Quantitative analysis of bacterial and mammalian proteomes using a combination of cysteine affinity tags and 15N-Metabolic labeling
Quantitative analysis of bacterial and mammalian proteomes using a combination of cysteine affinity tags and 15N-Metabolic labeling
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DOI：
10.1021/ac001487x
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发表时间：
2001-05-01
影响因子：
7.4
通讯作者：
Smith, RD
中科院分区：
文献类型：
--
作者：
Conrads, TP;Alving, K;Smith, RD
我们描述了结合使用n -15代谢标记和半胱氨酸活性生物素亲和标签，从耐辐射球菌和小鼠B16黑色素瘤细胞中分离和定量含半胱氨酸的多肽（Cys-polypeptides）。D，在天然同位素丰度和富n -15培养基中培养耐辐射菌。将两种培养物中等量的细胞结合并提取可溶性蛋白，使用市售的含有生物素基团的半胱氨酸反应试剂衍生出这种同位素不同的蛋白质混合物。胰酶消化后，用固定化亲和素分离得到修饰肽。利用毛细管反相液相色谱（LC）、离子阱质谱（RIS）和傅立叶变换离子回旋共振（FTICR）质谱对混合物进行分析，所得光谱包含许多对cys -多肽，其质量差对应于每个多肽中存在的氮原子数。n -15标记的多肽与n -14标记的多肽相比，其独特的同位素分布也促进了cys多肽对的指定。对培养的小鼠B16细胞的研究允许通过LC-FTICR分析观察数百个同位素不同的多肽对。同位素不同肽对的面积比显示了每个肽的N-14和N-15版本的预期1:1标记。该策略的另一个好处是，n -15标记的肽与n -14标记的肽相比，不会出现明显的同位素依赖色谱偏移，从而提高了定量肽丰度的精度。提出的方法提供了一种替代的，具有成本效益的策略，进行全球定量蛋白质组学测量。
We describe the combined use of N-15-metabolic labeling and a cysteine-reactive biotin affinity tag to isolate and quantitate cysteine-containing polypeptides (Cys-polypeptides) from Deinococcus radiodurans as well as from mouse B16 melanoma cells. D, radiodurans were cultured in both natural isotopic abundance and N-15-enriched media. Equal numbers of cells from both cultures were combined and the soluble proteins extracted, This mixture of isotopically distinct proteins was derivatized using a commercially available cysteine-reactive reagent that contains a biotin group. Following trypsin digestion, the resulting modified peptides were isolated using immobilized avidin. The mixture was analyzed by capillary reversed-phase liquid chromatography (LC) on-line with ion trap mass spectrometry (RIS) as well as Fourier transform ion cyclotron resonance (FTICR) MS. The resulting spectra contain numerous pairs of Cys-polypeptides whose mass difference corresponds to the number of nitrogen atoms present in each of the peptides. Designation of Cys-polypeptide pairs is also facilitated by the distinctive isotopic distribution of the N-15-labeled peptides versus their N-14-labeled counterparts, Studies with mouse B16 cells maintained in culture allowed the observation of hundreds of isotopically distinct pairs of peptides by LC-FTICR analysis. The ratios of the areas of the pairs of isotopically distinct peptides showed the expected 1:I labeling of the N-14 arid N-15 versions of each peptide. An additional benefit from the present strategy Is that the N-15-labeled peptides do not display significant isotope-dependent chromatographic shifts from their N-14-labeled counterparts, therefore improving the precision for quantitating peptide abundances. The methodology presented offers an alternate, cost-effective strategy for conducting global, quantitative proteomic measurements.