Acclaim1 pepmap 100 precolumn

We used two main LC‐MS/MS setups: System 1 and System 2. System 1 comprised an Easy nLC‐1000 (Thermo Fisher Scientific) coupled to a Q Exactive Plus mass spectrometer (Thermo Fisher Scientific, San Jose, CA). Here the peptides (∼1 μg) were initially loaded onto a trap column (Acclaim PepMap 100 precolumn, 75 μm i.d. × 2 cm, C18, 3 μm, 100 Å; ThermoFisher Scientific, San Jose, CA) and then separated on an analytical column (EASY‐Spray column, 75 μm i.d. × 25 cm, PepMap RSLC C18, 2 μm, 100 Å; ThermoFisher Scientific, San Jose, CA). System 2 comprised an Ultimate 3000 nLC (Thermo Scientific, San José, CA, USA, Bremen Germany) coupled to a Q Exactive HF‐X mass spectrometer (Thermo Scientific). For this case, the peptides (∼1 μg) were loaded in a trap column (Acclaim1 PepMap 100 precolumn, 75 μm, 2 cm, C18, 3 μm, 100 Å, Thermo Scientific) and then separated on an analytical column (EASY‐Spray column 25 or 50 cm, 75 μm i.d., PepMap RSLC C18, 2 μm, 100Å, Thermo Scientific). Both systems used a flow rate of 300 nL/min and a water/ACN gradient in 0.1% formic acid and samples were measured in DDA and DIA modes. The DIA‐MS Spectral library was built out of DDA‐LC‐MS/MS analyses of samples from tissue and cultured cell origin, with spiked in iRT peptides (Biognosis AG). This also included the analysis of the mixture of samples previously fractionated by HpH RP‐HPLC.

We used two main LC‐MS/MS setups. System 1 comprised an Easy nLC‐1000 (Thermo Fisher Scientific) coupled to a Q Exactive Plus mass spectrometer (Thermo Fisher Scientific). Here the peptides ( ´∼1 μg) were initially loaded onto a trap column (Acclaim PepMap 100 precolumn, 75 μm i.d. × 2 cm, C18, 3 μm, 100 Å; ThermoFisher Scientific) and then separated on an analytical column (EASY‐Spray column, 75 μm i.d. × 25 cm, PepMap RSLC C18, 2 μm, 100 Å; ThermoFisher Scientific). System 2 comprised an Ultimate 3000 nLC (Thermo Scientific Bremen Germany) coupled to a Q Exactive HF‐X mass spectrometer (Thermo Scientific). For this case the peptides (´∼1 μg) were loaded in a trap column (Acclaim1 PepMap 100 pre‐column, 75 μm, 2 cm, C18, 3 m, 100 Å, Thermo Scientific, San José, CA) and then separated on an analytical column (EASY‐Spray column 25 or 50 cm, 75 μm i.d., PepMap RSLC C18, 2 μm, 100Å, Thermo Scientific). Both systems used a flow rate of 300 nL/min and a water/ACN gradient in 0.1% formic acid and samples were measured in DDA and DIA modes. The DIA‐MS Spectral library was built out of DDA‐LC‐MS/MS analyses of samples from tissue and cultured cell origin, with spiked in iRT peptides (Biognosis AG). This also included the analysis of a mixture of samples previously fractionated by HpH RP‐HPLC.