| Autor: |
Garcia L; Université de Lyon, CNRS, Université Claude Bernard Lyon 1, Ecole Normale Supérieure de Lyon, Institut des Sciences Analytiques , UMR 5280, 5 rue de la Doua , F-69100 Villeurbanne , France., Girod M; Université de Lyon, CNRS, Université Claude Bernard Lyon 1, Ecole Normale Supérieure de Lyon, Institut des Sciences Analytiques , UMR 5280, 5 rue de la Doua , F-69100 Villeurbanne , France., Rompais M; Laboratoire de Spectrométrie de Masse Bio-Organique (LSMBO), IPHC , Université de Strasbourg, CNRS , UMR 7178, 25 rue Becquerel , 67087 Strasbourg , France., Dugourd P; Université de Lyon, Université Claude Bernard Lyon 1, CNRS, Institut Lumière Matière , F-69622 Villeurbanne , France., Carapito C; Laboratoire de Spectrométrie de Masse Bio-Organique (LSMBO), IPHC , Université de Strasbourg, CNRS , UMR 7178, 25 rue Becquerel , 67087 Strasbourg , France., Lemoine J; Université de Lyon, CNRS, Université Claude Bernard Lyon 1, Ecole Normale Supérieure de Lyon, Institut des Sciences Analytiques , UMR 5280, 5 rue de la Doua , F-69100 Villeurbanne , France. |
| Abstrakt: |
Thanks to comprehensive and unbiased sampling of all precursor ions, the interest to move toward bottom-up proteomic with data-independent acquisition (DIA) is continuously growing. DIA offers precision and reproducibility performances comparable to true targeted methods but has the advantage of enabling retrospective data testing with the hypothetical presence of new proteins of interest. Nonetheless, the chimeric nature of DIA MS/MS spectra inherent to concomitant transmission of a multiplicity of precursor ions makes the confident identification of peptides often challenging, even with spectral library-based extraction strategy. The introduction of specificity at the fragmentation step upon ultraviolet or visible laser-induced dissociation (LID) range targeting only the subset of cysteine-containing peptides (Cys-peptide) has been proposed as an option to streamline and reduce the search space. Here, we describe the first coupling between DIA and visible LID at 473 nm to test for the presence of Cys-peptides with a peptide-centric approach. As a test run, a spectral library was built for a pool of Cys-synthetic peptides used as surrogates of human kinases (1 peptide per protein). By extracting ion chromatograms of query standard and kinase peptides spiked at different concentration levels in an 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 for endogenous kinases in human cellular extract. |
