Kuzyk Michael A, Ohlund Leanne B, Elliott Monica H, Smith Derek, Qian Hong, Delaney Allen, Hunter Christie L, Borchers Christoph H
Genome British Columbia Proteomics Centre, University of Victoria, Victoria, BC, Canada.
Proteomics. 2009 Jun;9(12):3328-40. doi: 10.1002/pmic.200800412.
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 57 467 ESI-MS/MS and 26 085 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 S/N 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.
通过分析含有定义丰度（1:1、2.5:1、5:1和10:1）的iTRAQ标记肽段的相同大肠杆菌胰蛋白酶消化物，比较了基于肽段的液相色谱 - 电喷雾电离（QSTAR Elite）和液相色谱 - 基质辅助激光解吸电离（4800 MALDI TOF/TOF）的定量准确性和精密度。ProteinPilot软件用于定量分析的QSTAR光谱仅占51.4%，而液相色谱 - 基质辅助激光解吸电离光谱占66.7%。液相色谱 - 电喷雾电离和液相色谱 - 基质辅助激光解吸电离的平均蛋白质序列覆盖率分别为24.0%和18.2%（每个蛋白质分别为14.9和8.4个肽段）。对ProteinPilot从57467个电喷雾电离串联质谱和26085个基质辅助激光解吸电离串联质谱光谱中确定的基于iTRAQ的表达比率进行了测量准确性和重现性分析。当样品中肽段的相对丰度从1:1增加到10:1时，两个平台上计算的平均比率在平台之间仅相差0.7 - 6.7%。在10:1的实验中，液相色谱 - 电喷雾电离串联质谱光谱中高达64.7%的iTRAQ比率未达到信噪比阈值并被排除在定量分析之外，而等效的液相色谱 - 基质辅助激光解吸电离iTRAQ比率只有0.1%被拒绝。对存储5个月的存档液相色谱 - 基质辅助激光解吸电离样品集进行重新分析，生成了3715个用于定量分析的串联质谱光谱，与最初获得的3845个相比，平均比率仅相差3.1%。总体而言，离线液相色谱 - 基质辅助激光解吸电离基于串联质谱的肽段定量性能与在线液相色谱 - 电喷雾电离相当，而所需时间减少了两倍。然而，离线液相色谱 - 基质辅助激光解吸电离允许对存档的高效液相色谱分离样品进行重新分析。
