使用LTQ Orbitrap的HCD裂解定量iTRAQ标记肽段

　　简介：The primary goal of this work was to systematically optimize the experimental conditions for relative quantitation of iTRAQ labeled peptides using HCD on the LTQ Orbitrap XL. In a previous comprehensive study, we compared the performance of LTQ Orbitrap XL and QSTAR® Elite for peptide identifications in complex mixtures.10 In this study, peptides labeled with four iTRAQ mass tags were analyzed using both the LTQ Orbitrap XL (referred to in the text as LTQ Orbitrap) and QSTAR Elite (Applied Biosystems, Foster City, CA) (referred to in the text as QqTOF) to assess the performance of both platforms in a quantitative proteomics experiment. We deliberately chose a relatively simple peptide mixture with high peptide concentrations and limited dynamic range for these comparative experiments so not to disadvantage the slower scanning, less sensitive instrument (QqTOF) when assessing protein sequence coverage, quantitative precision and accuracy.10 The experiments run on the LTQ Orbitrap were performed at the Thermo Fisher Scientific labs in San Jose, CA. QqTOF analyses were performed by an independent third party laboratory. The experiments were conducted by a certified SCIEX®-trained operator to ensure the highest possible quality results.

　　仪器：LTQ Orbitrap

　　结论：

　　1. HCD with normalized collision energy can successfully generate high quality accurate mass (3 ppm RMS) MS/MS spectra that contain abundant iTRAQ reporter ions. This allows HCD spectra of iTRAQ labeled peptides to be used for simultaneous protein identification and quantitation.

　　2. The parallel acquisition in which Data Dependent MS/MS events are performed in the ion trap at the same time as accurate mass measurements of the precursor ions (3 ppm RMS) in the Orbitrap further increase the protein sequence coverage in HCD-based experiments.

　　3. The high resolution and low chemical noise of the Orbitrap spectra are indispensible when quantifying iTRAQ reporter ions. Isobaric interferences are common in the low mass region and require resolving power in excess of 11 000 FWHM to separate them from the iTRAQ reporter ions. This in turn significantly improves the accuracy and precision of quantitation. The benefits of using a resolving power of 16 000 were clearly observed (Figure 6) when analyzing relatively non-complex mixture. It can be inferred that the benefits would be considerable, and indeed essential for the quantitative analysis of low level peptides present within a complex peptide mixture (e.g. mudpit).

　　4. The accuracy of the iTRAQ ratios averaged 4% relative error at the protein level and 9% at the peptide level.

　　5. The relative variability (measure of precision) of iTRAQ ratios averaged 2.4% RSD at the protein level and 10% at the peptide level.

　　6. The LTQ Orbitrap outperforms the QqTOF for both qualitative and iTRAQ based quantitative protein analysis, as ndicated by better protein sequence coverage and significantly greater quantitative accuracy on protein level and precision and accuracy on peptide level. In particular the low precision for quantitation at the peptide level is likely attributed to the lack of routinely obtainable high resolving power and the high chemical noise of QqTOF mass analyzers making it difficult to separate iTRAQ reporters from their isobaric interferences.