iTRAQ定量蛋白质组分析中多级质谱采集策略对比分析

Comparison of multi-stage mass spectrometry in the iTRAQ-based quantitative proteomics

目的比较二级和三级质谱两种碎裂模式对基于同位素标记定量的蛋白质组学技术准确度的影响。方法采集斑马鱼在氧气浓度5%条件下,0,24,48 h取样斑马鱼脑组织蛋白样品,提取蛋白质进行i TRAQ标记,高p H值反相柱分离成15个组分,分别采用纳升液相色谱分离肽段,串级质谱(MS2)或三级质谱(MS3)两种方法碎裂肽段,利用MaxQuant定量蛋白质组分析软件,获得报告离子强度信息。结果 MS2碎裂和MS3碎裂分别鉴定到7 624个和6 940个蛋白质;MS3碎裂获得的差异蛋白质数量明显多于MS2,分别为173和641个,相同的蛋白质,MS3的差异比值距离1∶1的距离更远。虽然MS3鉴定的谱图数较低,但鉴定到的肽段和蛋白质数目降低不明显。结论对基于i TRAQ标记的定量蛋白质组研究策略,MS3质谱分析提供更为显著的差异分析结果,是更优的选择。

Objective To compare the effect of two fragmentation modes,MS/MS( MS2) and MS/MS/MS( MS3),on the accuracy of isotope-labeling-based quantitative proteomic techniques. Methods The brain tissue proteins of zebrafish treated under 5% hypoxia concentration for 0,24,48 h were labeled with i TRAQ,and the high p H reversed phase column was used to separate the peptides into15 fractions. The peptides were fragmented by MS2 or MS3. The MaxQuant software was used to search the database and obtain the report ion intensities. Results By MS2 or MS3 fragmentation,7 624 or 6 940 proteins were identified respectively. The number of differential proteins obtained by MS3 fragmentation was significantly higher than that of MS2,which were 641 and 173 respectively. The distance of the protein ratios from 1 ∶ 1 was larger by MS3 than that of MS2. Although the number of spectra identified by MS3 was lower,the number of identified peptides and proteins was not significantly reduced. Conclusion For the strategy of quantitative proteomics based on i TRAQ labeling,MS3 mass spectrometry provides more significantly differential results,and can be a better choice.