Conrads T P, Alving K, Veenstra T D, Belov M E, Anderson G A, Anderson D J, Lipton M S, Pasa-Tolić L, Udseth H R, Chrisler W B, Thrall B D, Smith R D
Environmental and Molecular Sciences Laboratory and Molecular Biosciences Department, Pacific Northwest National Laboratory, Richland, Washington 99352, USA.
Anal Chem. 2001 May 1;73(9):2132-9. doi: 10.1021/ac001487x.
We describe the combined use of 15N-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 15N-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) online with ion trap mass spectrometry (MS) as well as Fourier transform ion cyclotron resonance (FTICR) MS. The resulting spectra contain numerous pairs of Cyspolypeptides 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 15N-labeled peptides versus their 14N-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 1:1 labeling of the 14N and 15N versions of each peptide. An additional benefit from the present strategy is that the 15N-labeled peptides do not display significant isotope-dependent chromatographic shifts from their 14N-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.
我们描述了联合使用¹⁵N代谢标记和一种与半胱氨酸反应的生物素亲和标签，从耐辐射球菌以及小鼠B16黑色素瘤细胞中分离和定量含半胱氨酸的多肽（Cys-多肽）。耐辐射球菌在天然同位素丰度培养基和¹⁵N富集培养基中培养。将两种培养物中数量相等的细胞混合，并提取可溶性蛋白质。使用含有生物素基团的市售半胱氨酸反应试剂对这种同位素不同的蛋白质混合物进行衍生化。胰蛋白酶消化后，使用固定化抗生物素蛋白分离得到的修饰肽。混合物通过在线毛细管反相液相色谱（LC）与离子阱质谱（MS）以及傅里叶变换离子回旋共振（FTICR）MS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肽中存在的氮原子数。¹⁵N标记肽与其¹⁴N标记对应物的独特同位素分布也有助于确定Cys-多肽对。对培养的小鼠B16细胞进行的研究通过LC-FTICR分析观察到数百对同位素不同的肽。同位素不同的肽对的面积比显示了每种肽的¹⁴N和¹⁵N版本预期的1:1标记。本策略的另一个优点是，¹⁵N标记的肽与其¹⁴N标记的对应物相比，没有显示出明显的同位素依赖性色谱位移，因此提高了定量肽丰度的精度。所提出的方法为进行全面的定量蛋白质组学测量提供了一种替代的、具有成本效益的策略。
