Offline ERLIC-based Peptide Separation

To separate peptides, off-line ERLIC or ERLIC-based separation was performed (14 (link)). The following conditions were adapted and optimized to obtain the highest number of identified proteins from P2′ and SV samples. Mobile phase solvent preparation was as follows: Solvents were freshly prepared for each experiment using liquid chromatography/mass spectrometry grade acetonitrile (ACN), formic acid (FA), and water from Thermo Fisher Chemicals. Solvent A was prepared as follows: 90% ACN, 0.1% FA. Ammonium hydroxide (NH₄OH, 25% weight/weight [wt/wt] in water; Fluka) was then added to adjust pH at 4.5. Solvent B was prepared as follows: 30% ACN, 0.1% FA. The digested peptide mixture was resuspended with 20 µL of solvent A and injected into a weak anion exchange PolyWax column (1-mm inner diameter × 150 mm, 5-mm particle size, 300-Å pore size; PolyLC Inc.) using a PAL autosampler (CTC Analytics) for automatic injection and fractions collection, using a gradient mode (3 min solvent A, to 10% B in 7 min, 10% B to 25% B in 24 min, 25% B to 70% B in 16 min, 70% B to 81% B in 6 min, 81% B to 100% B in 3 min, with final wash at 100% B for 6 min and reequilibration at 100% A for 20 min) at a flow rate of 40 μL/min. Twenty-four fractions were collected every 3 min between 0 and 72 min and subsequently concentrated to dryness using a speed vacuum Genevac EZ-2 Elite (SP Scientific).