Quantitative Proteomic Analysis Pipeline
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Corresponding Organization :
Other organizations : Center for Excellence in Molecular Cell Science, University of Chinese Academy of Sciences, Peking University, Wuhan University, Chinese Academy of Sciences, Dalian Institute of Chemical Physics
Variable analysis
- Protein samples were tryptic digested
- Peptides eluted from the LC column were directly electrosprayed into a Q-Exactive plus mass spectrometer
- MS/MS spectra were searched against the UniProtKB/Swiss-Prot human database
- Protein/peptide quantification
- C18 column packed with 1.9umAqua C18 material (Phenomenex)
- Xcalibur data system (ThermoFisher) to control MS scan functions and HPLC solvent gradients
- Maxquant software (version 1.5.3.30) for feature detection, database searching, and protein/peptide quantification
- Minimum peptide length of seven amino acids and maximum peptide mass of 4600 Da
- Allowed missed cleavages for each peptide was two
- Second peptide search was activated to identify coeluting and cofragmented peptides from one MS/MS spectrum
- Both peptides and proteins were filtered with a maximum FDR of 0.01
- LFQ calculations were performed separately in each parameter group
- Both unique and razor peptides were selected for protein quantification
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