Improving proteome coverage on a LTQ-Orbitrap using design of experiments
In: Journal of the American Society for Mass Spectrometry, Jg. 22 (2010-09-24), Heft 4
Online
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Zugriff:
Design of experiments (DOE) was used to determine improved settings for a LTQ-Orbitrap XL to maximize proteome coverage of Saccharomyces cerevisiae. A total of nine instrument parameters were evaluated with the best values affording an increase of approximately 60% in proteome coverage. Utilizing JMP software, 2 DOE screening design tables were generated and used to specify parameter values for instrument methods. DOE 1, a fractional factorial design, required 32 methods fully resolving the investigation of six instrument parameters involving only half the time necessary for a full factorial design of the same resolution. It was advantageous to complete a full factorial design for the analysis of three additional instrument parameters. Measured with a maximum of 1% false discovery rate, protein groups, unique peptides, and spectral counts gauged instrument performance. Randomized triplicate nanoLC-LTQ-Orbitrap XL MS/MS analysis of the S. cerevisiae digest demonstrated that the following five parameters significantly influenced proteome coverage of the sample: (1) maximum ion trap ionization time; (2) monoisotopic precursor selection; (3) number of MS/MS events; (4) capillary temperature; and (5) tube lens voltage. Minimal influence on the proteome coverage was observed for the remaining four parameters (dynamic exclusion duration, resolving power, minimum count threshold to trigger a MS/MS event, and normalized collision energy). The DOE approach represents a time- and cost-effective method for empirically optimizing MS-based proteomics workflows including sample preparation, LC conditions, and multiple instrument platforms.
Titel: |
Improving proteome coverage on a LTQ-Orbitrap using design of experiments
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Autor/in / Beteiligte Person: | Dean, Ralph A. ; Muddiman, David C. ; Hawkridge, Adam M. ; Andrews, Genna L. |
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Zeitschrift: | Journal of the American Society for Mass Spectrometry, Jg. 22 (2010-09-24), Heft 4 |
Veröffentlichung: | 2010 |
Medientyp: | unknown |
ISSN: | 1879-1123 (print) |
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