下一代蛋白测序和个体离子质谱分析使白细胞介素-6的互补分析成为可能。

IF 3.8 2区 化学 Q1 BIOCHEMICAL RESEARCH METHODS
Kenneth A Skinner, Troy D Fisher, Andrew Lee, Taojunfeng Su, Eleonora Forte, Aniel Sanchez, Michael A Caldwell, Neil L Kelleher
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引用次数: 0

摘要

蛋白质组的巨大复杂性目前压倒了任何单一的分析技术,无法捕获蛋白质种类多样性的全谱。在这项研究中,我们评估了两种尖端蛋白质组学技术的互补性-单分子蛋白质测序和个体离子质谱-用于分析重组人IL-6 (rhIL-6)的氨基酸,肽和完整的蛋白质形式水平。对于单分子蛋白测序,我们使用了最近发布的Platinum仪器。基于铂的下一代蛋白测序(NGPS)利用n端氨基酸识别器结合和氨基肽酶裂解的循环来实现单个肽分子的平行测序。我们发现NGPS产生的单个氨基酸覆盖了IL-6的多个关键区域,包括螺旋A和C内的两个肽,其中含有据报道影响IL-6功能的残基。对于自上而下的蛋白质形态评估,我们使用了单个离子质谱(I2MS),这是一种高度并行的基于轨道阱的电荷检测质谱平台。单离子检测的气相破碎产物(I2MS2)在IL-6的关键区域提供了显著的序列覆盖,包括螺旋B和D内参与IL-6信号传导的两个区域。这些互补技术共同提供了52%的序列覆盖率,提供了比单独使用任何一种技术更完整的IL-6结构和功能多样性视图。本研究强调了这些蛋白质检测方法的互补性,可以更全面地覆盖与生物相互作用相关的蛋白质片段。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Next-generation protein sequencing and individual ion mass spectrometry enable complementary analysis of interleukin-6.

The vast complexity of the proteome currently overwhelms any single analytical technology in capturing the full spectrum of proteoform diversity. In this study, we evaluated the complementarity of two cutting-edge proteomic technologies-single-molecule protein sequencing and individual ion mass spectrometry-for analyzing recombinant human IL-6 (rhIL-6) at the amino acid, peptide, and intact proteoform levels. For single-molecule protein sequencing, we employed the recently released Platinum instrument. Next-generation protein sequencing (NGPS) on Platinum utilizes cycles of N-terminal amino acid recognizer binding and aminopeptidase cleavage to enable parallelized sequencing of single peptide molecules. We found that NGPS produces single amino acid coverage of multiple key regions of IL-6, including two peptides within helices A and C, which harbor residues that reportedly impact IL-6 function. For top-down proteoform evaluation, we used individual ion mass spectrometry (I2MS), a highly parallelized Orbitrap-based charge detection MS platform. Single ion detection of gas-phase fragmentation products (I2MS2) gives significant sequence coverage in key regions of IL-6, including two regions within helices B and D that are involved in IL-6 signaling. Together, these complementary technologies delivered a combined 52% sequence coverage, offering a more complete view of IL-6 structural and functional diversity than either technology alone. This study highlights the complementarity of these protein detection methods to cover protein segments relevant to biological interactions more comprehensively.

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来源期刊
CiteScore
8.00
自引率
4.70%
发文量
638
审稿时长
2.1 months
期刊介绍: Analytical and Bioanalytical Chemistry’s mission is the rapid publication of excellent and high-impact research articles on fundamental and applied topics of analytical and bioanalytical measurement science. Its scope is broad, and ranges from novel measurement platforms and their characterization to multidisciplinary approaches that effectively address important scientific problems. The Editors encourage submissions presenting innovative analytical research in concept, instrumentation, methods, and/or applications, including: mass spectrometry, spectroscopy, and electroanalysis; advanced separations; analytical strategies in “-omics” and imaging, bioanalysis, and sampling; miniaturized devices, medical diagnostics, sensors; analytical characterization of nano- and biomaterials; chemometrics and advanced data analysis.
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