Gas phase fractionation techniques have become popular in the field of mass spectrometry to improve results without using more sample. High field asymmetric waveform ion mobility spectrometry (FAIMS) coupled to liquid chromatography-mass spectrometry (LC-MS) has been shown to increase peptide and protein detections compared to LC-MS/MS alone. However, FAIMS has not been compared to other methods of gas phase fractionation, such as quadrupole gas phase fractionation, which leaves uncertainty about the mechanisms of improvement. The goal of this work was to assess whether FAIMS improves peptide identifications because 1) gas phase fractionation enables the analysis of less abundant signals by excluding more abundant precursors from filling the ion trap, 2) the use of FAIMS reduces co-isolation of peptides during the MS/MS process resulting in a reduction of chimeric spectra, or 3) a combination of both. To investigate these hypotheses, pooled human brain tissue samples were measured in triplicate using FAIMS gas phase fractionation, quadrupole gas phase fractionation, or no gas phase fractionation. To confirm the results, the experiment was reproduced on another instrument. On both instruments, FAIMS gas phase fractionation reduced co-isolation of peptides and generated fewer chimeric spectra even though it was less efficient at transmitting ions than quadrupole gas phase fractionation. This suggests that improvements can be made to the DDA acquisition process by excluding peptides for MS/MS that have more than one peptide isotope distribution in the isolation window.

This experiment was designed to replicate a previous experiment (https://doi.org/10.1021/acs.analchem.8b02233), introduce a new control, and test the reproducibility of results on more than one instrument. Eluted peptides were analyzed with two Orbitrap Eclipse Tribrids. The two instruments examined different pooled human brain tissue samples with slightly different LC setups and gradients. Sample preparation has been described in detail previously (https://doi.org/10.1038/s41597-023-02057-7), and the nanoLC conditions can be found in the Supporting Information. Experiments without FAIMS used a 240,000 resolving power MS1 survey scan, Standard AGC Target, and Auto Maximum Injection Time, followed by MS/MS of the most intense precursors for 1 second. The MS/MS analyses were performed by 0.7 m/z isolation with the quadrupole, normalized HCD (higher-energy collisional dissociation) energy of 30%, and analysis of fragment ions in the ion trap using the “Turbo” speed scanning from 200 to 1200 m/z. Dynamic exclusion was set to 10 seconds for the 1-hour analyses and was increased to 30 seconds for the 3-hour analyses. Monoisotopic precursor selection (MIPS) was set to Peptide, maximum injection time was 35 milliseconds, AGC target was 200%, unusual charge states (unknown, +1, or >+5) were excluded, the advanced peak determination was toggled on, and the internal mass calibration was off. For FAIMS experiments, the settings were identical except the FAIMS device was used between the electrospray source and the mass spectrometer. FAIMS separations were performed with a 100 °C inner electrode temperature, 100 °C outer electrode temperature, 4.7 L/min FAIMS carrier gas flow, and −5000 V dispersion voltage (DV). The FAIMS carrier gas was N2 only. For external stepping (i.e., single CV or single quadrupole fraction) experiments, the selected CV or quadrupole fraction was applied to all scans throughout the analysis. For internal stepping experiments, each of the 3 selected CVs or quadrupole fractions was applied to sequential survey scans. The MS/MS CV was always paired with the appropriate CV from the corresponding survey scan. For the 3-hour quantitative FAIMS experiments, the survey scan MS resolving power was reduced to 120,000 to permit a cycle time of 0.6 s. The 3 selected CVs (-50, -65, and -85) were chosen based on the results in Hebert et al (https://doi.org/10.1021/acs.analchem.8b02233). The 3 quadrupole fractions were sample-specific and were chosen based on splitting the number of peptide-like features in a normal LC-MS run into thirds.

Briefly, two 25 μm frozen sections of human brain tissue were resuspended in 120 μl of lysis buffer containing 5% SDS, 50mM triethylammonium bicarbonate (TEAB), 2mM MgCl2, 1X HALT phosphatase, and protease inhibitors. The suspension was vortexed and briefly sonicated with a Fisher sonic dismembrator model 100 set to setting 3 for 10 s. A microtube was loaded with 30 μl of lysate and capped with a micropestle. The sample was homogenized with a Barocycler 2320EXT (Pressure Biosciences Inc.) for a total of 20 minutes at 35°C with 30 cycles of 20 seconds at 45,000 psi and 10 seconds at atmospheric pressure. Protein concentration of the homogenate was measured with a BCA assay. Fifty micrograms were added to a process control of 800 ng of yeast enolase protein (Sigma), reduced with 20 mM DTT, and alkylated with 40 mM IAA. The lysate was prepared for S-trap column (Protifi) cleaning by adding 1.2% phosphoric acid and 350 μL of binding buffer (90% Methanol, 100 mM TEAB). The acidified lysate was bound to column incrementally, followed by 3 wash steps with binding buffer to remove SDS, 3 wash steps with 50:50 methanol:chloroform to remove lipids, and a final wash step with binding buffer. Trypsin (1:10) in 50mM TEAB was added to the S-trap column for digestion at 47°C for one hour. Hydrophilic peptides were eluted with 50 mM TEAB and hydrophobic peptides were eluted with a solution of 50% acetonitrile in 0.2% formic acid. Elutions were pooled, speed vacuumed and resuspended in 0.1% formic acid. For more sample details, see Merrihew et al. (https://doi.org/10.1038/s41597-023-02057-7).