Recent advances in mass spectrometry-based proteomics have revealed translation of previously nonannotated microproteins from thousands of small open reading frames (smORFs) in prokaryotic and eukaryotic genomes. Facile methods to determine cellular functions of these newly discovered microproteins are now needed. Here, we couple semiquantitative comparative proteomics with whole-genome database searching to identify two nonannotated, homologous cold shock-regulated microproteins in Escherichia coli K12 substr. MG1655, as well as two additional constitutively expressed microproteins. We apply molecular genetic approaches to confirm expression of these cold shock proteins (YmcF and YnfQ) at reduced temperatures and identify the noncanonical ATT start codons that initiate their translation. These proteins are conserved in related Gram-negative bacteria and are predicted to be structured, which, in combination with their cold shock upregulation, suggests that they are likely to have biological roles in the cell. These results reveal that previously unknown factors are involved in the response of E. coli to lowered temperatures and suggest that further nonannotated, stress-regulated E. coli microproteins may remain to be found. More broadly, comparative proteomics may enable discovery of regulated, and therefore potentially functional, products of smORF translation across many different organisms and conditions.

中文翻译：

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比较蛋白质组学能够鉴定大肠杆菌中未注释的冷激蛋白

基于质谱的蛋白质组学的最新进展表明，原核和真核基因组中成千上万个小的开放阅读框（smORF）都翻译了以前未注释的微蛋白。现在需要确定这些新发现的微蛋白的细胞功能的简便方法。在这里，我们将半定量比较蛋白质组学与全基因组数据库搜索结合起来，以鉴定大肠杆菌中两种非注释的，同源的冷激调节微蛋白。K12 substr。MG1655，以及另外两个组成性表达的微蛋白。我们应用分子遗传学方法来确认这些低温休克蛋白（YmcF和YnfQ）在降低的温度下的表达，并确定启动翻译的非规范ATT起始密码子。这些蛋白质在相关的革兰氏阴性细菌中是保守的，并被预测为结构化，再结合其冷激上调，表明它们可能在细胞中具有生物学作用。这些结果表明，以前未知的因素参与了大肠杆菌对降低的温度的响应，并表明了进一步的未注释的，压力调节的大肠杆菌微量蛋白可能仍有待发现。更广泛地说，比较蛋白质组学可以实现跨许多不同生物体和条件的smORF翻译的调控产物，因此具有潜在功能。