Department of the Geophysical Sciences, University of Chicago , 5734 South Ellis Avenue, Chicago, Illinois 60637, United States.
Anal Chem. 2017 Nov 7;89(21):11498-11504. doi: 10.1021/acs.analchem.7b02752. Epub 2017 Oct 23.
We present an analytical strategy, dimethylation-deuteration and oxygen-exchange IPTL (diDO-IPTL), for high-precision, broad-coverage quantitative proteomics. The diDO-IPTL approach combines two advances in isobaric peptide terminal labeling (IPTL) methodology: first, a one-pot chemical labeling strategy for attaching isotopic tags to both the N- and C-termini of tryptic peptides, and second, a search engine (based on the Morpheus algorithm) optimized for identification and quantification of twinned peaks from peptide fragment ions in MS spectra. The diDO-IPTL labeling chemistry uses only high-purity, relatively inexpensive isotopic reagents (O water and deuterated formaldehyde) and requires no postlabeling cleanup or isotopic impurity corrections. This strategy produces proteome-scale relative quantification results with high accuracy and precision, suitable for the detection of small protein abundance variations between complex biological samples. In a two-proteome mixture experiment, diDO-IPTL quantification discriminates 1.5-fold changes in abundance of over 1000 proteins with 88% accuracy. The diDO-IPTL methodology is a high-precision, economical approach to quantitative proteomics that is applicable to a wide variety of sample types.
我们提出了一种分析策略，即二甲基化-氘代和氧交换 IPTL（diDO-IPTL），用于高精度、广覆盖的定量蛋白质组学。diDO-IPTL 方法结合了同位肽末端标记（IPTL）方法学的两项进展：首先，是一种将同位素标记物同时连接到胰蛋白酶肽的 N-和 C-末端的一锅化学标记策略，其次，是一种基于 Morpheus 算法的搜索引擎，用于鉴定和定量 MS 谱中肽片段离子的双峰。diDO-IPTL 标记化学仅使用高纯、相对廉价的同位素试剂（O 水和氘代甲醛），且无需进行标记后清理或同位素杂质校正。该策略可产生具有高精度和高重复性的蛋白质组规模相对定量结果，适用于检测复杂生物样本中蛋白质丰度的微小变化。在两个蛋白质组混合物实验中，diDO-IPTL 定量可准确区分超过 1000 种蛋白质丰度 1.5 倍的变化，准确率为 88%。diDO-IPTL 方法是一种高精度、经济的定量蛋白质组学方法，适用于多种类型的样品。
