Re-evaluation of the rabbit myosin protein standard used to create the empirical statistical model for decoy library searching
| Autor: | Peihong Zhu, Jaimie Dufresne, Peter Bowden, Angelique Florentinus-Mefailoski, John Marshall |
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| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
Spectrometry Mass Electrospray Ionization Electrospray ionization Biophysics Myosins Tandem mass spectrometry Biochemistry Silver stain 03 medical and health sciences Tandem Mass Spectrometry Myosin Animals Quadrupole ion trap Molecular Biology Actin Detection limit Chromatography Models Statistical Chemistry Cell Biology Reference Standards 030104 developmental biology Immunoglobulin G Rabbits Decoy Peptides Chromatography Liquid |
| Zdroj: | Analytical biochemistry. 560 |
| ISSN: | 1096-0309 |
| Popis: | A Rabbit myosin standard, like that used to create the empirical statistical model, was randomly and independently sampled by liquid chromatography micro electrospray ionization and tandem mass spectrometry (LC-ESI-MS/MS) with a linear quadrupole ion trap. The rabbit myosin protein standard appeared pure by SDS-PAGE and CBBR staining but showed many other proteins by silver staining. The LC-MS intensity from myosin and IgG samples were above the 99% safe limit of detection and quantification computed from 36 blank LC-ESI-MS/MS runs. The myosin contained ≤406 Gene Symbols, open reading frames or loci where 79 protein types showed ≥3 peptides from X!TANDEM. Myosins, actin, troponin, other proteins showed 95%–100% homology between the rabbit versus the human decoy library. The myosin protein complex from STRING was true positive compared to random or noise spectra MS/MS with a low type I error (p-value) and low FDR (q-value) computed in R. SDS-PAGE, Western blot, comparison to random and noise MS/MS spectra, X!TANDEM p-values, FDR corrected q-values, and STRING all agreed that the error rate of LC-ESI-MS/MS with a quadrupole ion trap is far below that assumed a priori by the design of the empirical statistical model for decoy library searching. |
| Databáze: | OpenAIRE |
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