Reprosil pur 120 c18 aq reversed phase material

Just before MS analysis, the indexed retention time standard kit (Biognosys, Switzerland) was prepared according to manufacturer’s instructions and was added to each sample in a 1:100 ratio. Peptides were dissolved in 0.1% (v/v) acetic acid and loaded on an EASY-nLC II (Thermo Fisher Scientific, United States) system equipped with an in-house built 20 cm column (inner diameter 100 μm, outer diameter 360 μm) filled with ReproSil-Pur 120 C₁₈-AQ reversed-phase material (3 μm particles, Dr. Maisch GmbH, Germany). As previously described (Otto et al., 2016 (link)), elution of peptides was executed with a non-linear 80 min gradient from 1 to 99% solvent B (0.1% (v/v) acetic acid in acetonitrile) with a flow rate of 300 nl/min and injected online into a LTQ Orbitrap Velos Pro (Thermo Fisher Scientific, United States). The survey scan at a resolution of R = 30.000 and 1 × 10⁶ automatic gain control target in the Orbitrap with activated lock mass correction was followed by selection of the 20 most abundant precursor ions for fragmentation. Singly charged ions as well as ions without detected charge states were excluded from MS/MS analysis. All MS data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository (Vizcaíno et al., 2016 (link)) with the dataset identifier PXD010279.

Peptides were separated on an EASY-nLC II (Thermo Fisher Scientific, Waltham, MA, USA) system equipped with an in-house built 20 cm column (inner diameter 100 µm, outer diameter 360 µm) filled with ReproSil-Pur 120 C18-AQ reversed-phase material (3 µm particles, Dr. Maisch GmbH, Ammerbuch, Germany). The peptides were loaded with buffer A (0.1% acetic acid (v/v)) and subsequently eluted for 80 min using a 1% to 99% non-linear gradient with buffer B (0.1% acetic acid (v/v) in acetonitrile) at a flow rate of 300 nL/min. Eluted peptides were injected into a LTQ Orbitrap XL (Thermo Fisher Scientific, Waltham, MA, USA). A survey scan at a resolution of R = 30,000 was followed by selection of the five most abundant precursor ions for fragmentation. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository [16 (link)] with the dataset identifier PXD013412.

The enriched peptides were loaded on an EASY-nLC 1000 system (Thermo Fisher Scientific) equipped with an in-house built 20-cm column (inner diameter 100 mm, outer diameter 360 mm) filled with ReproSil-Pur 120 C18-AQ reversed-phase material (3 mm particles, Dr. Maisch GmbH, Germany). Elution of peptides was executed with a nonlinear 86 min gradient from 1 to 99% solvent B (0.1% [vol/vol] acetic acid in acetonitrile) with a flow rate of 300 nl/min and injected online into a QExactive mass spectrometer (Thermo Fisher Scientific). The survey scan at a resolution of R = 70,000 and 3 × 10⁶ automatic gain control target with activated lock mass correction was followed by selection of the 12 most abundant precursor ions for fragmentation. Data-dependent MS/MS scans were performed at a resolution of R = 17,500 and 1 × 10⁵ automatic gain control target with a normalized collision energy of 27.5. Singly charged ions as well as ions without detected charge states or charge states higher than six were excluded from MS/MS analysis. Dynamic exclusion for 30 s was activated.