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.
|Evidence ID||Analyze ID||Interactor||Interactor Systematic Name||Interactor||Interactor Systematic Name||Type||Assay||Annotation||Action||Modification||Phenotype||Source||Reference||Note|
|Evidence ID||Analyze ID||Gene||Gene Systematic Name||Gene Ontology Term||Gene Ontology Term ID||Qualifier||Aspect||Method||Evidence||Source||Assigned On||Reference||Annotation Extension|
|Evidence ID||Analyze ID||Gene||Gene Systematic Name||Phenotype||Experiment Type||Experiment Type Category||Mutant Information||Strain Background||Chemical||Details||Reference|
|Evidence ID||Analyze ID||Regulator||Regulator Systematic Name||Target||Target Systematic Name||Experiment||Conditions||Strain||Source||Reference|