Tryptic peptides were either directly separated on a one-dimensional fused silica capillary column (20 cm × 100 μm) packed with Jupiter resin (3-μm mean particle size, 300 Å pore size; Phenomenex, Torrance, CA, USA) or analyzed by MudPIT.
21 One-dimensional liquid chromatography was used with the following gradient at a flow rate of 0.5 μL/min: 0 to 2 minutes: 2% ACN (0.1% formic acid), 2 to 70 minutes: 2% to 35% ACN (0.1% formic acid), 70 to 90 minutes: 35% to 90% ACN (0.1% formic acid) balanced with 0.1% formic acid. The eluate was directly infused into a Velos Pro linear ion trap mass spectrometer (ThermoFisher, San Jose, CA, USA) equipped with a nanoelectrospray source. For MudPIT analysis, peptides were loaded onto a custom packed biphasic C18/SCX trap column (4 cm × 150 μm, Jupiter C18, 5 μm, 300 Å media followed by 4 cm × 150 μm, Luna SCX, 5 μm, 100 Å media; Phenomenex). The trap column was coupled to a capillary analytical column (20 cm × 100 μm, Jupiter C18, 3 μm, 300 Å media). MudPIT analysis was done with a 13-step salt pulse gradient (0 mM, 25 mM, 50 mM, 75 mM, 100 mM, 150 mM, 200 mM, 250 mM, 300 mM, 500 mM, 750 mM, 1 M, and 2 M ammonium acetate). Following each salt pulse, peptides were eluted from the analytical column with a 90-minute reverse-phase solvent gradient (2%–45% ACN, 0.1% formic acid; 2%–95% ACN, 0.1% formic acid for last two salt pulses). The eluate was directly electrosprayed into a Velos Pro mass spectrometer (ThermoFisher). The instrument was operated in a data-dependent mode with one precursor scan event to identify the top 15 most abundant ions in each MS scan, which were then selected for fragmentation. Dynamic exclusion (repeat count 1, exclusion list size 300, and exclusion duration 15 seconds) was enabled to allow detection of less abundant ions. For quantification of fatty acylated AQP0, the samples were run on a one-dimensional column as above with modified gradient (0–50 minutes: 2%–30% ACN, 50–65 minutes: 30%–95% ACN, 65–80 minutes: 95% ACN balanced with 0.1% formic acid) and the mass spectrometer was set to acquire one MS1 scan followed by targeted MS/MS scans for fatty acylated AQP0 peptides and unmodified AQP0 peptides. The iTRAQ-labeled samples were analyzed using MudPIT analysis as described above with eight salt pulse steps (0, 50 mM, 100 mM, 200 mM, 300 mM, 500 mM, 1 M, and 2 M ammonium acetate). Peptides were introduced via nano-electrospray into a Q Exactive mass spectrometer (Thermo Scientific, San Jose, CA, USA). The Q Exactive was operated in data-dependent mode acquiring higher-energy collisional dissociation (HCD) MS/MS scans (
R = 17,500) after each MS1 scan (
R = 70,000) on the 18 most abundant ions using an MS1 ion target of 1 × 10
6 ions and an MS2 target of 1 × 10
5 ions. The maximum ion time for MS/MS scans was set to 100 ms, the HCD-normalized collision energy was set to 26, dynamic exclusion was set to 30 seconds, and peptide match and isotope exclusion were enabled.