Reprosil pur 120 c18 aq

In Freiburg, sample preparation was performed in the same way as in Heidelberg except that, for desalting, Hypersep C18 SpinTips (Thermo Fisher Scientific) were used. For LC-MRM, a nanoflow Easy-nLC II system (Proxeon Biosystems; Odense, Denmark) was used, equipped with a trapping column (Fused Silica Capillary; 3 cm length, JR-T-7360-100, VICI Jour, Schenkon, Switzerland) and with an analytical Self-Pack PicoFrit column (40 cm length, 75 μm inner diameter, New Objective (Littleton, MA, USA)), both columns in-house packed with C18 particles (Dr. Maisch (Ammerbuch-Entringen, Germany), ReproSil-Pur 120 C18-AQ; 3 μm C18 particle size, 120 Å pore size) [39 (link)]. The samples were trapped at 220 bar with 100% of buffer A and separated using a linear gradient of buffer B in buffer A from 8 to 56% of B in 60 min with a column temperature of 60 °C and a flow rate of 250 nL/min (buffer A: 0.1% v/v FA; buffer B: 50% v/v ACN, 0.1% v/v FA). The Easy-nLC II system was coupled online to an ESI-TSQ Vantage triple quadrupole mass spectrometer via a Nanospray Flex Ion source (both Thermo Scientific).

Samples were automatically loaded at 3 μL/min onto a precolumn (150 μm i.d. and 3.5 cm in length) packed with ReproSil-Pur 120 C18-AQ stationary-phase material (5 μm in particle size, 120 Å in pore size, Dr. Maisch). The precolumn was connected to a 20-cm fused-silica analytical column (PicoTip Emitter, New Objective, 75 μm i.d.) packed with 3 μm C18 beads (ReproSil-Pur 120 C18-AQ, Dr. Maisch). The peptides were then resolved using a 180-min gradient of 2–45% acetonitrile in 0.1% formic acid, and the flow rate was maintained at 300 nL/min.
The mass spectrometer was operated in a data-dependent acquisition mode. Full-scan mass spectra were acquired in the range of m/z 350–1500 using the Orbitrap analyzer at a resolution of 70,000 at m/z 200. Up to 25 most abundant ions found in MS with a charge state of 2 or above were sequentially isolated and collisionally activated in the HCD cell with a normalized collision energy of 28 to yield MS/MS.

Samples were automatically loaded at 3 μL/min onto a precolumn (150 μm i.d. and 3.5 cm in length) packed with ReproSil-Pur 120 C18-AQ stationary-phase material (5 μm in particle size, 120 Å in pore size, Dr. Maisch). The precolumn was connected to a 20-cm fused-silica analytical column (PicoTip Emitter, New Objective, 75 μm i.d.) packed with 3 μm C18 beads (ReproSil-Pur 120 C18-AQ, Dr. Maisch). The peptides were then resolved using a 180-min gradient of 2–45% acetonitrile in 0.1% formic acid, and the flow rate was maintained at 300 nL/min.
The mass spectrometer was operated in a data-dependent acquisition mode. Full-scan mass spectra were acquired in the range of m/z 350–1500 using the Orbitrap analyzer at a resolution of 70,000 at m/z 200. Up to 25 most abundant ions found in MS with a charge state of 2 or above were sequentially isolated and collisionally activated in the HCD cell with a normalized collision energy of 28 to yield MS/MS.