Immunodepletion Plasma Proteomics by TripleTOF 5600 and Orbitrap Elite/LTQ-Orbitrap Velos/Q Exactive Mass Spectrometers

Plasma
proteomic experiments performed rapidly and economically
using several of the latest high-resolution mass spectrometers were
compared. Four quantitative hyperfractionated plasma proteomics experiments
were analyzed in replicates by two AB SCIEX TripleTOF 5600 and three
Thermo Scientific Orbitrap (Elite/LTQ-Orbitrap Velos/Q Exactive) instruments.
Each experiment compared two iTRAQ isobaric-labeled immunodepleted
plasma proteomes, provided as 30 labeled peptide fractions, and 480
LC–MS/MS runs delivered >250 GB of data in 2 months. Several
analysis algorithms were compared. At 1% false discovery rate, the
relative comparative findings concluded that the Thermo Scientific
Q Exactive Mass Spectrometer resulted in the highest number of identified
proteins and unique sequences with iTRAQ quantitation. The confidence
of iTRAQ fold-change for each protein is dependent on the overall
ion statistics (Mascot Protein Score) attainable by each instrument.
The benchmarking also suggested how to further improve the mass spectrometry
parameters and HPLC conditions. Our findings highlight the special
challenges presented by the low abundance peptide ions of iTRAQ plasma
proteome because the dynamic range of plasma protein abundance is
uniquely high compared with cell lysates, necessitating high instrument
sensitivity.

本研究对采用多款最新高分辨率质谱仪快速且经济高效地开展的血浆蛋白质组学实验进行了对比分析。本研究针对4次定量超分级血浆蛋白质组学实验进行了重复分析，所用仪器包括2台AB SCIEX TripleTOF 5600质谱仪与3台赛默飞世尔Orbitrap系列质谱仪（Elite/LTQ-Orbitrap Velos/Q Exactive）。每次实验均对比两份经iTRAQ（同重同位素标记相对和绝对定量）免疫耗竭处理的血浆蛋白质组，实验以30个标记肽段分级组分的形式开展，共计480次LC-MS/MS分析，在2个月内产生了超过250 GB的原始数据。本研究同时对多款分析算法进行了对比。在1%假发现率（false discovery rate）的筛选阈值下，相对定量分析结果显示，赛默飞世尔Q Exactive质谱仪在采用iTRAQ定量时，可鉴定到最多的蛋白质与独特肽段序列。各蛋白质的iTRAQ定量倍变化置信度，取决于各仪器可获取的整体离子统计数据，即Mascot蛋白质得分（Mascot Protein Score）。本次基准测试还提出了进一步优化质谱参数与高效液相色谱（HPLC，High Performance Liquid Chromatography）条件的可行方向。本研究结果凸显了iTRAQ标记血浆蛋白质组中低丰度肽段离子所面临的特殊挑战：相较于细胞裂解液，血浆蛋白质丰度的动态范围极高，因此对仪器灵敏度有着极高要求。