Robinson Noah E, Lampi Kirsten J, Speir J Paul, Kruppa Gary, Easterling Michael, Robinson Arthur B
Oregon Institute of Science and Medicine, Cave Junction, OR 97523, USA.
Mol Vis. 2006 Jun 21;12:704-11.
Human eye lenses at birth are primarily constructed of 12 distinct crystallins and two truncated crystallins. The molecular weights of these 14 proteins vary between about 20,000 and 30,000 Da. The relative amounts of these molecules and their post-synthetic changes with age are of substantial interest in the study of lens biochemistry and lens pathology. Fourier transform mass spectrometry of unfractionated lens homogenates now permits precise quantitative measurement of the relative amounts of lens crystallins. We report herein the measurement of the 14 crystallins in 10 pairs of lenses from humans between the ages of 2 and 300 days.
Eye lenses were obtained from human donors of various ages in the first year of life. These lenses were homogenized in 0.02 M phosphate buffer at pH 7.0 with 0.001 M EDTA, desalted by washing over a 3,000 Da filter, and injected directly into the nanospray source of a hybrid Fourier transform ion cyclotron resonance mass spectrometer, Qq-FT(ICR)MS, equipped with a 12 Tesla magnet. The crystallins were quantitatively ionized and mass analyzed in the ICR cell of the mass spectrometer. The detected signals of all of the isotopic and charge state species for each crystallin were normalized and summed to determine the protein quantities.
The relative amounts of the 14 crystallins are found to be quite similar from individual to individual at birth. These amounts are in integer ratios to one another that suggest important structural relations within the lens. In two cases, the relative amounts of alphaA- and betaB2-crystallin change proportionally to the logarithm of age during the first year, with alphaA- decreasing and betaB2-crystallin increasing. The changes in alphaA- and betaB2-crystallin are mutually offsetting, with alphaA-crystallin decreasing from 30% to 18% and betaB2-increasing from 12% to 24%.
These observations suggest that the human eye lens at birth is constructed of crystallins in which the numbers of crystallin molecules have regular integral relationships to each other. As the lens develops during the first year, some of these relationships change. While the functional significance of the reciprocal decrease in alphaA- and increase in betaB2-crystallin is not known, betaB2-crystallin may substitute for alphaA-crystallin in the lens structures synthesized during the year after birth. Direct injection FT(ICR)MS of unfractionated lens was found to be an excellent method for the quantitative measurement of lens crystallins.
人出生时的眼晶状体主要由12种不同的晶状体蛋白和两种截短的晶状体蛋白构成。这14种蛋白质的分子量在约20,000至30,000道尔顿之间变化。这些分子的相对含量及其随年龄的合成后变化在晶状体生物化学和晶状体病理学研究中具有重大意义。未分级晶状体匀浆的傅里叶变换质谱现在允许精确地定量测量晶状体蛋白的相对含量。我们在此报告了对2至300天大的人类10对晶状体中14种晶状体蛋白的测量。
从出生后第一年不同年龄的人类供体获取眼晶状体。将这些晶状体在含0.001 M乙二胺四乙酸（EDTA）的pH 7.0的0.02 M磷酸盐缓冲液中匀浆，通过3,000道尔顿滤膜洗涤进行脱盐，然后直接注入配备12特斯拉磁体的混合傅里叶变换离子回旋共振质谱仪Qq-FT(ICR)MS的纳喷雾源中。晶状体蛋白在质谱仪的离子回旋共振池中进行定量离子化和质量分析。对每种晶状体蛋白的所有同位素和电荷态物种的检测信号进行归一化并求和以确定蛋白质含量。
发现14种晶状体蛋白的相对含量在出生时个体之间非常相似。这些含量彼此呈整数比例，表明晶状体内部存在重要的结构关系。在两种情况下，αA-晶状体蛋白和βB2-晶状体蛋白的相对含量在出生后的第一年中与年龄的对数成比例变化，αA-晶状体蛋白减少而βB2-晶状体蛋白增加。αA-晶状体蛋白和βB2-晶状体蛋白的变化相互抵消，αA-晶状体蛋白从30%降至18%，βB2-晶状体蛋白从12%增至24%。
这些观察结果表明，人出生时的眼晶状体由晶状体蛋白构成，其中晶状体蛋白分子数量彼此具有规则的整数关系。随着晶状体在出生后的第一年中发育，其中一些关系发生变化。虽然αA-晶状体蛋白减少和βB2-晶状体蛋白增加的相互功能意义尚不清楚，但βB2-晶状体蛋白可能在出生后一年合成的晶状体结构中替代αA-晶状体蛋白。发现未分级晶状体的直接注入FT(ICR)MS是定量测量晶状体蛋白的一种出色方法。
