Fig. 4: Application of the micro-flow LC–MS/MS system to the analysis sub-proteomes.
From: Robust, reproducible and quantitative analysis of thousands of proteomes by micro-flow LC–MS/MS
a Recovery analysis of high-confidence interactors obtained by replicate analysis (n = 6; two biological replicates and three technical replicates) of affinity purifications performed using FLAG-tagged human MEPCE and EIF4A2, and BioID proximity labeled human LMNA, NIFK, and CTNNA1 and analyzed by one-shot micro-flow LC–MS/MS using 15 min gradients (AP-MS and BioID-MS), compared to results of the same experiments published previously or part of the Human Cell Map project. Numbers inside the bars represent the number of interactors identified by micro-flow LC–MS/MS vs those annotated in the aforementioned resources. b Interaction networks based on the ten most confident interaction partners in STRING for the bait proteins MEPCE (FLAG affinity purification). FC denotes the fold change values of an interaction partner (over control pulldowns) assessed by SAINTexpress analysis. Proteins without FC annotations indicate that they were not identified as high confident interactors in this micro-flow LC–MS/MS study. c Same as b but for NIFK (BioID proximity labeled). d Example dose-response curves of protein kinases that are the targets of the kinases inhibitor AT-9823 obtained by kinobeads competition pulldown experiments and analyzed by micro-flow LC–MS/MS system using a 15 min gradient for each drug dose. EC50 values (effective concentration 50) denote the drug concentration necessary to compete 50% of the binding of a protein to kinobeads. e Extracted ion chromatograms of the AURKA peptide QWALEDFEIGRPLGK from the kinobead experiment in d illustrating the quantification of this peptide as a function of the applied drug dose. f Dose–response curves akin to d but for four phosphopeptides containing the phosphorylation sites pS21 and pY279 of the protein kinase GSK3A, as well as the aggregated data for the entire protein.
