Department of Chemistry, University of Illinois at Chicago, 845 W Taylor St., SES 4500, M/C 111, Chicago, IL 60607, United States.
Department of Chemistry, University of Illinois at Chicago, 845 W Taylor St., SES 4500, M/C 111, Chicago, IL 60607, United States; Laboratory of Integrative Neuroscience, University of Illinois at Chicago, 840 W Taylor St., SEL 4311, M/C 067, Chicago, IL 60607, United States.
J Chromatogr B Analyt Technol Biomed Life Sci. 2015 Feb 15;981-982:33-9. doi: 10.1016/j.jchromb.2014.11.027. Epub 2014 Dec 25.
Analysis of blood proteins holds critical promise for in depth understanding of physiological states. Protein content of hemolymph from Drosophila melanogaster is of particular analytical interest because the insect open circulatory system involves chemical signaling through the hemolymph. The challenge of working with this sample, however, is the nanoliter volumes of solution available for analysis. In this study, we developed a novel hyphenated Agilent nano-HPLC chip column-MS method to obtain proteomic information from individual fruit fly hemolymph, using a low-volume sample collection technique established previously. The total amount of individual Drosophila hemolymph protein is determined around 0.798 ± 0.251 μg/100 nL based upon a Bradford assay with BSA. Hemolymph samples around 50 nL were collected from single flies and digested using a customized micro-scale digestion protocol. Mass spectral analysis shows a total of 19 proteins were identified from the hemolymph of individual flies. Of these findings, 6 novel proteins have been identified for the first time with evidence at the translation level. Detection of 13 proteins well-known in the literature speaks to the method's validity and demonstrates the ability to reproducibly analyze volume-limited samples from individual fruit flies for protein content. This nano-scale analysis method will facilitate future study of Drosophila and lead to a more complete understanding of the physiology of the fly model system.
血液蛋白质分析对于深入了解生理状态具有重要意义。黑腹果蝇（Drosophila melanogaster）血淋巴中的蛋白质含量是特别具有分析意义的，因为昆虫开放式循环系统涉及通过血淋巴进行化学信号传递。然而，处理这种样品的挑战在于可用于分析的溶液纳米升体积。在这项研究中，我们开发了一种新颖的 Agilent 纳升 HPLC 芯片柱-MS 方法，使用先前建立的低体积样品收集技术，从单个果蝇血淋巴中获得蛋白质组信息。根据 Bradford assay 与 BSA 的测定，个体果蝇血淋巴蛋白的总量约为 0.798 ± 0.251 μg/100 nL。从单个果蝇中收集约 50 nL 的血淋巴样品，并使用定制的微尺度消化方案进行消化。质谱分析显示，从单个果蝇的血淋巴中总共鉴定出 19 种蛋白质。其中 6 种新蛋白质是首次在翻译水平上鉴定出来的。检测到 13 种文献中熟知的蛋白质证明了该方法的有效性，并证明了能够从单个果蝇中可重复地分析体积有限的样品中的蛋白质含量。这种纳升分析方法将有助于未来对果蝇的研究，并深入了解果蝇模型系统的生理学。
