Reprosil 100 c18 resin

The affinity-enriched Kcr peptides obtained above were dissolved in 0.1% formic acid in water and directly loaded onto a reversed-phase HPLC column (10 cm length with 75 μm inner diameter) packed in-house with Reprosil 100 C18 resin (3 μm particle size, Dr. Maisch GmbH, Beim Brückle, Germany). The loaded samples were eluted with a gradient of 5–80% HPLC buffer B (0.1% formic acid in 90% acetonitrile, v/v) in buffer A (0.1% formic acid in water, v/v) at a flow rate of 200 nL min⁻¹ over 60 min by an EASY-nLC 1000 UHPLC (ThermoFisher Scientific, Waltham, MA). The samples were analyzed by a Q Exactive mass spectrometer (ThermoFisher Scientific). A data-dependent procedure that alternated between one full mass scan followed by the top 15 most intense precursor ions was applied with 25 second-dynamic exclusion. Intact peptides were detected with a resolution of 70 000, and the tandem mass spectra were acquired with a mass resolution of 17 500 at 27% normalized collision energy.

The enriched peptides obtained above were dissolved in 0.1% formic acid in water and loaded onto a
reversed-phase microcapillary column (10 cm length with 75 μm inner diameter) packed in-house with Reprosil 100 C18
resin (3 μm particle size, Dr. Maisch GmbH, Beim Brückle, Germany). The loaded samples were separated using a
gradient of 5% to 80% HPLC buffer B (0.1% formic acid in 90% acetonitrile, v/v) in buffer A
(0.1% formic acid in water, v/v) at a flow rate of 200 nL/min over 60 min by an EASY-nLC 1000 UPLC (ThermoFisher
Scientific, Waltham, MA). The samples were analyzed by a Q Exactive^™ hybrid quadrupole-orbitrap mass
spectrometer (ThermoFisher Scientific, Waltham, MA). A data-dependent procedure that alternated between one full mass scan
followed by the top 15 most intense precursor ions was applied with a 25 s dynamic exclusion. Intact peptides were detected
with a resolution of 70,000, and the tandem mass spectra were acquired with a mass resolution of 17,500 at 27%
normalized collision energy.