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. 2011 Feb 15;83(4):1469-74.
doi: 10.1021/ac102265w. Epub 2011 Jan 28.

Improved Precision of iTRAQ and TMT Quantification by an Axial Extraction Field in an Orbitrap HCD Cell

Free PMC article

Improved Precision of iTRAQ and TMT Quantification by an Axial Extraction Field in an Orbitrap HCD Cell

Peter Pichler et al. Anal Chem. .
Free PMC article


Improving analytical precision is a major goal in quantitative differential proteomics as high precision ensures low numbers of outliers, a source of false positives with regard to quantification. In addition, higher precision increases statistical power, i.e., the probability to detect significant differences. With chemical labeling using isobaric tags for relative and absolute quantitation (iTRAQ) or tandem mass tag (TMT) reagents, quantification is based on the extraction of reporter ions from tandem mass spectrometry (MS/MS) spectra. We compared the performance of two versions of the LTQ Orbitrap higher energy collisional dissociation (HCD) cell with and without an axial electric field with regard to reporter ion quantification. The HCD cell with the axial electric field was designed to push fragment ions into the C-trap and this version is mounted in current Orbitrap XL ETD and Orbitrap Velos instruments. Our goal was to evaluate whether the purported improvement in ion transmission had a measurable impact on the precision of MS/MS based quantification using peptide labeling with isobaric tags. We show that the axial electric field led to an increased percentage of HCD spectra in which the complete set of reporter ions was detected and, even more important, to a reduction in overall variance, i.e., improved analytical precision of the acquired data. Notably, adequate precision of HCD-based quantification was maintained even for low precursor ion intensities of a complex biological sample. These findings may help researchers in their design of quantitative proteomics studies using isobaric tags and establish HCD-based quantification on the LTQ Orbitrap as a highly precise approach in quantitative proteomics.


Figure 1
Figure 1
A HCD cell with an axial field decreases the fraction of HCD spectra with missing reporter ions and improves analytical precision. Analysis of a standard protein mixture on an LTQ Orbitrap equipped with the original HCD cell as compared to a HCD cell with an axial field. Panel A indicates the percentage of HCD spectra where one or more reporter ions failed to be detected. The geometric standard deviation of “duplicate” channels is depicted in panel B (for the ratio 115:114) and panel C (for the ratio 117:116). Panel D shows the fraction of the precursor ion currents that can be detected as reporter ion currents, as a measure of fragment ion generation and transmission. In each panel (A−D) individual measurements are shown on the left side, whereas arithmetic averages with one-sided error bars denoting 1 SD are shown on the right side. p-values calculated as a 2-sided paired t test for the comparison of the original HCD cell vs the HCD cell with an axial field.
Figure 2
Figure 2
The axial field improves reporter ion transmission, thereby increasing the overall precision of data acquired with the axial field. Scatter plots illustrating the variation of duplicate channel ratios in relation to reporter ion signals. Each data point represents a peptide-spectrum match. The log2 of duplicate channel ratios on the y-axis is plotted against a measure of reporter ion signal intensity. In panel A the x-axis is proportional to the log2 of the geometric mean of reporter ion areas 114 and 115, whereas in panel B it is proportional to the rank of the mean thus ensuring an even distribution of data points along the x-axis. Data are from the analyses of the iTRAQ 4-plex labeled HeLa sample (nocodazole sample split before labeling). Precision is high where the dispersion of the data points is narrow.

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