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.

中文翻译：

---

使用快速导电样品加热系统改进对啮齿动物下丘脑中成熟内源肽的鉴定和定量

通过质谱（MS）对组织样品中的内源肽进行测量，鉴定和定量分析有助于我们理解多种生物学现象的复杂分子机制。为了获得准确的结果，必须在进行肽组测量之前阻止样品中普遍存在的蛋白质的事后降解。这样做可确保蛋白质降解产物不会压倒通常以相对较低的丰度水平存在的内源肽的检测。已经显示出热稳定作用使组织样品中的酶失活，并使随后的肽提取物中蛋白质降解产物的存在最小化。但是，尚未充分证明使用不同的热处理来保持全长内源肽的完整性的功效。先前对热稳定方法的肽组学研究并未区分内源性肽的全长（成熟）形式和众多截短形式（可能的取样伪影）。我们展示了热样品处理通过快速传导热传递对于检测新鲜和冷冻啮齿动物脑组织中的成熟内源肽是有效的。与在热水浴中广泛使用的样品处理相比，使用商业化的Stabilizor T1热稳定系统进行的新鲜分离的组织处理可确定地鉴定出全长成熟神经肽多出65％的可信度。通过对选定神经肽的后续定量多反应监测MS分析验证了这一发现。通过确定总蛋白质含量来评估，Stabilizor T1提供的在部分真空中的快速传导加热有效地减少了蛋白质降解并降低了样品的化学复杂性。该系统启用了检测，识别，