Protein Identification by Mass Spectrometry, Supplemental

Protein Identification from SDS-PAGE:Protein in gel bands (self-made gel), have been reduced with DTT, alkylated with iodoacetomide, subjected to 3x alternating washes, and proteolysis with Trypsin (sequential grade, Promega) in 50mM TEAB buffer at 37C overnight. Peptides extracted and desalted on u-HLB Oasis plates, eluted with 60 acetonitrile/ 0.1%TFA, dried. Dry peptides re-constituted in 20uL acetonitrile/0.1uL,and analyzed by LC/MS/MS on Orbitrap Lumos-ETD. Resolution set to 120K, 30K for the precursor and fragment ions, respectively. HCD energy at 31 with a isolation window set to 0.8Da without offset. Injected 10% for samples 3 and 4. Data analysis: MS/MS raw data were searched via PD2.3 with Mascot 6.1, against RefSeq2017_83_human database (containing common contaminants). Mascot .dat files were validated with PD2.3-Percolator.