Multiplexed peptide analysis using data-independent acquisition and Skyline.
Multiplexed peptide analysis using data-independent acquisition and Skyline.
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DOI：
10.1038/nprot.2015.055
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发表时间：
2015-06
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影响因子：
14.8
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在这里，我们描述了在Q-Exactive质谱仪上使用数据独立采集(DIA)来使用Skyline靶向蛋白质组学环境(可在http://skyline.maccosslab.org).在线免费获得)来检测和定量复杂混合物中的多肽DIA系统地获取MS/MS光谱与DDA相反，后者获取的MS/MS光谱仅适用于鉴定一组随机采样的多肽。与选择性反应监测(SRM)类似，可以使用目标层析提取从DIA数据中定量多肽。与SRM不同，数据采集不限于预先确定的一组目标多肽。在该协议中，使用数据依赖采集(DDA)生成光谱库，并从库中所有多肽的DIA数据中提取色谱图。与SRM类似，DIA数据的定量是基于提取的MS/MS色谱图曲线下的面积。此外，详细介绍了一种适用于DIA的基于MS/MS定向采集的质量控制方法。不包括获取数据的时间和数据库搜索的时间，该过程大约需要1-2个小时才能完成。通常，数据采集需要每个样本大约1-4个小时，数据库搜索需要0.5-2个小时才能完成。
Here we describe the use of data independent acquisition (DIA) on a Q-Exactive mass spectrometer for the detection and quantification of peptides in complex mixtures using the Skyline Targeted Proteomics Environment (freely available on-line at http://skyline.maccosslab.org). The systematic acquisition of MS/MS spectra by DIA is in contrast to DDA where the acquired MS/MS spectra are only suitable for identification of a stochastically sampled set of peptides. Similar to selected reaction monitoring (SRM), peptides can be quantified from DIA data using targeted chromatogram extraction. Unlike SRM, data acquisition is not constrained to a pre-determined set of target peptides. In this protocol, a spectral library is generated using data dependent acquisition (DDA), and chromatograms are extracted from the DIA data for all peptides in the library. Similar to SRM, quantification using DIA data is based on the area under the curve of extracted MS/MS chromatograms. Additionally, a quality control method suitable for DIA based on targeted MS/MS acquisition is detailed. Not including time spent acquiring data, and time for database searching, the procedure takes about 1–2 hours to complete. Typically, data acquisition requires roughly 1–4 hours per sample and a database search will take 0.5–2 hours to complete.