Synovial fluid (SF) is of great interest for the investigation of orthopedic pathologies, as it is in close proximity to various tissues that are primarily altered during these disease processes and can be collected using minimally invasive protocols. Multi-“omic” approaches are commonplace, although little consideration is often given for multiple analysis techniques at sample collection. Nuclear magnetic resonance (NMR) metabolomics and liquid chromatography tandem mass spectrometry (LC-MS/MS) proteomics are two complementary techniques particularly suited to the study of SF. However, currently there are no agreed upon standard protocols that are published for SF collection and processing for use with NMR metabolomic analysis. Furthermore, the large protein concentration dynamic range present within SF can mask the detection of lower abundance proteins in proteomics. While combinational ligand libraries (ProteoMiner columns) have been developed to reduce this dynamic range, their reproducibility when used in conjunction with SF, or on-bead protein digestion protocols, has yet to be investigated. Here we employ optimized protocols for the collection, processing, and storage of SF for NMR metabolite analysis and LC-MS/MS proteome analysis, including a Lys-C endopeptidase digestion step prior to tryptic digestion, which increased the number of protein identifications and improved reproducibility for on-bead ProteoMiner digestion.

中文翻译：

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NMR 代谢组学和 LC-MS/MS 蛋白质组学的滑液收集和处理优化。

滑液 (SF) 对于骨科病理学的研究非常感兴趣，因为它靠近在这些疾病过程中主要改变的各种组织，并且可以使用微创方案收集。多“组学”方法很常见，尽管在样本采集时通常很少考虑多种分析技术。核磁共振 (NMR) 代谢组学和液相色谱串联质谱 (LC-MS/MS) 蛋白质组学是两种特别适合 SF 研究的互补技术。然而，目前还没有为 SF 收集和处理用于 NMR 代谢组学分析而发布的标准协议。此外，SF 中存在的大蛋白质浓度动态范围可以掩盖蛋白质组学中较低丰度蛋白质的检测。虽然已经开发了组合配体文库（ProteoMiner 柱）以降低此动态范围，但它们与 SF 或珠上蛋白质消化方案结合使用时的重现性仍有待研究。在这里，我们采用优化方案来收集、处理和存储 SF 以进行 NMR 代谢物分析和 LC-MS/MS 蛋白质组分析，包括胰蛋白酶消化前的 Lys-C 内肽酶消化步骤，这增加了蛋白质鉴定的数量并改进了珠上 ProteoMiner 消化的可重复性。它们与 SF 或珠上蛋白质消化方案结合使用时的重现性还有待研究。在这里，我们采用优化方案来收集、处理和存储 SF 以进行 NMR 代谢物分析和 LC-MS/MS 蛋白质组分析，包括胰蛋白酶消化前的 Lys-C 内肽酶消化步骤，这增加了蛋白质鉴定的数量并改进了珠上 ProteoMiner 消化的可重复性。它们与 SF 或珠上蛋白质消化方案结合使用时的重现性还有待研究。在这里，我们采用优化方案来收集、处理和存储 SF 以进行 NMR 代谢物分析和 LC-MS/MS 蛋白质组分析，包括胰蛋白酶消化前的 Lys-C 内肽酶消化步骤，这增加了蛋白质鉴定的数量并改进了珠上 ProteoMiner 消化的可重复性。