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Distribution of bovine and rabbit lens alpha-crystallin products by MALDI imaging mass spectrometry.

Grey AC, Schey KL - Mol. Vis. (2008)

Bottom Line: While alphaA-crystallin was extensively degraded in the lens core of both species, rabbit lenses exhibited a greater degree of larger molecular weight truncation products.In contrast, alphaB-crystallin degradation was limited in both species.The localization of multiple degradation products and specific regions of alpha-crystallin phosphorylation in bovine and rabbit lenses gives new insight into the program of lens fiber cell differentiation and normal lens function.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Molecular Pharmacology, Medical University of South Carolina, Charleston, SC 29425-5050, USA.

ABSTRACT

Purpose: To develop a general tissue preparation protocol for MALDI (Matrix-Assisted Laser Desorption Ionization) imaging mass spectrometry of ocular lens tissue, and to compare the spatial distributions of alpha-crystallin and its modified forms in bovine and rabbit lenses.

Methods: Frozen bovine and rabbit lenses were cryosectioned equatorially at -20 degrees C into 12 microm-thick tissue sections. Lens sections were mounted onto conductive glass slides by ethanol soft-landing to maintain tissue integrity. An ethanol/xylene washing procedure was applied to each section before matrix application to facilitate uniform matrix crystal formation across the entire tissue section. Molecular images of both alpha-crystallin subunits and their modified forms were obtained from mass spectral data acquired at 100 microm steps across both whole rabbit and half bovine lens sections.

Results: Distinct spatial patterns for the two subunits of alpha-crystallin and their modified forms were observed in the rabbit and bovine lens sections. While alphaA-crystallin was extensively degraded in the lens core of both species, rabbit lenses exhibited a greater degree of larger molecular weight truncation products. In contrast, alphaB-crystallin degradation was limited in both species. Interestingly, phosphorylation of alphaA- and alphaB-crystallin was most abundant in the middle cortex of both species.

Conclusions: An improved method for investigating the spatial distribution of alpha-crystallin in the ocular lens by MALDI imaging mass spectrometry has been developed. The localization of multiple degradation products and specific regions of alpha-crystallin phosphorylation in bovine and rabbit lenses gives new insight into the program of lens fiber cell differentiation and normal lens function.

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Related in: MedlinePlus

MALDI mass spectra of αA-crystallin truncation in bovine and rabbit lens. Extracted representative mass spectra from the outer cortex (A, D), middle cortex (B, E), and outer nucleus (C, F) of bovine (A-C) and rabbit (D-F) lenses are shown. Degradation products increased toward the nucleus of both species, although higher mass degradation products are more prominent in the rabbit lenses than in the bovine lenses.
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f1: MALDI mass spectra of αA-crystallin truncation in bovine and rabbit lens. Extracted representative mass spectra from the outer cortex (A, D), middle cortex (B, E), and outer nucleus (C, F) of bovine (A-C) and rabbit (D-F) lenses are shown. Degradation products increased toward the nucleus of both species, although higher mass degradation products are more prominent in the rabbit lenses than in the bovine lenses.

Mentions: Under the present experimental conditions, the most abundant ion signals observed in different regions of both bovine and rabbit lenses were for the two subunits of α-crystallin (Figure 1). In the outer and middle cortex regions, both species exhibited similar MALDI spectra. In the bovine lens, the most abundant ion signals in the outer cortex were for αA-crystallin at m/z 19838 and m/z 9922 (predicted [M+H]+=19833, [M+2H]2+=9918; Figure 1A). Similarly, the rabbit lens outer cortex exhibited little αA-crystallin degradation as ion signals for full-length αA-crystallin at m/z 19880 and m/z 9944 (predicted [M+H]+=19880, [M+2H]2+=9942) were most abundant (Figure 1D).


Distribution of bovine and rabbit lens alpha-crystallin products by MALDI imaging mass spectrometry.

Grey AC, Schey KL - Mol. Vis. (2008)

MALDI mass spectra of αA-crystallin truncation in bovine and rabbit lens. Extracted representative mass spectra from the outer cortex (A, D), middle cortex (B, E), and outer nucleus (C, F) of bovine (A-C) and rabbit (D-F) lenses are shown. Degradation products increased toward the nucleus of both species, although higher mass degradation products are more prominent in the rabbit lenses than in the bovine lenses.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2254960&req=5

f1: MALDI mass spectra of αA-crystallin truncation in bovine and rabbit lens. Extracted representative mass spectra from the outer cortex (A, D), middle cortex (B, E), and outer nucleus (C, F) of bovine (A-C) and rabbit (D-F) lenses are shown. Degradation products increased toward the nucleus of both species, although higher mass degradation products are more prominent in the rabbit lenses than in the bovine lenses.
Mentions: Under the present experimental conditions, the most abundant ion signals observed in different regions of both bovine and rabbit lenses were for the two subunits of α-crystallin (Figure 1). In the outer and middle cortex regions, both species exhibited similar MALDI spectra. In the bovine lens, the most abundant ion signals in the outer cortex were for αA-crystallin at m/z 19838 and m/z 9922 (predicted [M+H]+=19833, [M+2H]2+=9918; Figure 1A). Similarly, the rabbit lens outer cortex exhibited little αA-crystallin degradation as ion signals for full-length αA-crystallin at m/z 19880 and m/z 9944 (predicted [M+H]+=19880, [M+2H]2+=9942) were most abundant (Figure 1D).

Bottom Line: While alphaA-crystallin was extensively degraded in the lens core of both species, rabbit lenses exhibited a greater degree of larger molecular weight truncation products.In contrast, alphaB-crystallin degradation was limited in both species.The localization of multiple degradation products and specific regions of alpha-crystallin phosphorylation in bovine and rabbit lenses gives new insight into the program of lens fiber cell differentiation and normal lens function.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Molecular Pharmacology, Medical University of South Carolina, Charleston, SC 29425-5050, USA.

ABSTRACT

Purpose: To develop a general tissue preparation protocol for MALDI (Matrix-Assisted Laser Desorption Ionization) imaging mass spectrometry of ocular lens tissue, and to compare the spatial distributions of alpha-crystallin and its modified forms in bovine and rabbit lenses.

Methods: Frozen bovine and rabbit lenses were cryosectioned equatorially at -20 degrees C into 12 microm-thick tissue sections. Lens sections were mounted onto conductive glass slides by ethanol soft-landing to maintain tissue integrity. An ethanol/xylene washing procedure was applied to each section before matrix application to facilitate uniform matrix crystal formation across the entire tissue section. Molecular images of both alpha-crystallin subunits and their modified forms were obtained from mass spectral data acquired at 100 microm steps across both whole rabbit and half bovine lens sections.

Results: Distinct spatial patterns for the two subunits of alpha-crystallin and their modified forms were observed in the rabbit and bovine lens sections. While alphaA-crystallin was extensively degraded in the lens core of both species, rabbit lenses exhibited a greater degree of larger molecular weight truncation products. In contrast, alphaB-crystallin degradation was limited in both species. Interestingly, phosphorylation of alphaA- and alphaB-crystallin was most abundant in the middle cortex of both species.

Conclusions: An improved method for investigating the spatial distribution of alpha-crystallin in the ocular lens by MALDI imaging mass spectrometry has been developed. The localization of multiple degradation products and specific regions of alpha-crystallin phosphorylation in bovine and rabbit lenses gives new insight into the program of lens fiber cell differentiation and normal lens function.

Show MeSH
Related in: MedlinePlus