sox2 (blue).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0066026-g007: Qualitative analysis of identified hair cell markers by immunocytochemistry.Shown are cross sections of E18 chicken utricles (A and C) and transverse projections of utricle whole mounts (B and D). (A, B) Co-immunolabeling of the identified hair cell markers otoferlin (green), myosin VIIA (red), and Sox2 (blue), which is detectable in hair cells and supporting cells. (C, D) Co-immunolabeling with antibodies to otoferlin (green), the identified hair cell marker AIFM1 (red), and Sox2 (blue).
Mentions: Not surprisingly, we identified a number of proteins in hair cells and non-sensory cells that previously were known markers for these cell types. Otoferlin for example, is a known hair cell protein [26], [35] that was identified in our analysis as highly enriched in hair cells after Fisher exact analysis. Monoclonal antibody staining confirmed that otoferlin is detectable in E18 by chicken utricular hair cells, co-labeled with antibodies to myosin VIIA, and that otoferlin immunolabeling is absent from non-sensory cells (Fig. 7A,B). We used Sox2 immunostaining to distinguish sensory epithelium cells from mesenchymal stromal cells. In the E18 chicken utricle, Sox2 protein is expressed by supporting cells and hair cells (Fig. 7A,B). We also confirmed hair cell expression of the mitochondrial protein apoptosis-inducing factor 1 (AIFM1), which was identified by our mass spectrometry analysis as hair cell only protein (Fig. 7C,D). The protein was, however, also detectable albeit with lower intensity in non-sensory cells. This result revealed, as previously discussed, a limitation of the comparative analyses that is a general lack of sensitivity for proteins that are not highly abundant. AIFM1, for example appears to be strongly enriched in hair cells and was identified via two independent peptides in one of the mass spectrometry experiments (Table 1). The protein was not detected by mass spectrometry in the non-hair cell fraction. Immunolabeling revealed a clear difference in staining intensity between hair cells and non-sensory cells, highlighting the differential expression of AIFM1 in these two cell types, but it also demonstrated expression of AIFM1 in non-sensory and supporting cells. This result shows that absence of detection of a protein in mass spectrometry does not mean that the protein is not present. Mass spectrometry has detection limits, which has been elegantly shown and discussed in a recent quantitative study of hair bundle proteins [21]. Overall, as reported in these recent results, we also observed that the detection limit for spectra is limited, leveling out at about 104 per mass spectrometry run in the best cases. Particularly for abundant and large proteins, such a detection limit is not a big problem because the statistical likelihood that these proteins are represented by multiple peptides in a single run is quite high. For smaller proteins that are less abundant, the limit of detection might not be reached in a single run. In addition, it is reasonable to presume that simple biochemical features also limit the representation of certain groups of proteins – for example globular cytoplasmic versus membrane-spanning proteins, or detergent solubility, charge, protein degradation sensitivity, etc. For better representation and less variability, a substantial increase of the detection limit and methods for exclusion of abundant proteins would probably be the most efficient means.

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