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RATIONALE

There is interest in extending bottom-up proteomics to the smallest possible sample size. We investigated the performance of two modern mass spectrometers for the analysis of samples ranging from 1 ng to 1 µg of RAW 264.7 cell lysate digests.

METHODS

An ultra-performance liquid chromatography (UPLC) system coupled with either an LTQ-Orbitrap Velos or a Q-Exactive mass spectrometer was used for peptide separation and identification.

RESULTS

For 1–1000 ng RAW 264.7 cell lysate digests, the Q-Exactive generated 10–83% more protein groups and 11–109% more peptides than the LTQ-Orbitrap Velos (higher-energy collisional dissociation, HCD) with MASCOT database searching, due to its faster scan rate and higher resolution. In addition, HCD and collision-induced dissociation (CID) modes of the LTQ-Orbitrap Velos were compared. HCD produced higher peptide and protein group IDs than CID for 1–1000 ng RAW 264.7 cell lysate digests with MASCOT database searching. Database searching results from SEQUEST and MASCOT were also compared and comparable protein group IDs were obtained from the two search engines.

CONCLUSIONS

The Q-Exactive outperformed the LTQ-Orbitrap Velos for shotgun proteomics analysis of 1 to 1000 ng RAW 264.7 cell lysate digests in terms of obtained peptide and protein group IDs. Copyright © 2012 John Wiley & Sons, Ltd.