Combining Pulsed SILAC Labeling and Click-Chemistry for Quantitative Secretome Analysis
Combining Pulsed SILAC Labeling and Click-Chemistry for Quantitative Secretome Analysis
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DOI：
10.1007/978-1-4939-0944-5_7
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发表时间：
2014-01-01
期刊：
影响因子：
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通讯作者：
Krijgsveld, Jeroen
中科院分区：
文献类型：
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作者：
Eichelbaum, Katrin;Krijgsveld, Jeroen
分泌的蛋白质，如细胞因子、趋化因子和激素，在细胞间通讯中发挥中心作用，这对维持每个多细胞生物的稳态至关重要。鉴定分泌蛋白的常用方法是在用感兴趣的细胞类型调节后通过培养基的蛋白质组学分析。这优选在无血清条件下进行，以能够检测低丰度分泌因子，否则这些分泌因子将被血清蛋白掩蔽。然而，血清饥饿引入了使细胞处于应激或扰动状态的风险。一种上级方法采用从含血清的生长培养基中富集新合成和分泌的蛋白质。这是通过两种代谢标记的组合实现的：稳定的同位素标记的氨基酸用于可靠的定量，叠氮基高丙氨酸（AHA），甲硫氨酸的叠氮化物类似物，用于富集新合成和分泌的蛋白质。这种方法已用于比较多个细胞系的分泌组或分析在特定刺激下分泌的蛋白质。在这里，我们详细描述了新合成和分泌的蛋白质的富集和定量。
Secreted proteins, such as cytokines, chemokines, and hormones, exhibit central functions in intercellular communication, which is crucial to maintain homeostasis in every multicellular organism. A common approach to identify secreted proteins is by proteomic analysis of culture media after conditioning with a cell type of interest. This is preferably done in serum-free conditions to enable the detection of low-abundance secretory factors that would otherwise be masked by serum proteins. However, serum starvation introduces the risk of bringing cells in a stressed or perturbed state. A superior approach employs the enrichment of newly synthesized and secreted proteins from serum-containing growth medium. This is achieved by the combination of two metabolic labels: stable isotope-labeled amino acids for reliable quantification, and azidohomoalanine (AHA), an azide-bearing analogue of methionine, for the enrichment of newly synthesized and secreted proteins. This approach has been used to compare secretomes of multiple cell lines or to analyze proteins that are secreted upon a specific stimulation. Here we describe in detail the enrichment and quantification of newly synthesized and secreted proteins.