Buck Institute for Research on Aging, Novato, California 94945, USA.
Mol Cell Proteomics. 2012 May;11(5):202-14. doi: 10.1074/mcp.M112.017707. Epub 2012 Mar 26.
Despite advances in metabolic and postmetabolic labeling methods for quantitative proteomics, there remains a need for improved label-free approaches. This need is particularly pressing for workflows that incorporate affinity enrichment at the peptide level, where isobaric chemical labels such as isobaric tags for relative and absolute quantitation and tandem mass tags may prove problematic or where stable isotope labeling with amino acids in cell culture labeling cannot be readily applied. Skyline is a freely available, open source software tool for quantitative data processing and proteomic analysis. We expanded the capabilities of Skyline to process ion intensity chromatograms of peptide analytes from full scan mass spectral data (MS1) acquired during HPLC MS/MS proteomic experiments. Moreover, unlike existing programs, Skyline MS1 filtering can be used with mass spectrometers from four major vendors, which allows results to be compared directly across laboratories. The new quantitative and graphical tools now available in Skyline specifically support interrogation of multiple acquisitions for MS1 filtering, including visual inspection of peak picking and both automated and manual integration, key features often lacking in existing software. In addition, Skyline MS1 filtering displays retention time indicators from underlying MS/MS data contained within the spectral library to ensure proper peak selection. The modular structure of Skyline also provides well defined, customizable data reports and thus allows users to directly connect to existing statistical programs for post hoc data analysis. To demonstrate the utility of the MS1 filtering approach, we have carried out experiments on several MS platforms and have specifically examined the performance of this method to quantify two important post-translational modifications: acetylation and phosphorylation, in peptide-centric affinity workflows of increasing complexity using mouse and human models.
尽管代谢和后代谢标记方法在定量蛋白质组学方面取得了进展，但仍需要改进无标记方法。这种需求对于在肽水平上进行亲和富集的工作流程尤为紧迫，在后一种工作流程中，同量异位化学标记物（如相对和绝对定量的同位素标记物和串联质量标签）可能会出现问题，或者在细胞培养标记中无法轻易应用稳定同位素标记氨基酸的情况。Skyline 是一种免费的、开源的软件工具，用于定量数据处理和蛋白质组学分析。我们扩展了 Skyline 的功能，使其能够处理在 HPLC-MS/MS 蛋白质组学实验中从全扫描质谱数据 (MS1) 获得的肽分析物的离子强度色谱图。此外，与现有程序不同，Skyline MS1 过滤可与来自四个主要供应商的质谱仪一起使用，这允许直接在实验室之间比较结果。Skyline 中现在提供的新定量和图形工具专门支持对 MS1 过滤进行多次采集的查询，包括对峰选择的可视化检查以及自动和手动积分，这是现有软件中经常缺少的关键功能。此外，Skyline MS1 过滤会显示来自光谱库中包含的 MS/MS 数据的保留时间指示器，以确保正确选择峰。Skyline 的模块化结构还提供了定义明确的、可定制的数据报告，从而允许用户直接连接到现有的统计程序进行事后数据分析。为了证明 MS1 过滤方法的实用性，我们在几个 MS 平台上进行了实验，并特别研究了这种方法在使用小鼠和人类模型的肽中心亲和工作流程中定量两种重要的翻译后修饰（乙酰化和磷酸化）的性能。
