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Effect of mass spectrometric parameters on peptide and protein identification rates for shotgun proteomic experiments on an LTQ-orbitrap mass analyzer

Kalli, Anastasia and Hess, Sonja (2012) Effect of mass spectrometric parameters on peptide and protein identification rates for shotgun proteomic experiments on an LTQ-orbitrap mass analyzer. Proteomics, 12 (1). pp. 21-31. ISSN 1615-9853. doi:10.1002/pmic.201100464. https://resolver.caltech.edu/CaltechAUTHORS:20120222-080018464

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Abstract

The success of a shotgun proteomic experiment relies heavily on the performance and optimization of both the LC and the MS systems. Despite this, little consideration has, so far, been given to the importance of evaluating and optimizing the MS instrument settings during data-dependent acquisition mode. Moreover, during data-dependent acquisition, the users have to decide and choose among various MS parameters and settings, making a successful analysis even more challenging. We have systematically investigated and evaluated the effect of enabling and disabling the preview mode for FTMS scan, the number of microscans per MS/MS scan, the number of MS/MS events, the maximum ion injection time for MS/MS, and the automatic gain control target value for MS and MS/MS events on protein and peptide identification rates on an LTQ-Orbitrap using the Saccharomyces cerevisiae proteome. Our investigations aimed to assess the significance of each MS parameter to improve proteome analysis and coverage. We observed that higher identification rates were obtained at lower ion injection times i.e. 50–150 ms, by performing one microscan and 12–15 MS/MS events. In terms of ion population, optimal automatic gain control target values were at 5×10^5–1×10^6 ions for MS and 3×10^3–1×10^4 ions for MS/MS. The preview mode scan had a minimal effect on identification rates. Using optimized MS settings, we identified 1038 (±2.3%) protein groups with a minimum of two peptide identifications and an estimated false discovery rate of ∼1% at both peptide and protein level in a 160-min LC-MS/MS analysis.


Item Type:Article
Related URLs:
URLURL TypeDescription
http://dx.doi.org/10.1002/pmic.201100464DOIArticle
http://onlinelibrary.wiley.com/doi/10.1002/pmic.201100464/abstractPublisherArticle
ORCID:
AuthorORCID
Hess, Sonja0000-0002-5904-9816
Additional Information:© 2012 Wiley-VCH Verlag. Received: September 2, 2011. Revised: October 12, 2011. Accepted: October 17, 2011. This work was supported by the Betty and Gordon Moore Foundation and the Beckman Institute. The authors thank Raymond J. Deshaies for providing the yeast strain, Michael J. Sweredoski and Robert L. J. Graham for insightful discussions, and Geoffrey T. Smith for technical assistance. The authors have declared no conflict of interest.
Funders:
Funding AgencyGrant Number
Gordon and Betty Moore FoundationUNSPECIFIED
Caltech Beckman InstituteUNSPECIFIED
Subject Keywords:Data-dependent acquisition mode; Identification rates; LTQ-Orbitrap; MS parameters; Technology
Issue or Number:1
DOI:10.1002/pmic.201100464
Record Number:CaltechAUTHORS:20120222-080018464
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20120222-080018464
Official Citation:Kalli, A. and Hess, S. (2012), Effect of mass spectrometric parameters on peptide and protein identification rates for shotgun proteomic experiments on an LTQ-orbitrap mass analyzer. PROTEOMICS, 12: 21–31. doi: 10.1002/pmic.201100464
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:29402
Collection:CaltechAUTHORS
Deposited By: Ruth Sustaita
Deposited On:22 Feb 2012 17:22
Last Modified:09 Nov 2021 17:06

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