Expanding the dipeptidyl peptidase 4-regulated peptidome via an optimized peptidomics platform.
Expanding the dipeptidyl peptidase 4-regulated peptidome via an optimized peptidomics platform.
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DOI：
10.1021/ja909524e
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发表时间：
2010-03-24
影响因子：
15
通讯作者：
Saghatelian, Alan
中科院分区：
文献类型：
--
作者：
Tinoco, Arthur D.;Tagore, Debarati M.;Saghatelian, Alan
近年来，生物科学中用于定量测量细胞和组织的生物分子水平（即 RNA、蛋白质、代谢物）的方法（包括高通量全局方法）的发展激增。与生物分子定量一样重要的是揭示生物分子之间的调控和信号传导联系的方法的创建。我们的具体兴趣是了解生理环境中的肽代谢，这促使我们开发出一种多学科方法，整合遗传学、分析化学、合成化学、生物化学和化学生物学来识别体内肽酶的底物。为了实现这一目标，我们利用基于液相色谱-质谱 (LC-MS) 的肽组学平台来测量肽组（细胞、组织或生物体中的所有肽）随肽酶活性的变化。先前对缺乏二肽基肽酶 4（DPP4−/− 小鼠）（一种生物医学相关肽酶）的小鼠进行的分析，使用这种方法鉴定了一些新型内源性 DPP4 底物。在这里，我们利用来自 DPP4−/− 小鼠的这些底物和组织，通过优化工作流程中的关键步骤来提高肽组学平台的覆盖范围，在此过程中，总共发现了 70 种肾脏 DPP4 底物（从我们优化开始时的 7 种增加），覆盖范围提高了 10 倍。这些 DPP4 肽底物的序列支持 DPP4 在含脯氨酸肽分解代谢中的广泛作用，并加强了将氨肽酶和 DPP4 活性相互联系的生化模型。此外，改进的肽组覆盖范围还导致在分析肠道样本期间检测到更多数量的已知生物活性肽（例如肽激素），这表明这种优化的工作流程具有其他用途。这些结果共同增强了我们通过改善肽组覆盖范围来识别内源肽底物的能力，并证明了该肽组学平台的更广泛潜力。
In recent years, the biological sciences have seen a surge in the development of methods, including high-throughput global methods, for the quantitative measurement of biomolecule levels (i.e., RNA, proteins, metabolites) from cells and tissues. Just as important as quantitation of biomolecules has been the creation of approaches that uncover the regulatory and signaling connections between biomolecules. Our specific interest is in understanding peptide metabolism in a physiological setting, and this has led us to develop a multidisciplinary approach that integrates genetics, analytical chemistry, synthetic chemistry, biochemistry, and chemical biology to identify the substrates of peptidases in vivo. To accomplish this we utilize a liquid chromatography-mass spectrometry (LC-MS)-based peptidomics platform to measure changes in the peptidome–all peptides in a cell, tissue, or organism–as a function of peptidase activity. Previous analysis of mice lacking the enzyme dipeptidyl peptidase 4 (DPP4−/− mice), a biomedically relevant peptidase, using this approach identified a handful of novel endogenous DPP4 substrates. Here, we utilize these substrates and tissues from DPP4−/− mice to improve the coverage of the peptidomics platform by optimizing the key steps in the workflow, and in doing so, discover a total of 70 renal DPP4 substrates (up from 7 at the beginning of our optimization), a 10-fold improvement in our coverage. The sequences of these DPP4 peptide substrates support a broad role for DPP4 in proline-containing peptide catabolism and strengthen a biochemical model that interlinks aminopeptidase and DPP4 activities. Moreover, the improved peptidome coverage also led to the detection of greater numbers of known bioactive peptides (e.g., peptide hormones) during the analysis of gut samples suggesting additional uses for this optimized workflow. Together these results strengthen our ability to identify endogenous peptide substrates through improved peptidome coverage and demonstrate a broader potential of this peptidomics platform.
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影响因子：
3
作者：
Beinfeld MC;Funkelstein L;Foulon T;Cadel S;Kitagawa K;Toneff T;Reinheckel T;Peters C;Hook V
通讯作者：
Hook V
影响因子：
14.8
作者：
Tagore, Debarati M.;Nolte, Whitney M.;Neveu, John M.;Rangel, Roberto;Guzman-Rojas, Liliana;Pasqualini, Renata;Arap, Wadih;Lane, William S.;Saghatelian, Alan
通讯作者：
Saghatelian, Alan
影响因子：
1.8
作者：
McDonald, WH;Ohi, R;Yates, JR
通讯作者：
Yates, JR
影响因子：
4.8
作者：
MEDEIROS, MD;TURNER, AJ
通讯作者：
TURNER, AJ
影响因子：
2.9
作者：
Lortie, Mark;Bark, Steven;Hook, Vivian
通讯作者：
Hook, Vivian