Measuring protein synthesis by mass isotopomer distribution analysis (MIDA)
Measuring protein synthesis by mass isotopomer distribution analysis (MIDA)
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DOI：
10.1006/abio.1998.2958
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发表时间：
1999-02-01
影响因子：
2.9
通讯作者：
Hellerstein, MK
中科院分区：
文献类型：
--
作者：
Papageorgopoulos, C;Caldwell, K;Hellerstein, MK
通过同位素技术测量蛋白质动力学一直受到长期难以准确测量生物合成前体库（组织中的氨酰-tRNA）的同位素含量的阻碍。质量同位素异构体分布分析（MIDA）是一种稳定的同位素质谱（MS）技术，用于基于标记和未标记的单体亚基的组合概率，从对聚合产物的测量来测量生物合成前体富集。蛋白质由于其相对高的分子量而含有复杂的同位素异构体模式，然而，因此聚合产物中单个质量同位素异构体的解析（MIDA的分析要求）在技术上是困难的。本文描述并测试了一种通过MIDA测量蛋白质合成的方法：首先，在体外，使用人血清白蛋白中存在的合成肽;然后，在体内，用于大鼠中的白蛋白合成速率。用已知的天然丰度和[5，5，5-H-2（3）]亮氨酸的混合物通过固相肽合成法合成了人血清白蛋白（SVVLLLR）中所含的肽，理论上可从胰蛋白酶/胰凝乳蛋白酶蛋白水解中回收。此外，将富集的肽和天然丰度的肽在体外混合以模拟体内生物合成并解决仪器准确度、精密度和数据管理的问题。使用电喷雾电离（ESI）与磁扇区和四极杆质量分析仪的合成肽的质量同位素异构体模式进行了分析。磁扇区的分辨率是上级的四极仪器，但在质量同位素丰度和动力学参数的测量的准确性和精密度是可比的，都给值接近那些预测。接着，大鼠静脉输注[5，5，5-H-2（3）]亮氨酸，从胰蛋白酶消化的大鼠血清白蛋白（RHPDYSVSLLLR，1456 Da）中分离并纯化富含亮氨酸的肽，然后使用磁扇形仪器通过ESI-MS进行分析。计算前体库富集和部分合成速率（0.45 +/-0.03天（-1），t（1/2）= 1.53 +/-0.09天）。大鼠血清白蛋白的生物合成速率与先前发表的值一致。总之，使用蛋白水解衍生肽和ESI-MS分析，通过MIDA测量蛋白质合成和前体库富集在体内在技术上是可行和实用的。(C)北京：科学出版社.
The measurement of protein kinetics by isotopic techniques has been hindered by the long-standing difficulty of accurately measuring the isotope content of the biosynthetic precursor pool (aminoacyl-tRNA in tissues). Mass isotopomer distribution analysis (MIDA) is a stable isotope-mass spectrometric (MS) technique for measuring biosynthetic precursor enrichments from measurements on a polymeric product, based on combinatorial probabilities of labeled and unlabeled monomeric subunits, Proteins contain complex isotopomer patterns as a result of their relatively high molecular mass, however, so that resolution of the individual mass isotopomers in the polymeric product (an analytic requirement for MIDA) is technically difficult. An approach for measuring protein synthesis by MIDA is described and tested here: First, in vitro, using a synthetic peptide present in human serum albumin; and then, in vivo, for albumin synthetic rate in rats. A peptide contained in human serum albumin (SVVLLLR) and theoretically recoverable from trypsin/chymotrypsin proteolysis was synthesized by solid-phase peptide synthesis using known mixtures of natural abundance and [5,5,5-H-2(3)]leucine, Additionally, enriched and natural abundance peptides were mixed in vitro to simulate in vivo biosynthesis and to address problems of instrument accuracy, precision, and data management. The mass isotopomer patterns of the synthetic peptides were analyzed using electrospray ionization (ESI) with both magnetic sector and quadrupole mass analyzers. The resolution of the magnetic sector was superior to that of the quadrupole instrument, but accuracy and precision in the measurement of mass isotopomer abundances and kinetic parameters were comparable and both gave values close to those predicted. Next, rats were infused with [5,5,5-H-2(3)]leucine intravenously, and a leucine-rich peptide was isolated and purified from trypsin-digested rat serum albumin (RHPDYSVSLLLR, 1456 Da) and then analyzed by ESI-MS using a magnetic sector instrument. Precursor pool enrichments and fractional synthetic rates (0.45 +/- 0.03 day(-1), t(1/2) = 1.53 +/- 0.09 days) were calculated. Biosynthetic rates of rat serum albumin were congruent with previously published values. In summary, measurement of protein synthesis and precursor pool enrichments by MIDA is technically feasible and practical in vivo using proteolytically derived peptides and ESI-MS analysis. (C) 1999 Academic Press.