2014年9月5日 訊 /生物谷BIOON/ --近日，刊登在國際雜誌Nature Protocols和Cellular and Molecular Life Sciences上的兩篇研究論文中，來自昆士蘭大學等處的研究人員通過研究開發了一種鑑別人類機體蛋白質產生的新方法，其或許可以幫助研究者揭示阿爾茲海默氏症患者學習和記憶力被損傷的機制。
文章中，研究者開發的這種新方法可以利用新蛋白質產生的胺基酸來鑑別機體產生的蛋白質，通過對新生成的蛋白質進行標記，研究人員就可以確定蛋白質的產生是否是對內部刺激的反應，比如有規律的細胞運動或學習活動，該研究對於預防個體老齡化老年痴呆症非常重要。
研究者Gotz表示，我們開發的新技術可以應用於許多細胞過程中，可以用於揭示諸如神經變性及痴呆症等疾病的發病機制；理解個體的學習和記憶的發生非常重要，研究者希望有一天可以開發出緩解或者逆轉機體變性疾病的新型療法。一般認為蛋白質在學習和記憶形成過程中扮演著重要角色，研究者此前表示確定機體中的老齡化蛋白不太可能，但是本文中利用這種新型技術研究者就可以實現對機體新產生蛋白質的確定。
一種蛋白質是新生的還是已經生存了10年，利用常規的方法我們並不能確定其年齡，但是本文中研究人員開發的新型技術就可以對新生的蛋白質進行標記，來追蹤這些蛋白質的走向，並且進一步為揭示這種蛋白質在學習和記憶力發揮過程中的作用。（生物谷Bioon.com）
Bio-orthogonal labeling as a tool to visualize and identify newly synthesized proteins in Caenorhabditis elegans
Milena Ullrich, Vanessa Liang, Yee Lian Chew, Samuel Banister, Xiaomin Song, Thiri Zaw, Hong Lam, Slavica Berber, Michael Kassiou, Hannah R Nicholas & Jürgen Götz
In this protocol we describe the incorporation of bio-orthogonal amino acids as a versatile method for visualizing and identifying de novo–synthesized proteins in the roundworm Caenorhabditis elegans. This protocol contains directions on implementing three complementary types of analysis: 'click chemistry' followed by western blotting, click chemistry followed by immunofluorescence, and isobaric tags for relative and absolute quantification (iTRAQ) quantitative mass spectrometry. The detailed instructions provided herein enable researchers to investigate the de novo proteome, an analysis that is complicated by the fact that protein molecules are chemically identical to each other, regardless of the timing of their synthesis. Our protocol circumvents this limitation by identifying de novo–synthesized proteins via the incorporation of the chemically modifiable azidohomoalanine instead of the natural amino acid methionine in the nascent protein, followed by facilitating the visualization of the resulting labeled proteins in situ. It will therefore be an ideal tool for studying de novo protein synthesis in physiological and pathological processes including learning and memory. The protocol requires 10 d for worm growth, liquid culture and synchronization; 1–2 d for bio-orthogonal labeling; and, with regard to analysis, 3–4 d for western blotting, 5–6 d for immunofluorescence or ∼3 weeks for mass spectrometry.
Altered proteostasis in aging and heat shock response in C. elegans revealed by analysis of the global and de novo synthesized proteome
Vanessa Liang, Milena Ullrich, Hong Lam, Yee Lian Chew, Samuel Banister, Xiaomin Song, Thiri Zaw, Michael Kassiou, Jürgen Götz, Hannah R. Nicholas
Protein misfolding and aggregation as a consequence of impaired protein homeostasis (proteostasis) not only characterizes numerous age-related diseases but also the aging process itself. Functionally related to the aging process are, among others, ribosomal proteins, suggesting an intimate link between proteostasis and aging. We determined by iTRAQ quantitative proteomic analysis in C. elegans how the proteome changes with age and in response to heat shock. Levels of ribosomal proteins and mitochondrial chaperones were decreased in aged animals, supporting the notion that proteostasis is altered during aging. Mitochondrial enzymes of the tricarboxylic acid cycle and the electron transport chain were also reduced, consistent with an age-associated energy impairment. Moreover, we observed an age-associated decline in the heat shock response. In order to determine how protein synthesis is altered in aging and in response to heat shock, we complemented our global analysis by determining the de novo proteome. For that, we established a novel method that enables both the visualization and identification of de novo synthesized proteins, by incorporating the non-canonical methionine analogue, azidohomoalanine (AHA), into the nascent polypeptides, followed by reacting the azide group of AHA by 『click chemistry』 with an alkyne-labeled tag. Our analysis of AHA-tagged peptides demonstrated that the decreased abundance of, for example, ribosomal proteins in aged animals is not solely due to degradation but also reflects a relative decrease in their synthesis. Interestingly, although the net rate of protein synthesis is reduced in aged animals, our analyses indicate that the synthesis of certain proteins such as the vitellogenins increases with age.