Center for iPS Cell Research and Application , Kyoto University , Kyoto 606-8507 , Japan.
Graduate school of Pharmaceutical Sciences , Kyoto University , Kyoto 606-8501 , Japan.
J Proteome Res. 2019 Jun 7;18(6):2535-2544. doi: 10.1021/acs.jproteome.9b00078. Epub 2019 May 6.
Rapid progress in mass spectrometry (MS) has made comprehensive analyses of the proteome possible, but accurate quantification remains challenging. Isobaric tags for relative and absolute quantification (iTRAQ) is widely used as a tool to quantify proteins expressed in different cell types and various cellular conditions. The quantification precision of iTRAQ is quite high, but the accuracy dramatically decreases in the presence of interference peptides that are coeluted and coisolated with the target peptide. Here, we developed "removal of interference mixture MS/MS spectra (RiMS)" to improve the quantification accuracy of isobaric tag approaches. The presence of spectrum interference is judged by examining the overlap in the elution time of all scanned precursor ions. Removal of this interference decreased protein identification (11% loss) but improved quantification accuracy. Further, RiMS does not require any specialized equipment, such as MS instruments or an additional ion separation mode. Finally, we demonstrated that RiMS can be used to quantitatively compare human-induced pluripotent stem cells and human dermal fibroblasts, as it revealed differential protein expressions that reflect the biological characteristics of the cells.
质谱（MS）技术的快速发展使得对蛋白质组进行全面分析成为可能，但准确的定量仍然具有挑战性。相对和绝对定量同位素标记（iTRAQ）被广泛用作一种工具，用于定量不同细胞类型和各种细胞条件下表达的蛋白质。iTRAQ 的定量精度非常高，但在存在与目标肽共洗脱和共分离的干扰肽时，准确性会显著降低。在这里，我们开发了“去除干扰混合物 MS/MS 谱（RiMS）”以提高等压标记方法的定量准确性。通过检查所有扫描前体离子的洗脱时间重叠来判断谱干扰的存在。去除这种干扰会降低蛋白质鉴定（损失 11%），但会提高定量准确性。此外，RiMS 不需要任何特殊设备，例如 MS 仪器或额外的离子分离模式。最后，我们证明 RiMS 可用于定量比较人诱导多能干细胞和人真皮成纤维细胞，因为它揭示了反映细胞生物学特征的差异蛋白表达。
