High select fe nta phosphopeptides enrichment kit

Label-free proteomics analysis was performed by Applied Protein Technology (Shanghai, China). LC-MS/MS spectra were searched using a Q Exactive HF/HFX mass spectrometer coupled to Easy nLC (Thermo Fisher Scientific), which is controlled by IntelliFlow technology. Immobilized metal affinity chromatography (IMAC) was used to enrich phosphopeptides. According to the manufacturer’s instructions (Thermo Scientific), the enrichment was carried out using High-SelectTM Fe-NTA Phosphopeptides Enrichment Kit. The MS raw data for each sample were combined and searched using MaxQuant 1.5.3.17 software for the identification and quantification analysis. A false discovery rate <1% was applied. Proteomic samples were analyzed by LC-MS/MS as described in Supplement S1. Gene Ontology analysis was performed at https://david.ncifcrf.gov/home.jsp.

SDT (4%SDS, 100mM Tris-HCl, 1mM DTT, pH7.6) buffer was used for sample lysis and protein extraction. The digest peptides of each sample were desalted on C18 Cartridges, concentrated by vacuum centrifugation and reconstituted in 40 µL of 0.1% formic acid. The enrichment of phosphopeptides was carried out using High-Select^TM Fe-NTA Phosphopeptides Enrichment Kit (Thermo Scientific). After being lyophilized, the phosphopeptides peptides were resuspended in 20 µL loading buffer (0.1% formic acid).
LC-MS/MS analysis was performed on a timsTOF Pro mass spectrometer (Bruker) that was coupled to Nanoelute (Bruker Daltonics) for 60 min. The peptides were loaded on a C18-reversed phase analytical column in 0.1% formic acid and separated with a linear gradient of buffer mixed with 84% acetonitrile and 0.1% formic acid at a flow rate of 300 nl/min. The mass spectrometer was operated in positive ion mode. The quantitative information of the target protein set was normalized and then, R (version 3.4) was used to classify the two dimensions of sample and protein expression at the same time, and finally a hierarchical clustering heat map was generated.