Comparison of different signal thresholds on data dependent sampling in Orbitrap and LTQ mass spectrometry for the identification of peptides and proteins in complex mixtures

Abstract

We evaluate the effect of ion-abundance threshold settings for data-dependent acquisition on a hybrid LTQ-Orbitrap mass spectrometer, analyzing features such as the total number of spectra collected, the signal to noise ratio of the full MS scans, the spectral quality of the tandem mass spectra acquired, and the number of peptides and proteins identified from a complex mixture. We find that increasing the threshold for data-dependent acquisition generally decreases the quantity but increases the quality of the spectra acquired. This is especially true when the threshold setting is set above the noise level of the full MS scan. We compare two distinct experimental configurations: one where full MS scans are acquired in the Orbitrap analyzer while tandem MS scans are acquired in the LTQ analyzer, and one where both full MS and tandem MS scans are acquired in the LTQ analyzer. We examine the number of spectra, peptides, and proteins identified under various threshold conditions, and we find that the optimal threshold setting is at or below the respective noise level of the instrument regardless of whether the full MS scan is performed in the Orbitrap or in the LTQ analyzer. When comparing the high-throughput identification performance of the two analyzers, we conclude that, used at optimal threshold levels, the LTQ and the Orbitrap identify similar numbers of peptides and proteins. The higher scan speed of the LTQ, which results in more spectra being collected, is roughly compensated by the higher mass accuracy of the Orbitrap, which results in improved database searching and peptide validation software performance.

Figure 1

(a) Base peak chromatogram of solvent blank for a single phase run on the LTQ analyzer (the intensity of solvent impurity at about 3e5). (b) Amplified baseline of chromatogram between time points t = 21 minutes and t = 32 minutes. The average peak intensity is determined to be at approximately 50% of the baseline peak, or approximately 1e4 intensity units.

Figure 2

Comparison of number of spectra acquired by the LTQ (red) and Orbitrap (blue) analyzers as a function of threshold trigger level. Each data point was obtained by averaging over 3 replicate experiments.

Figure 3

Distribution of normalized quality scores for tandem mass spectra, as a function of threshold trigger level. (a) Precursor scan in the LTQ analyzer. (b) Precursor scan in the Orbitrap analyzer.

Figure 4

Distributions of signal to noise ratios (S/N) of the precursor ions for three replicate experiments in (a) the LTQ analyzer and (b) the Orbitrap analyzer, as a function of threshold trigger values.

Figure 5

Comparison of the total number of peptides confidently identified in the LTQ experiments (red) and the Orbitrap experiments (blue), as a function of threshold trigger level. Each data point was obtained by averaging over 3 replicate experiments.

Figure 6

Distribution of cross-correlation-based Z-scores and mass deviation values for peptide matches to spectra whose precursors were isolated in the Orbitrap analyzer. The black dots represent forward database matches, while the red dots represent decoy (reverse) database matches. The black line, calculated by discriminant analysis, separates the high confidence region (p > 0.9) above it from the low confidence region (p < 0.9) below it. Note that the Orbitrap confidently identifies many peptides whose scores are poor, but whose theoretical masses are within a few ppm of the experimental values.