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使用高分辨高质量精度的线性离子阱Orbitrap组合质谱对复杂样品中的目标肽段进行定量
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使用高分辨高质量精度的线性离子阱Orbitrap组合质谱对复杂样品中的目标肽段进行定量 |
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简介：Here we describe a targeted HR/AM peptide quantitation workflow on a Thermo Scientific LTQ Orbitrap Velos hybrid mass spectrometer7,8. Identities of targeted peptides present in a complex mixture are verified using selected ion monitoring (SIM) and scheduled MS/MS. In addition, we detail the utility of Thermo Scientific Pinpoint software for generating the targeted HR/AM instrument methods in an automated fashion from previously acquired raw data and/or known protein targets.
仪器：
结论：A workflow for simultaneous quantification and identity verification of targeted peptides using the LTQ Orbitrap Velos hybrid mass spectrometer and Pinpoint software was developed and evaluated. The workflow employs wide mass range SIM scanning to improve the sensitivity and scheduled MS/MS for peptide verification. The highresolution MS analysis (100,000 resolution FWHM) enables detection of co-eluting isobars that would interfere with quantitation. The detection limits were investigated by spiking isotopically labeled yeast peptides into a complex yeast digest matrix at different concentrations. The peptides were detected as low as 10 amol for all but one spiked heavy peptide and the quantitative dynamic range was 4 orders of magnitude.
The 77 peptides selected to represent the 25 targeted yeast proteins in the optimized instrument method were successfully quantified and verified with high analytical precision. Ninety-seven percent of the targeted peptides gave a CV of less than 10%, and the average CV was 4.7%. Workflow detection limits, precision and specificity were enhanced using wide SIM isolations and a timescheduled global MS/MS list.
