C18 pepmap100 pre column

Peptide samples were reconstituted in 5.0 μl 30% formic acid (FA) containing 10 fmol each of 4 synthetic standard peptides and diluted with 40 μl mobile phase A (98% H₂O, 2% ACN, 0.1% FA), as described previously [22 (link), 23 (link)]. LC-MS/MS analyses were conducted using a Dionex Ultimate 3000 nano LC-system coupled to a QExactive orbitrap mass spectrometer (Thermo Fisher Scientific, Austria). 5.0 μl of the peptide samples were pre-concentrated on a 2 cm x 75 μm C18 Pepmap100 pre-column (Thermo Fisher Scientific, Austria) at a flow rate of 10 μl/min using mobile phase A. Afterwards, peptides were eluted from the pre-column to a 50 cm x 75 μm Pepmap100 analytical column (Thermo Fisher Scientific, Austria) at a flow rate of 300 nl/min. Chromatographic separation was accomplished using a gradient from 8% to 40% mobile phase B (80% ACN, 20% H₂O, 0.1% FA) over 95 min.
All samples were analyzed in technical duplicates. MS scans were achieved in the range from m/z 400–1400 at a resolution of 70,000 (at m/z = 200), whereas MS/MS scans of the 8 most abundant ions were performed using HCD fragmentation at 30% normalized collision energy and analyzed in the orbitrap at a resolution of 17,500 (at m/z = 200).

Samples were reconstituted in 5 µl 30% formic acid containing 10 fmol each of 4 synthetic standard peptides and then immediately diluted with 40 µl mobile phase A (98% H₂O, 2% acetonitrile, and 0.1% formic acid). The synthetic peptides [Glu1-Fribrinopeptide B, EGVNDNEEGFFSAR; M28, TTPAVLDSDGSYFLYSK; HK0, VLETKSLYVR; HK1, VLETK(ε-AC)SLYVR] were spiked into each sample as an internal quality control for monitoring LC–MS instrument stability. Five microliters of the solution were injected into the nano HPLC-system (Dionex Ultimate 3000) loading peptides on a 2 cm × 75 µm C18 Pepmap100 pre-column (Thermo Fisher Scientific) at a flow rate of 10 µl/min using mobile phase A. Afterwards, peptides were eluted to a 50 cm × 75 µm Pepmap100 analytical column (Thermo Fisher Scientific) at a flow rate of 300 nl/min, using a gradient from 8 to 40% mobile phase B (80% acetonnitrile, 20% H2O, 0.1% formic acid) over 235 min. The nano-HPLC system was coupled to a QExactive orbitrap with a nanospray ion source (Thermo Fisher Scientific). MS scans were performed in the range from m/z 400 to 1400 at a resolution of 70,000 (at m/z = 200), MS/MS scans at a resolution of 17,500 (at m/z = 200), using a top 12 method and applying HCD fragmentation at 30% normalized collision energy.

Dried samples were reconstituted in 5 µL 30% FA containing 10 fmol each of four synthetic standard peptides and diluted with 40 µL mobile phase A (98% H₂O, 2% ACN, 0.1% FA). 5 µL of this solution was then injected into a Dionex Ultimate 3000 nano LC-system coupled to a QExactive orbitrap mass spectrometer equipped with a nanospray ion source (Thermo Fisher Scientific, Austria). As a pre-concentration step, peptides were loaded on a 2 cm x 100 µm C18 Pepmap100 pre-column (Thermo Fisher Scientific, Austria) at a flow rate of 10 µL/min using mobile phase A. Elution from the pre-column to a 50 cm ×  75 µm Pepmap100 analytical column (Thermo Fisher Scientific, Austria) and subsequent separation was achieved at a flow rate of 300 nL/min using a gradient of 8–40% mobile phase B (80% ACN, 2% H₂O, 0.1% FA) over 90 min. For mass spectrometric detection, MS scans were performed in the range from m/z 400–1400 at a resolution of 70,000 (at m/z = 200). MS/MS scans of the 8 most abundant ions were achieved through HCD fragmentation at 30% normalized collision energy and analyzed in the orbitrap at a resolution of 17,500 (at m/z = 200).