Mass spectrometry (MS)-based proteomics is a powerful tool for identifying and quantifying proteins. However, chimeric spectra caused by the fragmentation of multiple precursors within the same isolation window impair the accuracy of peptide identification and isobaric mass tag-based quantification. While there have been advances in computational deconvolution of chimeric spectra and methods to further separate the peptides by ion mobility or through MSn, the use of narrower isolation windows to decrease the fraction of chimeric species remains to be fully explored. We present results obtained on a SCIEX TripleTOF instrument where the quadrupole was optimized and tuned for precursor isolation at 0.1 Da (FWHH). Using a three-proteome model (trypsin digest of protein lysates from yeast, human and ) and 8-plex iTRAQ labeling to document the interference effect, we investigated the impact of co-fragmentation on spectral purity, identification accuracy and quantification accuracy. The narrow quadrupole isolation window significantly improved the spectral purity and reduced the interference of non-target precursors on quantification accuracy. The high-resolution isolation strategy also reduced the number of false identifications caused by chimeric spectra. While these improvements came at the cost of sensitivity loss, combining high-resolution isolation with other advanced techniques, including ion mobility, may result in improved accuracy in identification and quantification. Compared to standard-resolution quadrupole isolation (0.7 Da), high-resolution quadrupole isolation (0.1 Da) significantly improved the spectral purity and quantification accuracy while reducing the number of potential false identifications caused by chimeric spectra, thus showing excellent potential for further development to analyze clinical proteomics samples, for which high accuracy is essential.

中文翻译：

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高分辨率四极杆可提高光谱纯度并减少同量异位多重定量蛋白质组学中非目标离子的干扰


基于质谱 (MS) 的蛋白质组学是识别和定量蛋白质的强大工具。然而，由同一分离窗口内多个前体碎片引起的嵌合光谱损害了肽鉴定和基于同量异位质量标签的定量的准确性。虽然嵌合光谱的计算解卷积和通过离子淌度或 MSn 进一步分离肽的方法已经取得了进展，但使用更窄的分离窗口来减少嵌合物种的比例仍有待充分探索。我们展示了在 SCIEX TripleTOF 仪器上获得的结果，其中四极杆经过优化和调整，可实现 0.1 Da (FWHH) 的前体分离。我们使用三蛋白质组模型（酵母、人类和蛋白质裂解物的胰蛋白酶消化）和 8 重 iTRAQ 标记来记录干扰效应，研究了共断裂对光谱纯度、识别精度和定量精度的影响。窄四极杆隔离窗口显着提高了光谱纯度，减少了非目标前体对定量精度的干扰。高分辨率分离策略还减少了由嵌合谱引起的错误识别的数量。虽然这些改进是以灵敏度损失为代价的，但将高分辨率隔离与其他先进技术（包括离子淌度）相结合可能会提高识别和定量的准确性。与标准分辨率四极杆隔离 (0.7 Da) 相比，高分辨率四极杆隔离 (0.7 Da)1 Da）显着提高了光谱纯度和定量精度，同时减少了嵌合光谱引起的潜在错误鉴定的数量，从而显示出进一步开发分析临床蛋白质组学样品的巨大潜力，而高精度是至关重要的。