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Data-Independent Acquisition for the Orbitrap Q Exactive HF: A Tutorial

Reubsaet, Léon and Sweredoski, Michael J. and Moradian, Annie (2019) Data-Independent Acquisition for the Orbitrap Q Exactive HF: A Tutorial. Journal of Proteome Research, 18 (3). pp. 803-813. ISSN 1535-3893. doi:10.1021/acs.jproteome.8b00845.

[img] PDF (Supplementary 1: Calculation of the actual overhead time A and actual transient time. Supplementary 2: DIA on the Fusion Tribrid mass spectrometer. Supplementary 3: Quadrupole selection time versus isolation width. Supplementary 4: Description of the...) - Supplemental Material
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Data-independent acquisition (DIA) is a powerful mass spectrometric technique to perform both protein identification and quantification of complex protein samples. Setting up DIA methods on Orbitrap analyzers requires a thorough overview of the actions the Orbitrap mass spectrometers carry out. This Tutorial is written with the intention to give an overview of the important parameters to consider as well as which measurements to carry out to get the most out of your DIA method when setting it up on an Orbitrap mass analyzer. Instead of giving the optimal DIA settings, all steps in the construction and optimization of the DIA method are shown and discussed in a way that allows tailored DIA methods. They key steps are building the spectral library after sample fractionation, deciding upon the number of data points per chromatographic peak, determining the scan times of each mass spectrometric step, constructing various DIA methods using these data, and evaluating their performance. This proposed DIA method development strategy was tested on digested lysates from Pseudomonas aeruginosa and compared with conventional DDA analysis to put the DIA results into perspective.

Item Type:Article
Related URLs:
URLURL TypeDescription Information
Reubsaet, Léon0000-0001-5165-2701
Sweredoski, Michael J.0000-0003-0878-3831
Moradian, Annie0000-0002-0407-2031
Additional Information:© 2018 American Chemical Society. Published: December 17, 2018. We thank Prof. Dianna Newman and David Basta (California Institute of Technology) for the preparation of and supplying the Pseudomonas aeruginosa (strain PA14) digested lysates. We also thank Roxana Eggleston-Rangel from the Proteome Exploration Laboratory (Beckman Institute at the California Institute of Technology) for her technical assistance during the nano LC–MS/MS analyses. The Proteome Exploration Laboratory is supported by the Beckman Institute and NIH 1S10OD02001301. The authors declare no competing financial interest.
Funding AgencyGrant Number
Caltech Beckman InstituteUNSPECIFIED
Subject Keywords:data-independent acquisition, method development strategy, actual scan times, Orbitrap mass analyzer
Issue or Number:3
Record Number:CaltechAUTHORS:20190122-112737936
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Official Citation:Data-Independent Acquisition for the Orbitrap Q Exactive HF: A Tutorial. Léon Reubsaet, Michael J. Sweredoski, and Annie Moradian. Journal of Proteome Research 2019 18 (3), 803-813. DOI: 10.1021/acs.jproteome.8b00845
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:92396
Deposited By: Tony Diaz
Deposited On:22 Jan 2019 20:06
Last Modified:16 Nov 2021 03:50

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