Relative quantitation of peptides in wild-type and Cpefat/fat mouse pituitary using stable isotopic tags and mass spectrometry
Relative quantitation of peptides in wild-type and Cpefat/fat mouse pituitary using stable isotopic tags and mass spectrometry
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DOI：
10.1002/jms.742
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发表时间：
2005-02-01
影响因子：
2.3
通讯作者：
Fricker, LD
中科院分区：
文献类型：
--
作者：
Che, FY;Biswas, R;Fricker, LD
Cpe（fat/fat）小鼠在羧基肽酶E基因的编码区有一个点突变，使该酶失活。结果，这些小鼠降低了几种神经肽的水平，并大大增加了含有c端碱性残基的肽加工中间体的水平。然而，先前的研究只检测了相对少量的神经肽。在本研究中，我们使用稳定同位素标记的定量肽组学方法来检测CPe（脂肪/脂肪）小鼠相对于野生型小鼠的垂体肽水平。突变型和野生型小鼠的垂体提取物用含有9个氢原子或氘原子的稳定同位素标记[3-（2,5-二氧吡咯烷-1-酰基羰基）丙基]三甲基氯化铵进行标记。然后，将两种样品混合使用液相色谱/质谱（LC/MS）进行分析。肽的相对丰度是通过比较重峰和轻峰的强度来确定的。在CPe（脂肪/脂肪）和/或野生型小鼠垂体提取物中共检测到72条肽，其中53条通过MS/MS测序鉴定。在该分析中鉴定的几个肽代表了先前未描述的已知垂体前激素的翻译后加工产物。在垂体中检测到的72个多肽中，17个仅在Cpe（脂肪/脂肪）小鼠提取物中检测到；这些代表含有c端碱性残基的肽加工中间体。Cpe（脂肪/脂肪）和野生型小鼠所共有的多肽在Cpe（脂肪/脂肪）小鼠垂体提取物中的含量通常低2-5倍，尽管一些多肽在Cpe（脂肪/脂肪）小鼠垂体提取物中含量相同，并且有一种肽（乙酰β -内啡肽1-31）增加了近7倍。相比之下，与野生型小鼠相比，乙酰β -内啡肽1-26在Cpe（脂肪/脂肪）垂体中的水平降低了10倍。许多肽在Cpe（脂肪/脂肪）垂体中大量减少的发现与羧基肽酶E在许多神经肽的生物合成中的广泛作用是一致的。版权所有(C) 2005约翰威利股份有限公司
Cpe(fat/fat) mice have a point mutation in the coding region of the carboxypeptidase E gene that renders the enzyme inactive. As a result, these mice have reduced levels of several neuropeptides and greatly increased levels of the peptide processing intermediates that contain C-terminal basic residues. However, previous studies examined a relatively small number of neuropeptides. In the present study, we used a quantitative peptidomics approach with stable isotopic labels to examine the levels of pituitary peptides in CPe(fat/fat) mice relative to wild-type mice. Pituitary extracts from mutant and wild type mice were labeled with the stable isotopic label [3-(2,5-dioxopyrrolidin-1-yloxycarbonyl)propyl]trimethylammonium chloride containing nine atoms of hydrogen or deuterium. Then, the two samples were pooled and analyzed by liquid chromatography/mass spectrometry (LC/MS). The relative abundance of peptides was determined from a comparison of the intensities of the heavy and light peaks. Altogether, 72 peptides were detected in the CPe(fat/fat) and/or wild-type mouse pituitary extracts of which 53 were identified by MS/MS sequencing. Several peptides identified in this analysis represent previously undescribed post-translational processing products of known pituitary prohormones. Of the 72 peptides detected in pituitary, 17 were detected only in the Cpe(fat/fat) mouse extracts; these represent peptide processing intermediates containing C-terminal basic residues. The peptides common to both Cpe(fat/fat) and wild-type mice were generally present at 2-5-fold lower levels in the Cpe(fat/fat) mouse pituitary extracts, although some peptides were present at equal levels and one peptide (acetyl beta-endorphin 1-31) was increased similar to7-fold in the Cpe(fat/fat) pituitary extracts. In contrast, acetyl beta-endorphin 1-26 was present at similar to10-fold lower levels in the Cpe(fat/fat) pituitary, compared with wild-type mice. The finding that many peptides are substantially decreased in Cpe(fat/fat) pituitary is consistent with the broad role for carboxypeptidase E in the biosynthesis of numerous neuropeptides. Copyright (C) 2005 John Wiley Sons, Ltd.