Proteomic stable isotope probing with an upgraded Sipros algorithm for improved identification and quantification of isotopically labeled proteins.

IF 13.8 1区 生物学 Q1 MICROBIOLOGY Microbiome Pub Date : 2024-08-08 DOI:10.1186/s40168-024-01866-1
Yi Xiong, Ryan S Mueller, Shichao Feng, Xuan Guo, Chongle Pan
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Abstract

Background: Proteomic stable isotope probing (SIP) is used in microbial ecology to trace a non-radioactive isotope from a labeled substrate into de novo synthesized proteins in specific populations that are actively assimilating and metabolizing the substrate in a complex microbial community. The Sipros algorithm is used in proteomic SIP to identify variably labeled proteins and quantify their isotopic enrichment levels (atom%) by performing enrichment-resolved database searching.

Results: In this study, Sipros was upgraded to improve the labeled protein identification, isotopic enrichment quantification, and database searching speed. The new Sipros 4 was compared with the existing Sipros 3, Calisp, and MetaProSIP in terms of the number of identifications and the accuracy and precision of atom% quantification on both the peptide and protein levels using standard E. coli cultures with 1.07 atom%, 2 atom%, 5 atom%, 25 atom%, 50 atom%, and 99 atom% 13C enrichment. Sipros 4 outperformed Calisp and MetaProSIP across all samples, especially in samples with ≥ 5 atom% 13C labeling. The computational speed on Sipros 4 was > 20 times higher than Sipros 3 and was on par with the overall speed of Calisp- and MetaProSIP-based pipelines. Sipros 4 also demonstrated higher sensitivity for the detection of labeled proteins in two 13C-SIP experiments on a real-world soil community. The labeled proteins were used to trace 13C from 13C-methanol and 13C-labeled plant exudates to the consuming soil microorganisms and their newly synthesized proteins.

Conclusion: Overall, Sipros 4 improved the quality of the proteomic SIP results and reduced the computational cost of SIP database searching, which will make proteomic SIP more useful and accessible to the border community. Video Abstract.

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利用升级版 Sipros 算法进行蛋白质组稳定同位素探测,改进同位素标记蛋白质的鉴定和定量。
背景:蛋白质组稳定同位素探针(SIP)用于微生物生态学,以追踪非放射性同位素从标记底物到特定种群中从头合成蛋白质的过程,这些种群在复杂的微生物群落中积极同化和代谢底物。在蛋白质组 SIP 中使用 Sipros 算法识别可变标记蛋白质,并通过富集分辨数据库搜索量化其同位素富集水平(原子%):本研究对 Sipros 进行了升级,以提高标记蛋白质识别、同位素富集量化和数据库搜索速度。使用 13C 富集度分别为 1.07 原子%、2 原子%、5 原子%、25 原子%、50 原子% 和 99 原子%的标准大肠杆菌培养物,比较了新的 Sipros 4 与现有的 Sipros 3、Calisp 和 MetaProSIP 在多肽和蛋白质水平上的鉴定数量以及原子%定量的准确性和精确性。在所有样本中,Sipros 4 的性能均优于 Calisp 和 MetaProSIP,尤其是在 13C 标记≥ 5 个原子%的样本中。Sipros 4 的计算速度是 Sipros 3 的 20 倍以上,与基于 Calisp 和 MetaProSIP 的管道的总体速度相当。在对真实世界土壤群落进行的两次 13C-SIP 实验中,Sipros 4 也表现出了更高的标记蛋白质检测灵敏度。标记蛋白质被用来追踪 13C 甲醇和 13C 标记植物渗出物中的 13C 含量,以及消耗的土壤微生物和它们新合成的蛋白质:总之,Sipros 4 提高了蛋白质组 SIP 结果的质量,降低了 SIP 数据库搜索的计算成本,这将使蛋白质组 SIP 对边境社区更有用、更易获取。视频摘要
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来源期刊
Microbiome
Microbiome MICROBIOLOGY-
CiteScore
21.90
自引率
2.60%
发文量
198
审稿时长
4 weeks
期刊介绍: Microbiome is a journal that focuses on studies of microbiomes in humans, animals, plants, and the environment. It covers both natural and manipulated microbiomes, such as those in agriculture. The journal is interested in research that uses meta-omics approaches or novel bioinformatics tools and emphasizes the community/host interaction and structure-function relationship within the microbiome. Studies that go beyond descriptive omics surveys and include experimental or theoretical approaches will be considered for publication. The journal also encourages research that establishes cause and effect relationships and supports proposed microbiome functions. However, studies of individual microbial isolates/species without exploring their impact on the host or the complex microbiome structures and functions will not be considered for publication. Microbiome is indexed in BIOSIS, Current Contents, DOAJ, Embase, MEDLINE, PubMed, PubMed Central, and Science Citations Index Expanded.
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