Orbitrap/LTQ signal threshold

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.

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