SCIEX, Vaughan, Ontario, L4K 4V8, Canada; Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Sinai Health, Toronto, Ontario, M5G 1X5, Canada; NHC Key Laboratory of Human Stem Cell and Reproductive Engineering, School of Basic Medical Sciences, Central South University, Changsha, Hunan, 410075, China; Clinical Research Center for Reproduction and Genetics in Hunan Province, Reproductive and Genetic Hospital of CITIC-XIANGYA, Changsha, Hunan, 410000, China.
SCIEX, Vaughan, Ontario, L4K 4V8, Canada.
Anal Chim Acta. 2024 Oct 9;1325:343135. doi: 10.1016/j.aca.2024.343135. Epub 2024 Aug 22.
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 E. coli) 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.
基于质谱（MS）的蛋白质组学是一种用于鉴定和定量蛋白质的强大工具。然而，在同一分离窗口内多个前体的碎裂会产生嵌合谱，从而影响肽鉴定的准确性和基于等压质量标签的定量准确性。虽然在计算上对嵌合谱进行解卷积以及通过离子淌度或 MSn 进一步分离肽的方法方面已经取得了进展，但使用更窄的分离窗口来降低嵌合物种的比例仍有待充分探索。
我们在 SCIEX TripleTOF 仪器上获得了结果，该仪器对四极杆进行了优化和调整，以便在 0.1 Da（半峰全宽）下进行前体分离。使用三蛋白组模型（酵母、人类和大肠杆菌蛋白质裂解物的胰蛋白酶消化物）和 8 重 iTRAQ 标记来记录干扰效应，我们研究了共碎裂对光谱纯度、鉴定准确性和定量准确性的影响。窄四极杆隔离窗口显著提高了光谱纯度，并减少了非目标前体对定量准确性的干扰。高分辨率隔离策略还减少了嵌合谱引起的假鉴定数量。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包括离子淌度在内的其他先进技术相结合，可能会提高鉴定和定量的准确性。
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（0.1 Da）显著提高了光谱纯度和定量准确性，同时减少了嵌合谱引起的潜在假鉴定数量，因此具有进一步开发的巨大潜力，可用于分析临床蛋白质组学样本，因为高准确性至关重要。
