Silicatip emitter

A 5 μL aliquot of the peptide resuspension was injected into nano-Acquity LC for MS analysis. The nano-Acquity LC (Waters Corporation, Milford, MA, USA) was fitted with HSS T3 75 μm × 100 μm, 1.8 μm column and a flow rate of 0.5 μL/min of a gradient of solution A and B was used to separate the peptides. Solution A was composed of 0.1% formic acid in MS grade water and solution B was composed of 0.1% formic acid in acetonitrile. Peptides were eluted from the column with a gradient of 2%–20% of solution B in 28 min, then 20%–40% solution B for another 13 min before ramping up to 85% solution B in another 3 min to clean the column. The Orbitrap Fusion Lumos was equipped with a Nanospray Flex electrospray ion source (Thermo Fisher Scientific, San Jose, CA, USA). Peptide ions sprayed from a 10 μm SilicaTip emitter (New Objective, Woburn, MA, USA) into the ion source were targeted and isolated in the quadrupole. These were then fragmented by HCD and ion fragments were detected in the Orbitrap (resolution of 60,000, mass range 150–1,200 m/z). Monitoring of hydrophilic peptides (SSRcalc <9, all without leucine) for peptide profiling was performed on a HSS T3 300 μm × 100 μm, 1.8 mm column at a flow rate of 4 μl/min with an elution occurring with a 2%–12% solution B gradient and a spray operating on a 30 mm SilicaTip emitter.

High resolution LC-MS/MS analysis was carried out using an Ultimate 3000 RSLCnano system (Dionex, Germering, Germany) coupled to a Q Exactive Plus mass spectrometer (Thermo Fisher Scientific, Bremen, Germany). Prior to analysis, tryptic peptides were mixed with iRT peptides (Biognosys) (1:10 v/v), desalted on the trap column and separated on the analytical column (PepMap™ RSLC C18, 50 cm × 75 μm). Ionization was achieved using nanospray Flex ion source (Thermo Fisher Scientific, Bremen, Germany) equipped with a 10 μm-inner diameter SilicaTip emitter (New Objective, Littleton, MA, USA).