Enhanced Ion Mobility Separation and Characterization of Isomeric Phosphatidylcholines Using Absorption Mode Fourier Transform Multiplexing and Ultraviolet Photodissociation Mass Spectrometry

Abstract

The structural diversity of phospholipids plays a critical role in cellular membrane dynamics, energy storage, and cellular signaling. Despite its importance, the extent of this diversity has only recently come into focus, largely owing to advances in separation science and mass spectrometry methodology and instrumentation. Characterization of glycerophospholipid (GP) isomers differing only in their acyl chain configurations and locations of carbon–carbon double bonds (CC) remains challenging due to the need for both effective separation of isomers and advanced tandem mass spectrometry (MS/MS) technologies capable of double-bond localization. Drift tube ion mobility spectrometry (DTIMS) coupled with MS can provide both fast separation and accurate determination of collision cross section (CCS) of molecules but typically lacks the resolving power needed to separate phospholipid isomers. Ultraviolet photodissociation (UVPD) can provide unambiguous double-bond localization but is challenging to implement on the timescales of modern commercial drift tube time-of-flight mass spectrometers. Here, we present a novel method for coupling DTIMS with a UVPD-enabled Orbitrap mass spectrometer using absorption mode Fourier transform multiplexing that affords simultaneous localization of double bonds and accurate CCS measurements even when isomers cannot be fully resolved in the mobility dimension. This method is demonstrated on two- and three-component mixtures and shown to provide CCS measurements that differ from those obtained by individual analysis of each component by less than 1%.

Separation and Collision Cross Section Measurements of Protein Complexes Afforded by a Modular Drift Tube Coupled to an Orbitrap Mass Spectrometer

“…FT-IMS acquisitions commonly require ≥5 minutes sweeps to adequately sample CCS distributions. ,,,, Shorter acquisitions down to 1 min were recently shown to be possible by artificially extending the XICs (Figure S1b) using zero-padding and apodization (Figure S1c). Here, the 8–10 min sweeps collected for all proteins listed in Table S1 were truncated to 1 min and 3 min and reprocessed using the aFT method as described previously.…”

“…The PCB-DT with a 10 cm desolvation region and a 10 cm drift region was assembled as previously described. ,, A schematic representation of the instrument coupled to a prototype Thermo Scientific Q Exactive Plus UHMR Orbitrap mass spectrometer (Bremen, Germany) is shown in Figure S1a, and FT-IMS parameters are listed in Table S1. Further details about the modular drift tube can be found in the Supporting Information.…”

“…Thermo.raw files were converted to .mzXML using MSConvert and analyzed using a custom Matlab (R2020a) program as previously described. , Briefly, extracted ion chromatograms (XICs) were generated for protein ions (Figure S1b) and averaged across five replicates then zero-padded and apodized (Figure S1c). Resultant XICs were then transformed to arrival time distributions (ATDs) using aFT and baseline-corrected (Figure S1d).…”

“…Our group has adopted an ambient drift tube module constructed from printed circuit boards (PCB) based on an open-source design and integrated it with Orbitrap mass spectrometers modified with an excimer laser for 193 nm ultraviolet photodissociation (UVPD). , Conformer-specific fragments generated from UVPD of lipid double-bond isomers allowed postmobility separation of isobaric ions that were not fully resolved using IMS alone . UVPD has also been shown to generate conformer-dependent fragmentation of proteins and peptides, , underscoring the capabilities of exploring conformational landscapes of gas-phase ions by coupling IMS with UVPD.…”

Nanohydrophobic Interaction Chromatography Coupled to Ultraviolet Photodissociation Mass Spectrometry for the Analysis of Intact Proteins in Low Charge States

“…All data were collected using a modified, research-grade Thermo Scientific Q Exactive HF-X Biopharma mass spectrometer (Bremen, Germany) that was modified by the addition of a 500 Hz, 193 nm coherent ExciStar excimer laser (Santa Cruz, CA) to perform UVPD in the HCD collision cell, as described previously. , For all direction infusion experiments, an nESI NanoFlex source was utilized. The same source was used for interfacing of a nano-LC column eluent into the mass spectrometer.…”