Easy pepmap rslc c18 column

Procedures were performed essentially as described previously (Lytvynenko et al., 2019 (link)) with the exception that peptide separation was performed on as EASY PepMapTM RSLC C18 column (2μm, 100Å, 75 μm x 50cm, Thermo Scientific, San Jose, CA), and ions were created with an EASY Spray source (Thermo Scientific, San Jose, CA) held at 50°C using a voltage of 1.9 kV. All flow rates were 250 nL/min on this column and peptides were eluted using a gradient of 5%–25% solvent B (80/20 acetonitrile/water, 0.1% formic acid) in 180 min, followed by 25%–44% solvent B in 60 min, 44%–80% solvent B in 0.10 min, a 5 min hold of 80% solvent B, a return to 5% solvent B in 0.10 min, and finally a 20 min hold of solvent B. Tandem mass spectra were searched as previously described (Lytvynenko et al., 2019 (link)) and the protein and peptide identification results were also visualized with Scaffold v 4.7.1 (Proteome Software Inc., Portland OR), a program that relies on various search engine results (i.e.: Sequest, X!Tandem, MASCOT) and which uses Bayesian statistics to reliably identify more spectra (Keller et al., 2002 (link)). Proteins were accepted that passed a minimum of two peptides identified at 1% peptide and protein FDR, within Scaffold.

Tryptic peptide samples were run on a Fusion Tribrid mass spectrometer (Thermo Scientific) with an EASY PepMapTM RSLC C18 column. Precise run gradients and conditions are available in the SI.

Samples were solubilized in 1% trifluoroacetic acid and desalted using ZipTips (Millipore), according to the manufacturer’s instructions. Peptides were online eluted into a Fusion Tribrid mass spectrometer (Thermo Scientific) from an EASY PepMapTM RSLC C18 column (2 mm, 100 Å, 75 mm × 50 cm, Thermo Scientific). The precise LC gradient is available in the Supporting Information. Ions were created at 1.9 kV using an EASY Spray source (Thermo Scientific) held at 50 °C. A synchronous precursor selection (SPS)-MS3 mass spectrometry method was used based on the work of Ting et al. (see Supporting Information).^{34 (link)}