Analysis of tandem mass spectra by FTMS for improved large-scale proteomics with superior protein quantification

Abstract

Using a newly developed dual-cell quadrupole linear ion trap-orbitrap hybrid mass spectrometer (dcQLT-orbitrap), we demonstrate the utility of collecting high-resolution tandem mass spectral data for large-scale shotgun proteomics. Multiple nanoLC-MS/MS experiments on both an older generation quadrupole linear ion trap-orbitrap hybrid (QLT-orbitrap) and the dcQLT-orbitrap, using both resonant-excitation CAD and beam-type CAD (HCD), were performed. Resulting from various technological advances (e.g., a stacked ring ion guide AP inlet, a dual cell QLT), the dcQLT-orbitrap exhibited increased duty cycle (approximately 1.5-2 times) and sensitivity for both CAD (ion trap detection) and HCD (orbitrap detection) methods. As compared to the older system, the dcQLT-orbitrap produced significantly more unique peptide identifications for both methods (approximately 30% improvement for CAD and approximately 115% improvement for HCD). The sizable improvement of the HCD method on the dcQLT-orbitrap system outperforms the current standard method of CAD with ion trap detection for large-scale analysis. Finally, we demonstrate that the increased HCD performance translates to a direct and substantial improvement in protein quantitation precision using isobaric tags.

Figure 1

Two example single-scan tandem mass spectra collected using a dcQLT-orbitrap mass spectrometer. The same precursor was interrogated in both spectra. The top spectrum was produced using resonant-excitation CAD with QLT m/z analysis of fragment ions. The bottom spectrum was collected using beam-type CAD (HCD) with orbitrap m/z analysis of fragment ions.

Figure 2

The average number of MS ²events that followed the MS¹survey scan during a 30-minute interval in the middle of the LC gradient.

Figure 3

The mass accuracy of all the fragment ions detected using the dcQLT -orbitrap during the three (A) CAD-QLT and (B) the three HCD-orbitrap analyses.

Figure 4

Plots of identifications from the target database (blue), decoy database (red) and the difference between the two (black) as a function of identification expectation value. Plots are broken down into the (A) CAD -QLT experiment, (B) HCD-orbitrap experiment, and (C) the HCD-orbitrap experiment searched with a product tolerance of ±0.5Th.

Figure 5

The measured iTRAQ product ion ratios of samples that were mixed in known amounts. The samples were analyzed using PQD and HCD.