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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

Bovine lens α-crystallin phosphorylation. A shows the optical scan of a bovine lens equatorial cryosection before MALDI matrix deposition. B illustrates the distribution of full-length αA-crystallin. C: Singly-phosphorylated full-length αA-crystallin (observed m/z=19920) shows higher abundance in the middle cortex. D: The dual color image shows the relationship between the spatial distributions of full-length αA-crystallin (red) and phosphorylated αA-crystallin (green). E: The distribution of full-length αB-crystallin (residues 1–175, observed m/z=20084) showed higher abundance toward the edge of the lens and in the inner cortex. Singly- phosphorylated (F) and doubly- phosphorylated (G) αB-crystallin (observed m/z=20163 and 20248, respectively) are more abundant in the middle cortex. H: The dual color image shows the relationship between the spatial distributions of full-length αB-crystallin (red) and doubly-phosphorylated αB-crystallin (green). P/2P=phosphorylated forms of protein. Scale bar=2 mm.
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f5: Bovine lens α-crystallin phosphorylation. A shows the optical scan of a bovine lens equatorial cryosection before MALDI matrix deposition. B illustrates the distribution of full-length αA-crystallin. C: Singly-phosphorylated full-length αA-crystallin (observed m/z=19920) shows higher abundance in the middle cortex. D: The dual color image shows the relationship between the spatial distributions of full-length αA-crystallin (red) and phosphorylated αA-crystallin (green). E: The distribution of full-length αB-crystallin (residues 1–175, observed m/z=20084) showed higher abundance toward the edge of the lens and in the inner cortex. Singly- phosphorylated (F) and doubly- phosphorylated (G) αB-crystallin (observed m/z=20163 and 20248, respectively) are more abundant in the middle cortex. H: The dual color image shows the relationship between the spatial distributions of full-length αB-crystallin (red) and doubly-phosphorylated αB-crystallin (green). P/2P=phosphorylated forms of protein. Scale bar=2 mm.

Mentions: The molecular images of unphosphorylated and phosphorylated forms of bovine α-crystallin are presented in Figure 5. Unphosphorylated αA-crystallin was most abundant in a narrow zone at the edge of equatorial lens sections (Figure 5B) while phosphorylated αA-crystallin at m/z 19920 and m/z 9962 (predicted [M+H]+=19914, [M+2H]2+=9959) was most abundant in the lens middle cortex (Figure 5C). Both signals decreased dramatically in the lens nucleus. A dual-color overlay of unphosphorylated (red) and phosphorylated (green) αA-crystallin is presented in Figure 5D. In contrast, unphosphorylated intact αB-crystallin was more ubiquitous in the lens but was most abundant toward the edge and in the outer nucleus (Figure 5E). Both singly- phosphorylated αB-crystallin at m/z 20165 (predicted [M+H]+=20161) and doubly- phosphorylated αB-crystallin at m/z 20243 (predicted [M+H]+=20241) were most abundant in the lens middle cortex and decreased dramatically in the lens nucleus (Figure 5F,G, respectively). Furthermore, the increase in the abundance of phosphorylated αB-crystallin coincided with a relative decrease in unphosphorylated αB-crystallin in the lens middle cortex. A dual-color overlay of unphosphorylated (red) and doubly-phosphorylated (green) αB-crystallin indicates the complementarity of the spatial distribution of phosphorylated and unphosphorylated αB-crystallin (Figure 5H).


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

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

Bovine lens α-crystallin phosphorylation. A shows the optical scan of a bovine lens equatorial cryosection before MALDI matrix deposition. B illustrates the distribution of full-length αA-crystallin. C: Singly-phosphorylated full-length αA-crystallin (observed m/z=19920) shows higher abundance in the middle cortex. D: The dual color image shows the relationship between the spatial distributions of full-length αA-crystallin (red) and phosphorylated αA-crystallin (green). E: The distribution of full-length αB-crystallin (residues 1–175, observed m/z=20084) showed higher abundance toward the edge of the lens and in the inner cortex. Singly- phosphorylated (F) and doubly- phosphorylated (G) αB-crystallin (observed m/z=20163 and 20248, respectively) are more abundant in the middle cortex. H: The dual color image shows the relationship between the spatial distributions of full-length αB-crystallin (red) and doubly-phosphorylated αB-crystallin (green). P/2P=phosphorylated forms of protein. Scale bar=2 mm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f5: Bovine lens α-crystallin phosphorylation. A shows the optical scan of a bovine lens equatorial cryosection before MALDI matrix deposition. B illustrates the distribution of full-length αA-crystallin. C: Singly-phosphorylated full-length αA-crystallin (observed m/z=19920) shows higher abundance in the middle cortex. D: The dual color image shows the relationship between the spatial distributions of full-length αA-crystallin (red) and phosphorylated αA-crystallin (green). E: The distribution of full-length αB-crystallin (residues 1–175, observed m/z=20084) showed higher abundance toward the edge of the lens and in the inner cortex. Singly- phosphorylated (F) and doubly- phosphorylated (G) αB-crystallin (observed m/z=20163 and 20248, respectively) are more abundant in the middle cortex. H: The dual color image shows the relationship between the spatial distributions of full-length αB-crystallin (red) and doubly-phosphorylated αB-crystallin (green). P/2P=phosphorylated forms of protein. Scale bar=2 mm.
Mentions: The molecular images of unphosphorylated and phosphorylated forms of bovine α-crystallin are presented in Figure 5. Unphosphorylated αA-crystallin was most abundant in a narrow zone at the edge of equatorial lens sections (Figure 5B) while phosphorylated αA-crystallin at m/z 19920 and m/z 9962 (predicted [M+H]+=19914, [M+2H]2+=9959) was most abundant in the lens middle cortex (Figure 5C). Both signals decreased dramatically in the lens nucleus. A dual-color overlay of unphosphorylated (red) and phosphorylated (green) αA-crystallin is presented in Figure 5D. In contrast, unphosphorylated intact αB-crystallin was more ubiquitous in the lens but was most abundant toward the edge and in the outer nucleus (Figure 5E). Both singly- phosphorylated αB-crystallin at m/z 20165 (predicted [M+H]+=20161) and doubly- phosphorylated αB-crystallin at m/z 20243 (predicted [M+H]+=20241) were most abundant in the lens middle cortex and decreased dramatically in the lens nucleus (Figure 5F,G, respectively). Furthermore, the increase in the abundance of phosphorylated αB-crystallin coincided with a relative decrease in unphosphorylated αB-crystallin in the lens middle cortex. A dual-color overlay of unphosphorylated (red) and doubly-phosphorylated (green) αB-crystallin indicates the complementarity of the spatial distribution of phosphorylated and unphosphorylated αB-crystallin (Figure 5H).

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