Improved identification and quantitation of mature endogenous peptides in the rodent hypothalamus using a rapid conductive sample heating system.
Improved identification and quantitation of mature endogenous peptides in the rodent hypothalamus using a rapid conductive sample heating system.
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DOI：
10.1039/c7an01358b
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发表时间：
2017-11-20
期刊：
影响因子：
--
通讯作者：
Sweedler JV
中科院分区：
文献类型：
--
作者：
Yang N;Anapindi KDB;Romanova EV;Rubakhin SS;Sweedler JV
通过质谱 (MS) 测量、鉴定和定量组织样品中的内源肽有助于我们理解众多生物现象的复杂分子机制。为了获得准确的结果，在进行肽组学测量之前，必须阻止样品中普遍存在的蛋白质的死后降解。这样做可以确保内源肽（通常以相对较低的丰度水平存在）的检测不会被蛋白质降解产物淹没。热稳定已被证明可以使组织样品中的酶失活，并最大限度地减少后续肽提取物中蛋白质降解产物的存在。然而，不同热处理对于保持全长内源肽完整性的功效尚未得到充分记录。先前的热稳定方法的肽组学研究并未区分内源肽的全长（成熟）和许多截短（可能的采样伪影）形式。我们表明，通过快速传导热传递对样品进行热处理可有效检测新鲜和冷冻啮齿动物脑组织中的成熟内源肽。与广泛使用的热水浴样品处理相比，使用商用 Stabilizor T1 热稳定系统处理新鲜分离的组织，全长成熟神经肽的鉴定结果增加了 65%。这一发现通过对选定神经肽的后续定量多重反应监测 MS 分析得到了验证。 Stabilizor T1 在部分真空中提供快速传导加热，可有效减少蛋白质降解并降低样品的化学复杂性（通过测定总蛋白质含量进行评估）。该系统能够检测、鉴定和定量与个体大鼠下丘脑和视交叉上核中表达的 22 种激素原相关的神经肽。
Measurement, identification, and quantitation of endogenous peptides in tissue samples by mass spectrometry (MS) contribute to our understanding of the complex molecular mechanisms of numerous biological phenomena. For accurate results, it is essential to arrest the postmortem degradation of ubiquitous proteins in samples prior to performing peptidomic measurements. Doing so ensures that the detection of endogenous peptides, typically present at relatively low levels of abundance, is not overwhelmed by protein degradation products. Heat stabilization has been shown to inactivate the enzymes in tissue samples and minimize the presence of protein degradation products in the subsequent peptide extracts. However, the efficacy of different heat treatments to preserve the integrity of full-length endogenous peptides has not been well documented; prior peptidomic studies of heat stabilization methods have not distinguished between the full-length (mature) and numerous truncated (possible artifacts of sampling) forms of endogenous peptides. We show that thermal sample treatment via rapid conductive heat transfer is effective for detection of mature endogenous peptides in fresh and frozen rodent brain tissues. Freshly isolated tissue processing with the commercial Stabilizor T1 heat stabilization system resulted in the confident identification of 65% more full-length mature neuropeptides compared to widely used sample treatment in a hot water bath. This finding was validated by a follow-up quantitative multiple reaction monitoring MS analysis of select neuropeptides. The rapid conductive heating in partial vacuum provided by the Stabilizor T1 effectively reduces protein degradation and decreases the chemical complexity of the sample, as assessed by determining total protein content. This system enabled the detection, identification, and quantitation of neuropeptides related to 22 prohormones expressed in individual rat hypothalami and suprachiasmatic nuclei.
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影响因子：
7.4
作者：
Do TD;Comi TJ;Dunham SJ;Rubakhin SS;Sweedler JV
通讯作者：
Sweedler JV
DOI：
10.1073/pnas.95.7.3972
发表时间：
1998-03-31
影响因子：
11.1
作者：
Garden, RW;Shippy, SA;Sweedler, JV
通讯作者：
Sweedler, JV
影响因子：
3.4
作者：
Colgrave, Michelle L.;Xi, Li;Wijffels, Gene
通讯作者：
Wijffels, Gene
影响因子：
3
作者：
Ahmed, Md Mahiuddin;Gardiner, Katheleen J.
通讯作者：
Gardiner, Katheleen J.
DOI：
10.1007/s13361-015-1251-6
发表时间：
2015-12-01
影响因子：
3.2
作者：
Hook, Vivian;Bandeira, Nuno
通讯作者：
Bandeira, Nuno