Mass spec raw data.xlsx

Agilent 1260 Infinity nHPLC stack and Thermo Orbitrap Velos Pro hybrid mass spectrometer were used for analysis of 8-µl samples with a C-18 column (75 μm x 15 cm; 300 Å; 5 μm; Phenomenex). All data were acquired in collision-induced dissociation mode. The phase A was 0.1% FA in ddH2O and phase B was 0.1% formic acid (FA) in 15% ddH2O/85% acetonitrile). The mobile phase gradient was: 10 min at 2% phase B, 90 min at 5-40% phase B, 5 min at 70% phase B and 10 min at 0% phase B. The MS detection included a full scan (m/z 300 -1200) with resolution at 60k and data-dependent MS2 scans on the top abundant ions (15 ions). The MS data files were converted to MzXML using ReAdW (v. 3.5.1). MzXML2 Search was used to create a Mascot generic format file. Data were analyzed using the SEQUEST engine and searches were performed using the Uniref100 database. The peptide ID lists were then further analyzed by Scaffold viewer. The mass spectrometry peptide identifications were filtered by Scaffold. In short, protein probabilities were set to ≥0.99 with false discovery rate <1% cutoff, minimum two peptides assigned per protein, and individual peptide probabilities were ≥0.8. Quantification across experiments was based on normalized spectral counting between samples for MS spectra assigned to each protein.