Single-lysine precursor ion labelling in KARMA assays for a subset of proteins (10 NUP baits)

Published: 16 October 2020| Version 1 | DOI: 10.17632/wxvtnnczcs.1
Contributor:
Evgeny Onishchenko

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Supplementary analyzed data from Manuscript: TITLE: Maturation kinetics of a multiprotein complex revealed by metabolic labeling. JOURNAL: CELL. Article Type: Research Article Authors: Evgeny Onischenko*, Elad Noor*, Jonas S. Fischer*, Ludovic Gillet, Matthias Wojtynek, Pascal Vallotton, Karsten Weis *Equally Contributing Authors Corresponding Authors: Evgeny Onischenko and Karsten Weis Related to Figure S2; Table S4 Single-lysine precursor ion labelling in KARMA assays for a subset of proteins (10 NUP baits). The target protein complex is isolated from cell lysates by affinity pulldowns at several time points following the onset of metabolic labeling, tryptically digested and analyzed with LC-MS on an Orbitrap mass-spectrometer. The MS2 fragmentation spectra are acquired in a DIA mode for all samples. Zero time point samples (containing only light lysine) are additionally analyzed in a DDA mode to produce assay spectral libraries. Peptide intensities are extracted from the DIA datasets with Spectronaut software (Biognosis) using complementary assay spectral libraries. Individual plots: Protein labeling H/(H+L) in each post-labeling sample (red track) is determined based on the summed intensities of light (L) and heavy (H) constituent high quality precursor ions (green tracks). Low-quality precursor ions are excluded from quantification (gry tracks). y-axis: fractional labeling H/(H+L); x-axis: individual samples (10 handles, 3 replicates, 3 timepoints).

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Affinity Separation Applications, Maturation, Nuclear Pore, Metabolic Flux Analysis Application, Proteomics Experimental Approach, Bioprocess Kinetics, Stable Isotopes Technique, Mass Spectrometry

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