A comparison of MS/MS-based, stable-isotope-labeled, quantitation performance on ESI-quadrupole TOF and MALDI-TOF/TOF mass spectrometers
A comparison of MS/MS-based, stable-isotope-labeled, quantitation performance on ESI-quadrupole TOF and MALDI-TOF/TOF mass spectrometers
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DOI：
10.1002/pmic.200800412
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发表时间：
2009-06-01
期刊：
影响因子：
3.4
通讯作者：
Borchers, Christoph H.
中科院分区：
文献类型：
--
作者：
Kuzyk, Michael A.;Ohlund, Leanne B.;Borchers, Christoph H.
通过分析含有规定丰度（1：1、2.5：1、5：1和10：1）的iTRAQ标记肽的相同大肠杆菌胰蛋白酶，比较了LC-ESI（QSTAR Elite）和LC-MALDI（4800 MALDI TOF/TOF）基于肽的定量准确度和精密度。仅51.4%的QSTAR光谱用于ProteinPilot软件定量，而LC-MALDI光谱为66.7%。LC-ESI和LC-MALDI的平均蛋白质序列覆盖率分别为24.0和18.2%（每个蛋白质14.9和8.4个肽）。分析了ProteinPilot根据57467 ESI-MS/MS和26085 MALDI-MS/MS光谱测定的基于iTRAQ的表达率的测量准确度和重现性。当样品中肽的相对丰度从1：1增加到10：1时，在两种仪器上计算的平均比值在平台之间仅相差0.7-6.7%。在10：1实验中，来自LC-ESI MS/MS光谱的高达64.7%的iTRAQ比率不符合SIN阈值，并从定量中排除，而仅0.1%的等效LC-MALDI iTRAQ比率被拒绝。储存5个月的存档LC-MALDI样品集的再分析生成3715个MS/MS光谱用于定量，与最初获得的3845个相比，平均比率仅相差3.1%。总体而言，离线LC-MALDI的基于MS/MS的肽定量性能与在线LC-ESI相当，在线LC-ESI需要的时间少三倍。然而，离线LC-MALDI允许对存档的HPLC分离样品进行重新分析。
The peptide-based quantitation accuracy and precision of LC-ESI (QSTAR Elite) and LC-MALDI (4800 MALDI TOF/TOF) were compared by analyzing identical Escherichia coli tryptic digests containing iTRAQ-labeled peptides of defined abundances (1:1, 2.5:1, 5:1, and 10:1). Only 51.4% of QSTAR spectra were used for quantitation by ProteinPilot Software versus 66.7% of LC-MALDI spectra. The average protein sequence coverages for LC-ESI and LC-MALDI were 24.0 and 18.2% (14.9 and 8.4 peptides per protein), respectively. The iTRAQ-based expression ratios determined by ProteinPilot from the 57467 ESI-MS/MS and 26085 MALDI-MS/MS spectra were analyzed for measurement accuracy and reproducibility. When the relative abundances of peptides within a sample were increased from 1:1 to 10:1, the mean ratios calculated on both instruments differed by only 0.7-6.7% between platforms. In the 10:1 experiment, up to 64.7% of iTRAQ ratios from LC-ESI MS/MS spectra failed SIN thresholds and were excluded from quantitation, while only 0.1% of the equivalent LC-MALDI iTRAQ ratios were rejected. Re-analysis of an archived LC-MALDI sample set stored for 5 months generated 3715 MS/MS spectra for quantitation, compared with 3845 acquired originally, and the average ratios differed by only 3.1%. Overall, MS/MS-based peptide quantitation performance of offline LC-MALDI was comparable with on-line LC-ESI, which required threefold less time. However, offline LC-MALDI allows the re-analysis of archived HPLC-separated samples.