Proxeon easy nlc 1000 lc pump

Data were collected on an Orbitrap Eclipse mass spectrometer (Thermo Fisher Scientific) coupled to a Proxeon EASY-nLC 1000 LC pump (Thermo Fisher Scientific). Peptides were separated using a 90 min gradient at 500 nL/min on a 30 cm column (i.d. 100 μm, Accucore, 2.6 μm, 150 Å) packed in-house. High-field asymmetric-waveform ion mobility spectroscopy (FAIMS) was enabled during data acquisition with compensation voltages (CVs) set as −40 V, −60 V, and −80 V (Schweppe et al., 2019 (link)). MS1 data were collected using the Orbitrap (60,000 resolution; maximum injection time 50 ms; AGC 4 × 10⁵). Determined charge states between 2 and 6 were required for sequencing, and a 60 s dynamic exclusion window was used. Data-dependent mode was set as cycle time (1 s). MS2 scans were performed in the Orbitrap with HCD fragmentation (isolation window 0.5 Da; 50,000 resolution; NCE 36%; maximum injection time 86 ms; AGC 1 × 10⁵).

Mass spectrometry data were acquired using an Orbitrap Lumos coupled with a Proxeon EASY-nLC 1000 LC pump (Thermo Fisher Scientific, San Jose, CA). Peptides were separated on a 75 mm inner diameter microcapillary column packed with 0.5 cm of Magic C4 resin (5 mm, 100 A°, Michrom Bioresources) followed by approximately 20 cm of GP118 resin (1.8 mm, 120 A°, Sepax Technologies). Peptides were separated using a 3 hr gradient of 6 to 30% acetonitrile in 0.125% formic acid at a flow rate of 300 nl/min. Each analysis used an MS3-based TMT method (McAlister et al., 2012 (link), Ting et al., 2011 (link)). The scan sequence began with an MS1 spectrum (Orbitrap analysis, resolution 120,000, 350-1400 Th, AGC target 5 x 10⁵, maximum injection time 100 ms). ‘Rapid’ was selected for MS2 analysis, which consisted of CID (quadrupole ion trap analysis, AGC 1.8 x 10⁴, NCE 35, maximum injection time 120 ms). For MS3 analysis, precursors were fragmented by HCD prior to Orbitrap analysis (NCE 55, max AGC 2 x 10⁵, maximum injection time 150 ms, isolation specificity 0.7 Th, resolution 50,000).

All data were collected on an Orbitrap Eclipse mass spectrometer (Thermo Fisher Scientific) coupled to a Proxeon EASY-nLC 1000 LC pump (Thermo Fisher Scientific). Peptides were separated using a 90-min gradient at 500 nl/min on a 30-cm column (i.d. 100 mm, Accucore, 2.6 mm, 150 Å) packed inhouse. Data were collected as follows: High-field asymmetric-waveform ion mobility spectroscopy (FAIMS) was enabled during data acquisition with compensation voltages (CVs) set as 40 V, 60 V, and 80 V [33 (link)]. MS1 data were collected using the Orbitrap (60,000 resolution; maximum injection time 50 ms; AGC 4 3 105). Determined charge states between 2 and 6 were required for sequencing, and a 60 s dynamic exclusion window was used. Data dependent mode was set as cycle time (1 s). MS2 scans were performed in the Orbitrap with HCD fragmentation (isolation window 0.5 Da; 50,000 resolution; NCE 36%; maximum injection time 86 ms; AGC 1 3 105).