Reprosil pure aq c18 phase

An EASY-nLC 1000 LC system (Thermo Fisher Scientific) interfaced via nanoSpray Flex ion source to an Orbitrap Fusion Lumos MS (Thermo Fisher Scientific) was used for MS and MS/MS analyses. A single analytical column setup using PicoFrit Emitters (New Objectives, 75 µm inner diameter) custom packed with Reprosil-Pure-AQ C18 phase (Dr. Maisch, 1.9-µm particle size, 19–21 cm column length) was applied in nLC. 2 μL of each sample was injected onto the column, followed by elution with a gradient of Solvent B from 2% to 25% at 200 nL/min for 45 min (Solvent A: 100% H₂O+ 0.1% (v/v) formic acid; Solvent B: 100% acetonitrile +0.1% (v/v) formic acid). With the nominal resolution setting of 120,000, precursors of MS1 scan (m/z 350-2,000) were obtained. Then HCD-MS2 of the five most abundant multiply charged precursors in the MS1 spectrum was acquired at the nominal resolution setting of 60,000. To trigger data-dependent fragmentation events, the minimum MS1 signal threshold was 50,000. Targeted MS/MS analysis was performed by setting up a targeted MSn (tMSn) Scan Properties panel. 30 targeted entries were included in the Mass List Table.

LC–MS/MS site-specific O-glycopeptide analysis of mucin TRs was performed on EASY-nLC 1000 UHPLC interfaced via nanoSpray Flex ion source to an Orbitrap Fusion MS or EASY-nLC 1000 UHPLC interfaced via New Objectives ion source to an Orbitrap Fusion MS (Thermo Fisher Scientific). Briefly, the nLC was operated in a single analytical column set up using PicoFrit Emitters (New Objectives; inner diameterof 75 mm) packed in-house with Reprosil-Pure-AQ C18 phase (Dr Maisch; particle size of 1.9 mm, column length of 19–21 cm). Each sample was injected onto the column and eluted in gradients from 3 to 32% B for glycopeptides and 10 to 40% for released and labeled glycans in 45 min at 200 nl/min (solvent A, 100% H₂O; solvent B, 100% ACN; both containing 0.1% [v/v] FA). A precursor MS1 scan (m/z 350–2000) of intact peptides was acquired in the Orbitrap at the nominal resolution setting of 120,000, followed by Orbitrap higher-energy collisional dissociation–MS2 and electron-transfer dissociation–MS2 at the nominal resolution setting of 60,000 of the five most abundant multiply charged precursors in the MS1 spectrum; a minimum MS1 signal threshold of 50,000 was used for triggering data-dependent fragmentation events. Targeted MS/MS analysis was performed by setting up a targeted MSn Scan Properties pane.