Evaluation and optimization of mass spectrometric settings during data-dependent acquisition mode: focus on LTQ-Orbitrap mass analyzers

Abstract

Mass-spectrometry-based proteomics has evolved as the preferred method for the analysis of complex proteomes. Undoubtedly, recent advances in mass spectrometry instrumentation have greatly enhanced proteomic analysis. A popular instrument platform in proteomics research is the LTQ-Orbitrap mass analyzer. In this tutorial, we discuss the significance of evaluating and optimizing mass spectrometric settings on the LTQ-Orbitrap during CID data-dependent acquisition (DDA) mode to improve protein and peptide identification rates. We focus on those MS and MS/MS parameters that have been systematically examined and evaluated by several researchers and are commonly used during DDA. More specifically, we discuss the effect of mass resolving power, preview mode for FTMS scan, monoisotopic precursor selection, signal threshold for triggering MS/MS events, number of microscans per MS/MS scan, number of MS/MS events, automatic gain control target value (ion population) for MS and MS/MS, maximum ion injection time for MS/MS, rapid and normal scan rate, and prediction of ion injection time. We furthermore present data from the latest generation LTQ-Orbitrap system, the Orbitrap Elite, along with recommended MS and MS/MS parameters. The Orbitrap Elite outperforms the Orbitrap Classic in terms of scan speed, sensitivity, dynamic range, and resolving power and results in higher identification rates. Several of the optimized MS parameters determined on the LTQ-Orbitrap Classic and XL were easily transferable to the Orbitrap Elite, whereas others needed to be reevaluated. Finally, the Q Exactive and HCD are briefly discussed, as well as sample preparation, LC-optimization, and bioinformatics analysis. We hope this tutorial will serve as guidance for researchers new to the field of proteomics and assist in achieving optimal results.

Figure 1

Suggested optimized MS and MS/MS parameters for the LTQ-Orbitrap. Values were determined on the LTQ-Orbitrap Classic and XL and are taken from references [13], [15] and [16].

Figure 2

Mass resolving power of the Orbitrap mass spectrometers is not steady across the m/z scan range and decreases as the square root of the m/z ratio. Data from Orbitrap Classic at a set mass resolving power of 60,000 (A) and Orbitrap Elite at a set mass resolving power of 120,000 (B).

Figure 3

Effect of MS/MS maximum ion injection time on identification rates observed for the Orbitrap Elite.

Figure 4

Effect of MS resolving power on identification rates observed for the Orbitrap Elite.

Figure 5

Identification rates obtained in the LTQ-Orbitrap Classic and Orbitrap Elite under optimized settings. (LTQ-Orbitrap Classic: 12 MS/MS events, 1 microscan, preview mode for FTMS scan enabled, ion injection time for MS/MS 50 ms, AGC target value for MS/MS 5 × 10³, AGC target value for MS 5 × 10⁵, mass resolving power 60, 000, Orbitrap Elite: 15 MS/MS events, 1 microscan, preview mode for FTMS scan not enabled, ion injection time for MS/MS 50 ms, AGC target value for MS/MS 5 × 10³, AGC target value for MS 1 × 10⁶, mass resolving power 120, 000).

Figure 6

Identified proteins as a function of their cellular expression levels observed on the LTQ-Orbitrap Classic and Orbitrap-Elite using the Saccharomyces cerevisiae proteome as the model system. Protein cellular expression levels were obtained from reference [37]. The number of identified proteins displayed in the Figure is a sum over two replicate analyses.