Quantitative measurement of young human eye lens crystallins by direct injection Fourier transform ion cyclotron resonance mass spectrometry.
Quantitative measurement of young human eye lens crystallins by direct injection Fourier transform ion cyclotron resonance mass spectrometry.
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发表时间：
2006-06
期刊：
影响因子：
2.2
通讯作者：
N. Robinson;K. Lampi;J. Speir;G. Kruppa;M. Easterling;A. B. Robinson
中科院分区：
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作者：
N. Robinson;K. Lampi;J. Speir;G. Kruppa;M. Easterling;A. B. Robinson
人类出生时的晶状体主要由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20，000和30，000 Da之间变化。这些分子的相对量及其合成后随年龄的变化在透镜生物化学和透镜病理学的研究中具有重要意义。未分级透镜匀浆的傅立叶变换质谱法现在允许精确定量测量透镜晶体蛋白的相对量。本文报道了2 ~ 300日龄人10对晶状体中14种晶体蛋白的测定结果。方法眼晶状体从不同年龄的人类供体在生命的第一年获得。将这些晶状体在含有0.001 M EDTA的pH 7.0的0.02 M磷酸盐缓冲液中均质化，通过在3，000 Da过滤器上洗涤脱盐，并直接注入配备有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的纳米喷雾源中。晶体蛋白在质谱仪的ICR池中定量电离和质量分析。将每种晶状体蛋白的所有同位素和电荷状态物质的检测信号归一化并求和以确定蛋白质量。结果14种晶体蛋白在出生时的相对含量在个体间差异不显著。这些量彼此呈整数比，这表明透镜内的重要结构关系。在两种情况下，α A-和β B2-晶状体蛋白的相对量在第一年期间与年龄的对数成比例地变化，其中α A-晶状体蛋白减少而β B2-晶状体蛋白增加。α A-和β B2-晶状体蛋白的变化相互抵消，α A-晶状体蛋白从30%降低到18%，β B2-从12%增加到24%。结论人出生时的透镜是由晶体蛋白构成的，晶体蛋白分子的数目之间存在有规律的积分关系。随着透镜在第一年的发展，其中一些关系发生了变化。虽然α A-晶状体蛋白的相互减少和β B2-晶状体蛋白的增加的功能意义尚不清楚，但β B2-晶状体蛋白可能在出生后一年内合成的透镜结构中取代α A-晶状体蛋白。直接进样透镜的傅立叶变换离子回旋共振质谱法是一种很好的定量测定透镜晶体蛋白的方法。
PURPOSE 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. METHODS 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. RESULTS 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%. CONCLUSIONS 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.