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The hybrid FT mass spectrometers (i.e., LTQ Orbitrap) can be used in several different data acquisition modes. The accurate mass measurement of the precursor peptide ion is a prerequisite for confident protein identification, and should not be compromised. But MS/MS fragmentation spectra can be acquired using either an ion trap (low resolution/low mass accuracy—fast) or an orbitrap (high resolution/mass accuracy—slower) analyser as a detector. Moreover, different fragmentation techniques can be employed, such as collisionally induced dissociation (CID) (in an ion trap) or higher energy collisional dissociation (HCD) (in a C-trap). One would expect these parameters to impact significantly on the results of protein identification by database searching or de novo sequencing.
We carried out an extensive evaluation of the datasets acquired on LTQ Orbitrap using various method settings. The particular questions we addressed in this study were:
Which method delivers maximum sequence coverage for detected proteins?
Which method provides data most suitable for automated de novo sequencing?
What is the performance of PEAKS for automated de novo sequencing?
These questions were answered in the context of LC-MS analyses of a protein digest sample of medium complexity (six proteins).
The results showed that performing MS/MS fragmentation in the ion trap (CID with the ion-trap detection) and in the C-trap (HCD with the orbtitrap detection) are roughly equivalent with respect to the total protein sequence coverage obtained. However, when automated de novo sequencing is to be employed, the latter method afforded far superior results. Twice as many complete correct sequences were obtained for HCD data compared to LTQ fragmentation spectra.