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, which 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, resolving power and resulting 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.

The Mediator Subunit MED16 Transduces NRF2-Activating Signals into Antioxidant Gene Expression

“…The data acquisition for every sample was done for 60 min after a 50-min LC gradient was started, where MS 1 scans from m/z 321 to 1,600 were carried out in the Orbitrap device, with the resolution set at 60,000 and a lock mass at m/z 445.120025, followed by top-15 MS 2 acquisition by collision-induced dissociation (CID) in the ion trap in the normal resolution mode. The settings for the MS 2 scans were as follows: minimal signal intensity required ϭ 500, AGC target ϭ 5,000, and maximum ion injection time ϭ 50 ms (22). The raw data files derived from samples in the same SDS-PAGE gel lane were converted together into a single MASCOT generic format file and used for database searches by MASCOT (version 2.5.1; Matrix Science) against the mouse proteins in Swiss-Prot (August 2015) and a custom database including contaminant proteins.…”

JNK-associated Leucine Zipper Protein Functions as a Docking Platform for Polo-like Kinase 1 and Regulation of the Associating Transcription Factor Forkhead Box Protein K1

“…The samples were desalted using Vivapure C18 microspin columns and lyophilized. Mass spectrometric analysis of the lyophilized peptides was performed by the Proteome Exploration Laboratory, California Institute of Technology as described previously (23). Raw files were analyzed by MaxQuant (v. 1.4.1.2) (24, 25) in a manner similar to that previously described (26).…”

Bioorthogonal Noncanonical Amino Acid Tagging (BONCAT) Enables Time-Resolved Analysis of Protein Synthesis in Native Plant Tissue

“…Trypsin-digested samples were subjected to LC-MS/MS analysis on a nanoflow LC system, EASY-nLC II, (Thermo Fisher Scientific) coupled to a Orbitrap Elite Hybrid Ion Trap-Orbitrap Mass Spectrometer (Thermo Fisher Scientific) equipped with a nanospray Flex ion source, essentially as described previously (Kalli et al, 2013).…”