Faims pro interface

Mass spectrometry was performed utilizing an EASY-nLC 1200 HPLC system (SCR: 014993, Thermo Fisher Scientific) coupled to an Exploris 480™ mass spectrometer with a FAIMSpro interface (Thermo Fisher Scientific). One-fifth of each fraction was loaded onto a 25 cm IonOpticks-TS column (Ionopticks Aurora Ultimate TS 25 cm) at 350 nL/min. The gradient was held at 5% B for 5 min (mobile phases A: 0.1% formic acid (FA), water; B: 0.1% FA, 80% acetonitrile (Thermo Fisher Scientific Cat No: LS122500)), then increased from 4 to 30% B over 98 min, 30 to 80% B over 10 min, held at 80% for 2 min, and dropped from 80 to 4% B over the final 5 min. The mass spectrometer was operated in positive ion mode, default charge state of 2, advanced peak determination on, and lock mass of 445.12003. Three FAIMS CVs were utilized (−40 CV; −55 CV; −70 CV), each with a cycle time of 1.3 s and with identical MS and MS2 parameters. Precursor scans (m/z 375–1500) were performed with an orbitrap resolution of 120,000, RF lens% 40, automatic maximum inject time, standard AGC target, minimum MS2 intensity threshold of 5 × 10³, MIPS mode to peptide, including charges of 2 to 7 for fragmentation with 30 sec dynamic exclusion. MS2 scans were performed with a quadrupole isolation window of 1.6 m/z, 30% HCD CE, 15,000 resolution, standard AGC target, automatic maximum IT, and fixed first mass of 110 m/z.

Desalted samples (1.2 µg) were combined with synthetic isotope-labelled peptides (6 fmol). Five-sixths of this mixture (1 µg of total peptide and 5 fmol of each synthetic peptide) was loaded onto EvoTip trapping columns before separation with the EvoSep One nanoLC system (EvoSep, Odense, Denmark) coupled to an Orbitrap Fusion Lumos mass spectrometer with a FAIMS-PRO interface (Thermo Fisher). Peptides were eluted over a 44-min gradient, from 7 to 30% acetonitrile (on-column), at a flow rate of 500 nL/min (Supplemental Materials and Methods). The FAIMS-PRM experiment employed higher-energy collisional dissociation (HCD) fragmentation with an isolation window of 0.7 mass-to-charge ratio (m/z), a target automatic gain control of 1E6 ions, and a maximum injection time of 100 ms. Tandem MS (MS/MS) scans were acquired in centroid mode with the Orbitrap detector, using 30 K resolution at 200 m/z (unless otherwise stated it the text). FAIMS was operated at the standard resolution, with no additional FAIMS gas.

Digested samples were separated by nanoflow HPLC (Evosep One, Evosep, Odense, Denmark) and analyzed on a Orbitrap Fusion Lumos mass spectrometer with a FAIMS-PRO interface (Thermo) as previously described [19 (link)]. Briefly, a total of 1 µg of total protein digest with 5 fmol of spiked heavy peptide standards was loaded onto Evotips and separated using the 30SPD standardized method (44 min gradient). Optimized FAIMS compensation voltages were used to reduce background ions and selected ions were fragmented by higher-energy collisional dissociation and detected at 30 k resolution. Full experimental details on method development, optimized parameters, and standard curve generation have been reported [19 (link)]. The target protein concentration (in units of amol/µg) was determined by multiplying the peak area ratios (light/heavy transition) by the heavy spike (5 fmol per ug total protein).