Protein stoichiometry of a multiprotein complex, the human spliceosomal U1 small nuclear ribonucleoprotein -: Absolute quantification using isotope-coded tags and mass spectrometry
Protein stoichiometry of a multiprotein complex, the human spliceosomal U1 small nuclear ribonucleoprotein -: Absolute quantification using isotope-coded tags and mass spectrometry
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DOI：
10.1074/jbc.m409587200
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发表时间：
2005-01-28
影响因子：
4.8
通讯作者：
Lottspeich, F
中科院分区：
文献类型：
--
作者：
Hochleitner, EO;Kastner, B;Lottspeich, F
人U1 snRNP（小核核糖核蛋白）是剪接体的一部分，由U1 snRNA和10种不同的蛋白质组成：7种Sm蛋白13/1399 D1、D2、D3、Eq F和G以及3种U1特异性蛋白U1-70 K、U1-A、U1-C。为了确定所有十种蛋白质的化学计量，将复合物变性，用内切蛋白酶完全消化，并用胺特异性标签标记。合成相应的肽并用含有较重同位素的相同标签标记。然后用规定量的合成肽掺入消化物，并通过质谱法定量分析所得同位素肽对。质谱提供了有关起始蛋白质混合物中每种组分绝对量的信息。使用同位素编码的胺特异性试剂丙酰基-N-氧琥珀酰亚胺和烟酰基-N-氧琥珀酰亚胺进行了化学计量测定评价;烟酰基试剂被认为是有利的，因为它具有更高的质谱灵敏度。测量所有十种蛋白质的绝对量，显示U1剪接体snRNP中所有十种蛋白质的数量相等。这些数据表明，定量质谱测定多蛋白质复合物的化学计量有很大的潜力。
The human U1 snRNP (small nuclear ribonucleoprotein), which is a part of the spliceosome, consists of U1 snRNA and ten different proteins: seven Sm proteins 13/1399 D1, D2, D3, Eq F, and G and the three U1-specific proteins U1-70 K, U1-A, U1-C. To determine the stoichiometry of all ten proteins, the complex was denatured, digested completely with an endoproteinase and labeled with an amine-specific tag. Corresponding peptides were synthesized and labeled with the same tag containing heavier isotopes. The digest was then spiked with defined amounts of the synthetic peptides, and the resulting isotopic peptide pairs were analyzed quantitatively by mass spectrometry. The mass spectra provided information about the absolute amount of each component in the starting protein mixture. The use of the isotope-coded, amine-specific reagents propionyl-N-oxysuceinimide and nicotinoyl-N-oxysuceinimide was evaluated for stoichiometry determination; the nicotinoyl reagent was found to be advantageous because of its greater mass spectrometric sensitivity. Absolute quantities of all ten proteins were measured, showing equal numbers of all ten proteins in the U1 spliceosomal snRNP. These data demonstrate that quantitative mass spectrometry has great potential for the determination of the stoichiometry of multiprotein complexes.