REFERENCE POINTS FOR COMPARISONS OF 2-DIMENSIONAL MAPS OF PROTEINS FROM DIFFERENT HUMAN CELL-TYPES DEFINED IN A PH SCALE WHERE ISOELECTRIC POINTS CORRELATE WITH POLYPEPTIDE COMPOSITIONS
REFERENCE POINTS FOR COMPARISONS OF 2-DIMENSIONAL MAPS OF PROTEINS FROM DIFFERENT HUMAN CELL-TYPES DEFINED IN A PH SCALE WHERE ISOELECTRIC POINTS CORRELATE WITH POLYPEPTIDE COMPOSITIONS
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DOI：
10.1002/elps.1150150171
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发表时间：
1994-03-01
期刊：
影响因子：
2.9
通讯作者：
CELIS, JE
中科院分区：
文献类型：
--
作者：
BJELLQVIST, B;BASSE, B;CELIS, JE
一个高度可重复的，商业和非线性，宽范围的固定化pH梯度（IPG）被用来产生二维（2-D）凝胶图的[S-35]蛋氨酸标记的蛋白质从非培养的，未分级的正常人表皮角质形成细胞。在2-D凝胶图中鉴定了41种蛋白质，其为大多数人类细胞类型所共有并记录在人类角质形成细胞2-D凝胶蛋白质数据库中，并使用窄范围IPG测定其等电点（pI）。后者建立了一个pH标度，允许在第一维中使用其他IPG或使用不同的人类蛋白质样本生成的2-D凝胶图之间进行比较。在鉴定的41种蛋白质中，18种蛋白质的子集被定义为适合于评价具有已知组成的多肽的计算pI值与实验pI值之间的相关性。这些蛋白质的计算pI值与实验pI值之间的差异计算方差为0.001 pH单位。通过t检验（配对检验）对相关样本的值进行比较，p水平为0.49，表明计算的pi值与实验pi值之间无显著差异。计算值的精确度取决于蛋白质的缓冲能力，平均而言，随着缓冲能力的增加而提高。如本文所示，不能先验地假设广泛可用的蛋白质序列信息足以计算pI值，因为翻译后修饰，特别是N-末端阻断，会造成主要问题。在本研究中分析的36种蛋白质中，发现18-20种是N-末端封闭的，并且其中只有6种在数据库中如此指示。N-末端阻断的概率取决于N-末端基团的性质。26种蛋白质的N-末端氨基酸为M、S或A，其中17-19种被阻断。只有1/10的蛋白质含有其他N-末端基团被封闭。
A highly reproducible, commercial and nonlinear, wide-range immobilized pH gradient (IPG) was used to generate two-dimensional (2-D) gel maps of [S-35]methionine-labeled proteins from noncultured, unfractionated normal human epidermal keratinocytes. Forty one proteins, common to most human cell types and recorded in the human keratinocyte 2-D gel protein database were identified in the 2-D gel maps and their isoelectric points (pI) were determined using narrow-range IPGs. The latter established a pH scale that allowed comparisons between 2-D gel maps generated either with other IPGs in the first dimension or with different human protein samples. Of the 41 proteins identified, a subset of 18 was defined as suitable to evaluate the correlation between calculated and experimental pI values for polypeptides with known composition. The variance calculated for the discrepancies between calculated and experimental pI values for these proteins was 0.001 pH units. Comparison of the values by the t-test for dependent samples (paired test) gave a p-level of 0.49, indicating that there is no significant difference between the calculated and experimental pi values. The precision of the calculated values depended on the buffer capacity of the proteins, and on average, it improved with increased buffer capacity. As shown here, the widely available information on protein sequences cannot, a priori, be assumed to be sufficient for calculating pI values because post-translational modifications, in particular N-terminal blockage, pose a major problem. Of the 36 proteins analyzed in this study, 18-20 were found to be N-terminally blocked and of these only 6 were indicated as such in databases. The probability of N-terminal blockage depended on the nature of the N-terminal group. Twenty six of the proteins had either M, S or A as N-terminal amino acids and of these 17-19 were blocked. Only 1 in 10 proteins containing other N-terminal groups were blocked.