Laser-Induced Dissociation combined with Data Independent acquisition (DIA-LID) : a proof of concept study covering human kinome expression
Résumé
Experimental setup Conclusions-Building of a LID peptide spectral library covering the human kinome .-DIA-LID method able to identify and quantify derivatized cysteine-containing peptides in a repeatable way on 3 decades and at low concentrations (down to 1 ng/100 µg) in biological matrix.-Capability of DIA-LID to target and detect endogenous kinases in human cellular extract.-Nano-chromatography and kinase-specific enrichment step required in order to improve the detection specificity in a biomarker discovery approach. Methods Overview The experimental setup consists of a hybrid quadrupole-orbitrap mass spectrometer (Q-exactive, Thermo Fisher Scientific) coupled to a 473 nm continuous wavelength (cw) excitation laser [2]. Orbitrap Specific detection of labeled peptides by DIA-LID Simplification of DIA-MS/MS spectra Identification of derivatized cysteine-containing peptides with the spectral library Good S/N observed in DIA-LID thanks to the specificity References Repeatability and response linearity Linearity over 3 decades was obtained between 1 ng and 1000 ng / 100 µg of E.Coli with R² > 0.99 (greatly preserved for the lowest concentrations with R² > 0.999). First coupling between Data Independent Analysis (DIA) and visible Laser Induced Dissociation (LID) at 473 nm to test for the presence of cysteine-containing peptides. A spectral library in LID was built for synthetic peptide surrogates of human kinases grafted with a dabcyl chromophore. By extracting ion chromatograms of query standard and kinase peptides spiked at different concentration levels in a Escherichia coli proteome lysate, DIA-LID demonstrates a dynamic range of detection of at least 3 decades and coefficients of precision better than 20%. Finally, the spectral library was used to search 354 kinases in human cellular extract [1].
Domaines
Chimie analytique
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