Core N: MOLECULAR PROFILING

定量蛋白质组学研究是通过使用LC-MS分析偶联的胰蛋白酶多肽来完成的 用化学试剂标记或代谢标记培养中的细胞。目前的大多数化学品 用N-羟基琥珀酰亚胺标记赖氨酸N-末端和表氨基上的多肽 化学反应。同量同量标记物在生物标志物发现实验中尤其流行 因为标记形式之间的质量差异发生在碎片离子而不是母体分析物(3)上。 然而，这种应用的一个限制是，同位素标记是在蛋白质提取后引入的，这意味着 必须非常小心，以确保在样品制备过程中不会引入误差。我们已经向您展示了 使用SILAC标记的蛋白质作为可应用于任何来源材料的SILAP标准的效用(1)。 对于培养中的细胞(4；5)和血清样本，获得了该方法应用的原理证据。 来自胰腺癌患者(6)。在后一项研究中，Capan-2胰腺癌细胞系被标记为 SILAC和条件培养上清液中的分泌蛋白。然后将分泌的标记蛋白 用作SILAP标准，添加到汇集的胰腺癌或对照血清中超过100 确定了差异表达的生物标记物候选。这一方法被扩展到研究 Apcmin小鼠结肠癌模型(7)中早产宫颈阴道液样本的生物标志物 研究(8)，以及精神分裂症患者突触后密度中差异蛋白的表达 正常脑组织标本(9例)。 临床上有用的生物标志物的开发是一个分两个阶段的过程。最初的生物分析验证阶段 在适当的生物体液中使用对照样品来建立定量的分析限值并确定误差 通过通常在常规生物分析验证研究中使用的样品处理引入(10)。这 阶段之后是临床验证，使用患者样本来确定检测方法？S灵敏度来检测 对某一特定疾病的药物反应或歧视。我们的重点是LC-MS方法学的使用 用于DNA加合物、谷胱甘肽加合物、硫醇、类固醇、叶酸、葡萄糖醛酸苷和硫酸盐的分析 代谢物，因为它适用于更广泛的生物标志物的分析(11-16)。分析了几个问题 非甾体抗炎药需要严格的验证和质量控制程序，以确保水平是通过 精确度和精密度高。这是通过使用稳定的同位素稀释方法来实现的 执行严格的标准作业程序(10)。

Quantitative proteomics studies are accomplished by the use of LC-MS analysis of tryptic peptides coupled with tagging with chemical reagents or metabolic labeling of cells in culture. The majority of current chemical tagging techniques label peptides on the N-terminus and epsilon-amino group of lysine using Nhydroxysuccinimide chemistry. Isobaric mass tags are particularly popular for biomarker discovery experiments since the mass difference between labeled forms occur on fragment ions rather than on the parent analyte (3). A limitation to this application, however, is that the isotope label is introduced post-protein extraction, meaning extreme care must be taken to ensure error is not introduced during sample preparation. We have shown the utility of using the SILAC labeled proteins as SILAP standards which can be applied to any source material (1). Proof of principle for the utility of this approach was obtained for cells in culture (4;5) and for serum samples from pancreatic cancer patients (6). In this latter study, the CAPAN-2 pancreatic cancer cell line was labeled by SILAC and secreted proteins from the conditioned media collected. The secreted labeled proteins were then used as a SILAP standard that was added to pooled pancreatic cancer or control serum Over one hundred differentially expressed biomarker candidates were identified. This approach was extended to study biomarkers in the Apcmin mouse, a colon cancer model (7), for cervicovaginal fluid samples in a preterm birth study (8), and differential protein expression in the post-synaptic density from schizophrenic compared with normal brain samples (9). The development of clinically useful biomarkers is a two-stage process. The initial bioanalytical validation stage uses control samples in appropriate biofluids to establish analytical limits of quantification and determine errors introduced by sample handling that are typically used in conventional bioanalytical validation studies (10). This stage is followed by clinical validation using patient samples to determine the assay¿s sensitivity to detect the drug response or discrimination of a particular disease. We have focused on the use of LC-MS methodology for the analysis of DNA-adducts, glutathione-adducts, thiols, steroids, folates, and glucuronide and sulfate metabolites because it is applicable to the analysis of a wider range of biomarkers (11-16). The analysis of NSAIDs requires rigorous validation and quality control procedures to ensure that levels are determined with high accuracy and precision. This is accomplished by the use of stable isotope dilution methodology coupled with the implementation of rigorous standard operating procedures (10).