This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. 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. (J Am Soc Mass Spectrom 2009, xx, xxx) ? 2009 Published by Elsevier Inc. on behalf of American Society for Mass Spectrometry