Bottari Patricia, Aebersold Ruedi, Turecek Frantisek, Gelb Michael H
Department of Chemistry, University of Washington, Seattle, Washington 98195, USA.
Bioconjug Chem. 2004 Mar-Apr;15(2):380-8. doi: 10.1021/bc034174s.
Identification of proteins in complex mixtures by mass spectrometry is most useful when quantitative data is also obtained. We recently introduced isotope-coded affinity tags (ICAT reagents) for the relative quantification of proteins present in two or more biological samples. In this report, we describe a new generation of ICAT reagents that contain the following additional features: (1) a visible tag that allows the electrophoretic position of tagged peptides during separation to be easily monitored; (2) a photocleavable linker that allows most of the tag to be removed prior to mass spectrometric analysis; (3) an isotope tag that contains carbon-13 and nitrogen-15 atoms instead of deuterium to ensure precise comigration of light and heavy tagged peptides by reverse-phase HPLC. These reagents contain an iodoacetyl group that selectively reacts with peptide cysteine residues. Peptide modification chemistry is also reported that allows tagging of peptides that are devoid of cysteine. The synthesis of these visible isotope-coded affinity tags (VICAT reagents), and their reaction with peptides are described in this report. VICAT reagents containing a carbon-14 visible probe or an NBD fluorophore are described. These reagents are most useful for the determination of the absolute quantity of specific target proteins in complex protein mixtures such as serum or cell lysates.
当同时获得定量数据时，通过质谱法鉴定复杂混合物中的蛋白质最为有用。我们最近引入了同位素编码亲和标签（ICAT试剂），用于对两个或更多生物样品中存在的蛋白质进行相对定量。在本报告中，我们描述了新一代ICAT试剂，其具有以下附加特征：（1）一个可见标签，可轻松监测分离过程中标记肽的电泳位置；（2）一个光可裂解连接子，可在质谱分析前去除大部分标签；（3）一个同位素标签，含有碳-13和氮-15原子而非氘，以确保轻、重标记肽在反相HPLC中精确共迁移。这些试剂含有一个碘乙酰基团，可与肽的半胱氨酸残基选择性反应。还报道了肽修饰化学方法，可对不含半胱氨酸的肽进行标记。本报告描述了这些可见同位素编码亲和标签（VICAT试剂）的合成及其与肽的反应。描述了含有碳-14可见探针或NBD荧光团的VICAT试剂。这些试剂对于测定复杂蛋白质混合物（如血清或细胞裂解物）中特定靶蛋白的绝对量最为有用。
