We describe a protocol for multiplexed proteomic analysis using neutron-encoded (NeuCode) stable isotope labeling of amino acids in cells (SILAC) or mice (SILAM). This method currently enables simultaneous comparison of up to nine treatment and control proteomes. Another important advantage over traditional SILAC/SILAM is that shorter labeling times are required. Exploiting the small mass differences that correspond to subtle differences in the neutron-binding energies of different isotopes, the amino acids used in NeuCode SILAC/SILAM differ in mass by just a few milliDaltons. Isotopologs of lysine are introduced into cells or mammals, via the culture medium or diet, respectively, to metabolically label the proteome. Labeling time is ∼2 weeks for cultured cells and 3-4 weeks for mammals. The proteins are then extracted, relevant samples are combined, and these are enzymatically digested with lysyl endopeptidase (Lys-C). The resultant peptides are chromatographically separated and then mass analyzed. During mass spectrometry (MS) data acquisition, high-resolution MS¹ spectra (≥240,000 resolving power at m/z = 400) reveal the embedded isotopic signatures, enabling relative quantification, while tandem mass spectra, collected at lower resolutions, provide peptide identities. Both types of spectra are processed using NeuCode-enabled MaxQuant software. In total, the approximate completion time for the protocol is 3-5 weeks.

中文翻译：

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在细胞和小鼠中进行中子编码的稳定同位素标记的蛋白质组分析。

我们描述了使用中子编码的（NeuCode）稳定同位素标记的细胞（SILAC）或小鼠（SILAM）中的氨基酸进行蛋白质组分析的协议。该方法目前可以​​同时比较多达9种治疗和对照蛋白质组。与传统的SILAC / SILAM相比，另一个重要的优点是需要较短的标签时间。利用与不同同位素的中子结合能的细微差异相对应的微小质量差异，NeuCode SILAC / SILAM中使用的氨基酸质量差异仅几毫尔顿。赖氨酸的同系物分别通过培养基或饮食引入细胞或哺乳动物中，以代谢方式标记蛋白质组。对于培养的细胞，标记时间约为2周，对于哺乳动物，标记时间为3-4周。然后提取蛋白质，合并相关样品，并用赖氨酰内肽酶（Lys-C）酶消化。色谱分离得到的肽，然后进行质量分析。在质谱（MS）数据采集过程中，高分辨率MS^1个光谱（在m / z = 400时≥240,000的分辨力）揭示了嵌入的同位素特征，可实现相对定量，而以较低分辨率收集的串联质谱可提供肽段身份。两种类型的光谱均使用启用了NeuCode的MaxQuant软件进行处理。总体而言，该方案的大致完成时间为3-5周。