创新数据非依赖性采集用于复杂基质目标蛋白质的定量分析

Quantification Analysis of Targeted Proteins in Complex Sample by Novel Data Independent Acquisition

数据非依赖性采集（ DIA）是随着定量蛋白质组学而建立的质谱扫描技术。 DIA能够获得扫描范围内所有母离子及二级子离子信息,不会造成低丰度离子信息的丢失,同时突破了高分辨质谱二级定量的通量限制。本研究基于静电场轨道阱Q-qIT-OT三合一质谱,发展了经典DIA方法以及WiSIM-DIA和Full MS-DIA两种全新DIA方法,并对Hela细胞全蛋白中添加的10条低浓度肽段进行定量分析,考察方法的线性、重现性和灵敏度。结果表明,3种方法的定量限均低至amol （14～435 amol）,并展示出良好的线性和定性确证可靠性。其中,WiSIM-DIA基于超高分辨一级监测定量,与经典DIA优势互补；Full MS-DIA的选择窗口仅3 amu,能够直接进行搜库鉴定,实现了数据依赖性采集（ DDA）和DIA的统一,摆脱了DIA依赖于DDA建立谱图库的局限性。

Data independent acquisition （ DIA ） is a novel MS scan mode for quantitative proteomics, acquiring all precursors as well as fragments without any loss of low abundant ions, and breaks the throughput limitation of product ion quantification by high-resolution MS. Here we developed three DIA methods on quadrupole-linear ion trap-Orbitrap （ Q-qIT-OT ） Tribrid MS, classic DIA, as well as novel wide isolation window SIM scan （ WiSIM）-DIA and full scan-DIA （ Full MS-DIA） . Quantitative analysis of 10 low abundant peptides spiked in Hela cell digest was performed by the three methods for linearity, reproducibility and sensitivity evaluation. The results showed that the LOQs reached amol level （ 14-435 amol ） with good linearity and effective MS/MS confirmation. WiSIM-DIA utilizes ultra-high resolution SIM scan for quantification complementary with classic DIA. The isolation window of Full MS-DIA was down to 3 amu, and the data could be directly used for database searching, thus realizing the integration of data dependent acquisition （ DDA） and DIA, and avoiding the limitation of using spectra library.