Proteome Analysis Two Ways: A Direct Comparison of Q-Exactive…

Two contemporary mass spectrometers offer the sensitivity, resolution, and efficiency necessary for optimum shotgun analysis of the proteome: the Q-Exactive and LTQ-Orbitrap Velos (both from Thermo Scientific). While both of these instruments offer benefits to researchers, few studies have been performed to evaluate the relative performance of these tools for proteomics-based research with samples of differing sizes.

Sun et al.¹ prepared RAW 264.7 cell lysate for analysis using the Q-Exactive and the LTQ-Orbitrap Velos in both HCD and CID modes. For the Q-Exactive, full MS scans were obtained with a range of m/z 350 to 1800, a mass resolution of 70,000 at m/z 200, and a target value of 1.00E+06. HCD collision was performed on the 12 most significant peaks, and tandem mass spectra were acquired at a mass resolution of 35,000 at m/z 200 and a target value of 1.00E+06. Researchers set the ion threshold at 1.00E+05 counts with maximum allowed ion accumulation at 250 ms for full MS and 120 ms for tandem mass spectra. For the 1- and 5-ng cell lysate samples, an ion selection threshold of 1.00E+04 counts and a maximum ion accumulation time of 250 ms for MS/MS were also used. The dynamic exclusion time was 15 s.

For the LTQ-Orbitrap Velos (HCD mode) analysis, full MS scans were obtained in the Orbitrap with a range of m/z 350 to 1800, a mass resolution of 30,000 at m/z 400, and target value of 1.00E+06. HCD collision was performed on the 10 most significant peaks, and tandem mass spectra were acquired in the Orbitrap at a mass resolution of 7500 and a target value of 5.00E+04. Researchers set the ion threshold at 5000 counts, and the maximum allowed ion accumulation times of 500 ms for full scans and 250 ms for HCD were used. CID analysis was performed with a range of m/z 350 to 1800, a mass resolution of 60,000 at m/z 400, and a target value of 5.00E+05. Sequencing and fragmenting were performed on the 20 most significant peaks with activation q = 0.25, activation time of 10 ms, and a target value of 1.00E+04. Researchers set the ion selection threshold at 500 counts and the maximum allowed ion accumulation times at 500 ms for full scans and 100 ms for CID.

The researchers used Proteome Discoverer 1.3 to interrogate the databases with a low FDR of peptide identification (less than 1%). The proteins were also grouped with strict adherence to the parsimony principle. At a sample loading value of 1000 ng, the Q-Exactive produced a 10% increase in identification rate for protein groups, peptides, and peptide spectrum matches. The Q-Exactive also exhibited a slightly higher rate of identification with sample loading values from 10 to 500 ng. This instrument identified 31% to 74% more protein groups, 32% to 88% more peptides, 46% to 109% more peptide spectrum matches, and 36% to 78% more MS/MS spectra. The researchers point to a smaller maximum injection time for MS/MS, faster scan rate, and increased MS/MS spectra for the Q-Exactive. The Q-Exactive also had higher resolution, which produced a low mass error for both precursor and product ions and positively limited the peptide matches during database interrogation.

When the sample loading value was limited to 1 ng and the “120 ms and 1.00E+05 counts” method was applied to the Q-Exactive, the LTQ-Orbitrap Velos identified more protein groups, peptides, peptide spectrum matches, and MS/MS spectra. However, the application of the “250 ms and 1.00E+04 counts method” to the Q-Exactive data set resulted in a dramatic increase in all four areas of identification that exceeded those of the LTQ-Orbitrap Velos. Manual evaluation confirmed that, for very small sample sizes, a lower intensity threshold and maximum MS/MS injection time (250 ms) was necessary for good spectra production with the Q-Exactive instrument.

The researchers also investigated the link between loading value and identifications using a descriptive formula. They found a higher asymptotic value for protein and peptide identifications for the Q-Exactive but comparable asymptotic values for protein identification from the databases. For duplicate runs with loading values between 1 and 1000 ng, both instruments produced an overlap of identification of 50% to 80% for protein groups and 40% to 70% for peptides with the lower overlaps derived from the runs with lower sample sizes.

When one specific peptide (TPEELSAIK) from a known high-abundance protein was used to evaluate the link between peptide intensity and loading value, researchers found that, for both instruments, the former value increased with the loading value, although the relationship was not linear due to the varying LC gradients used for analysis. At all loading values, the peptide intensity produced by the Q-Exactive remained significantly higher than that produced by the LTQ-Orbitrap Velos.

Overall, Sun et al.¹ found that, for shotgun proteome analysis of the specific cell lysate digests evaluated, the Q-Exactive outperformed the LTQ-Orbitrap Velos in HCD mode and that, for the LTQ instrument, HCD mode outperformed CID mode. The increased identification of both peptide and protein groups by the Q-Exactive was likely due to an increased scan rate and higher resolution. However, the researchers assert that the instrumental superiority demonstrated in this study does not indicate that the Q-Exactive is an overall replacement for the LTQ-Orbitrap Velos since the multiple dissociation modes (HCD, CID, and ETD) available with the latter instrument offer analytical variation important for PTM analysis and large-scale proteomics in general.

References

1. Sun, L., et al. (2013) ‘Comparison of the LTQ-Orbitrap Velos and the Q-Exactive for proteomic analysis of 1-1000 ng RAW 264.7 cell lysate digests‘, Rapid Communications in Mass Spectrometry, 27 (1), (pp. 157–162)