Fig. 1: Pipeline for hybrid spectral library construction and phosphosite quantification. | Nature Communications

Fig. 1: Pipeline for hybrid spectral library construction and phosphosite quantification.

From: A data-independent acquisition-based global phosphoproteomics system enables deep profiling

Fig. 1

a For the construction of the spectral library, non-small cell lung cancer (NSCLC) cells and tissues were lysed, digested, and fractionated by high pH reversed-phase (HpRP) chromatography in StageTip or in column (HPLC), and phosphopeptides were enriched by iron-based immobilized metal affinity chromatography (Fe-IMAC) and analyzed by the data-dependent acquisition (DDA) and data-independent acquisition (DIA) modes. The indexed retention time (iRT) standard peptides were spiked for normalization in retention time. b A phosphopeptide reference library was constructed from both DDA datasets (n = 156 raw files) and DIA datasets (n = 24 raw files), which were processed by Spectronaut Pulsar. Lung cancer proteome library was constructed from 191 DDA raw files processed with MaxQuant search. All data were filtered at 1% false discovery rate (FDR) at peptide spectrum match (PSM)/Precursor, Peptide, and Protein. c Single-shot DIA was acquired and processed by both library-based DIA (libDIA) and direct DIA (dirDIA) approach by Spectronaut. Phosphosite-level quantification was obtained by an in-house customized R program.

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