Age-related changes in the spatial distribution of human lens alpha-crystallin products by MALDI imaging mass spectrometry.
Age-related changes in the spatial distribution of human lens alpha-crystallin products by MALDI imaging mass spectrometry.
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DOI：
10.1167/iovs.09-3522
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发表时间：
2009-09
影响因子：
4.4
通讯作者：
Schey KL
中科院分区：
文献类型：
--
作者：
Grey AC;Schey KL
建立基质辅助激光解吸电离（MALDI）成像质谱法，用于绘制人透镜组织中α-晶状体蛋白及其修饰形式的分布与透镜年龄和白内障的关系。将冷冻的人晶状体沿赤道和轴向冷冻切片成20 μ m厚的切片，并通过甲醇软着陆将切片固定在导电载玻片上。乙醇洗涤程序促进了均匀的矩阵晶体形成的两步矩阵沉积程序，以产生高质量的质谱数据。从正常和白内障透镜切片中以100 μm步长获得的质谱数据中获得修饰和未修饰α-晶状体蛋白亚基的分子图像。从透镜切片中提取的蛋白质用内切蛋白酶Glu-C消化，并进行质谱分析以鉴定修饰。完整的α-晶状体蛋白信号主要在29岁以下的晶状体的外皮质纤维细胞中检测到。观察到α-晶状体蛋白的多个截短产物，其丰度随着进入透镜的距离和透镜年龄的增加而增加。磷酸化α B-晶状体蛋白形式在老年晶状体的皮质区最丰富。在轴向切片中，未观察到显著的前后极变异。在年龄匹配的白内障患者透镜中检测到一种以前未报道的α A-晶状体蛋白突变。本研究建立了一种利用MALDI成像质谱分析人类透镜α-晶体蛋白随年龄变化的方法，包括白内障透镜中一种新的L52 F α A-晶体蛋白突变。这种空间分辨蛋白质组学技术在透镜生物学中的应用增强了对老化和患病人类晶状体中α-晶状体蛋白质加工的理解。
To develop a protocol for MALDI (matrix-assisted laser desorption ionization) imaging mass spectrometry for mapping the distributions of α-crystallin and its modified forms in human lens tissue as a function of lens age and cataract. Frozen human lenses were cryosectioned equatorially and axially into 20-μm-thick sections, and the sections were mounted onto conductive glass slides by methanol soft-landing. An ethanol washing procedure facilitated uniform matrix crystal formation by a two-step matrix deposition procedure to produce high-quality mass spectral data. Molecular images of modified and unmodified α-crystallin subunits were obtained from mass spectral data acquired in 100-μm steps across normal and cataractous lens sections. Proteins extracted from the lens sections were digested with endoproteinase Glu-C and subjected to mass spectrometric analysis for identification of modifications. Intact α-crystallin signals were detected primarily in the outer cortical fiber cells in lenses up to 29 years of age. Multiple truncation products were observed for α-crystallin that increased in abundance, both with distance into the lens and with lens age. Phosphorylated αB-crystallin forms were most abundant in the cortical region of older lenses. In axial sections, no significant anterior–posterior pole variation was observed. A previously unreported αA-crystallin mutation was detected in an age-matched cataractous human lens. A method has been developed to spatially map the age-related changes of human lens α-crystallin by MALDI imaging mass spectrometry including a novel L52F αA-crystallin mutation in a cataractous lens. Application of this spatially resolved proteomic technique to lens biology enhances the understanding of α-crystallin protein processing in aging and diseased human lenses.
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影响因子：
5.6
作者：
Ahmad, Md. Falz;Raman, Bakthisaran;Rao, Ch. Mohan
通讯作者：
Rao, Ch. Mohan
影响因子：
4.8
作者：
Hayes, Victoria H.;Devlin, Glyn;Quinlan, Roy A.
通讯作者：
Quinlan, Roy A.
影响因子：
2
作者：
IFEANYI, F;TAKEMOTO, L
通讯作者：
TAKEMOTO, L
影响因子：
4.1
作者：
Kumar, P. Anil;Kumar, M. Satish;Reddy, G. Bhanuprakash
通讯作者：
Reddy, G. Bhanuprakash
影响因子：
3.4
作者：
Hanson, SRA;Hasan, A;Smith, JB
通讯作者：
Smith, JB