Mass spectrometry in coupling with affinity capture-release and isotope-coded affinity tags for quantitative protein analysis.
Mass spectrometry in coupling with affinity capture-release and isotope-coded affinity tags for quantitative protein analysis.
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DOI：
10.1002/jms.275
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发表时间：
2002
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影响因子：
--
通讯作者：
F. Tureček
中科院分区：
文献类型：
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作者：
F. Tureček
亲和捕获-释放电喷雾离子化质谱（ACESIMS）和同位素编码亲和标签（ICAT）是两种新近引入的用于定量蛋白质活性和含量的技术，分别应用于临床酶学和功能蛋白质组学。这些方法的一个共同特征是它们使用生物素化的标签，其用作分子手柄，用于从复杂的生物混合物（例如细胞匀浆和亚细胞器）中高度选择性和可逆地亲和捕获缀合物。ACESIMS使用合成底物缀合物特异性靶向细胞酶，当缺乏时，是遗传疾病的原因。酶活性的多重测定用于诊断溶酶体贮积病。ICAT方法依赖于蛋白质中半胱氨酸巯基的选择性缀合，然后进行酶消化并通过质谱法对肽缀合物进行定量分析。ACESIMS和ICAT方法的另一个共同特征是两者都使用用稳定重同位素标记的缀合物作为定量的内标。ACESIMS和ICAT技术的选定应用，包括在儿童遗传性疾病的分子水平诊断和细胞中蛋白质表达的定量测定。
Affinity capture-release electrospray ionization mass spectrometry (ACESIMS) and isotope-coded affinity tags (ICAT) are two recently introduced techniques for the quantitation of protein activity and content with applications to clinical enzymology and functional proteomics, respectively. One common feature of these methods is that they use biotinylated tags that function as molecular handles for highly selective and reversible affinity capture of conjugates from complex biological mixtures such as cell homogenates and sub-cellular organelles. ACESIMS uses synthetic substrate conjugates specifically to target cellular enzymes that, when deficient, are the cause of genetic diseases. Multiplex determination of enzyme activities is used for the diagnosis of lysosomal storage diseases. The ICAT method relies on selective conjugation of cysteine thiol groups in proteins, followed by enzymatic digestion and quantitative analysis of peptide conjugates by mass spectrometry. Another common feature of the ACESIMS and ICAT approaches is that both use conjugates labeled with stable heavy isotopes as internal standards for quantitation. Selected applications of the ACESIMS and ICAT techniques are presented that include molecular-level diagnosis of genetic diseases in children and quantitative determination of protein expression in cells.