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Evaluation and Optimization of Mass Spectrometric Settings during Data-dependent Acquisition Mode: Focus on LTQ-Orbitrap Mass Analyzers

Kalli, Anastasia and Smith, Geoffrey T. and Sweredoski, Michael J. and Hess, Sonja (2013) Evaluation and Optimization of Mass Spectrometric Settings during Data-dependent Acquisition Mode: Focus on LTQ-Orbitrap Mass Analyzers. Journal of Proteome Research, 12 (7). pp. 3071-3086. ISSN 1535-3893. PMCID PMC3748959. doi:10.1021/pr3011588.

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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.

Item Type:Article
Related URLs:
URLURL TypeDescription DOIArticle CentralArticle
Sweredoski, Michael J.0000-0003-0878-3831
Hess, Sonja0000-0002-5904-9816
Additional Information:© 2013 American Chemical Society. Published: May 5, 2013. We thank Tonya P. Second, Rolly Singh, Anjum A. Khan and Martin Zeller at Thermo Fisher Scientific for their valuable discussions and help. The Proteome Exploration Laboratory is supported by the Gordon and Betty Moore Foundation through Grant GBMF775, the Beckman Institute, and an instrumentation grant from NIH (10565784).
Funding AgencyGrant Number
Gordon and Betty Moore FoundationGBMF775
Caltech Beckman InstituteUNSPECIFIED
Subject Keywords:shotgun proteomics, CID data-dependent acquisition mode, identification rates, LTQ-Orbitrap, MS and MS/MS parameters
Issue or Number:7
PubMed Central ID:PMC3748959
Record Number:CaltechAUTHORS:20130906-153141418
Persistent URL:
Official Citation:Evaluation and Optimization of Mass Spectrometric Settings during Data-dependent Acquisition Mode: Focus on LTQ-Orbitrap Mass Analyzers Anastasia Kalli, Geoffrey T. Smith, Michael J. Sweredoski, and Sonja Hess Journal of Proteome Research 2013 12 (7), 3071-3086
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:41153
Deposited By: Tony Diaz
Deposited On:17 Sep 2013 20:50
Last Modified:10 Nov 2021 04:26

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