Measurement of collision cross section (CCS), a parameter reflecting an ion’s size and shape, alongside high-resolution mass analysis extends the depth of molecular analysis by providing structural information beyond molecular mass alone. Although these measurements are most commonly undertaken using a dedicated ion mobility cell coupled to a mass spectrometer, alternative methods have emerged to extract CCSs directly by analysis of the decay rates of either time-domain transient signals or the FWHM of frequency domain peaks in FT mass analyzers. This information is also accessible from FTMS mass spectra obtained in commonly used workflows directly without the explicit access to transient or complex Fourier spectra. Previously, these experiments required isolation of individual charge states of ions prior to CCS analysis, limiting throughput. Here we advance Orbitrap CCS measurements to more users and applications by determining CCSs from commonly available mass spectra files as well as estimating CCS for multiple charge states simultaneously and showcase these methods by the measurement of CCSs of fragment ions produced from collisional activation of proteins.

Glycerophospholipids (GPLs), one of the main components of bacterial cell membranes, exhibit high levels of structural complexity that are directly correlated with biophysical membrane properties such as permeability and fluidity. This structural complexity arises from the substantial variability in the individual GPL structural components such as the acyl chain length and headgroup type and is further amplified by the presence of modifications such as double bonds and cyclopropane rings. Here we use liquid chromatography coupled to high-resolution and high-mass-accuracy ultraviolet photodissociation mass spectrometry for the most in-depth study of bacterial GPL modifications to date. In doing so, we unravel a diverse array of unexplored GPL modifications, ranging from acyl chain hydroxyl groups to novel headgroup structures. Along with characterizing these modifications, we elucidate general trends in bacterial GPL unsaturation elements and thus aim to decipher some of the biochemical pathways of unsaturation incorporation in bacterial GPLs. Finally, we discover aminoacyl-PGs not only in Gram-positive bacteria but also in Gram-negative C. jejuni, advancing our knowledge of the methods of surface charge modulation that Gram-negative organisms may adopt for antibiotic resistance.

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%.

The direct correlation between proteoforms and biological phenotype necessitates the exploration of mass spectrometry (MS)-based methods more suitable for proteoform detection and characterization. Here, we couple nano-hydrophobic interaction chromatography (nano-HIC) to ultraviolet photodissociation MS (UVPD-MS) for separation and characterization of intact proteins and proteoforms. High linearity, sensitivity, and sequence coverage are obtained with this method for a variety of proteins. Investigation of collisional cross sections of intact proteins during nano-HIC indicates semifolded conformations in low charge states, enabling a different dimension of separation in comparison to traditional, fully denaturing reversed-phase separations. This method is demonstrated for a mixture of intact proteins from Escherichia coli ribosomes; high sequence coverage is obtained for a variety of modified and unmodified proteoforms.

The measurement of collision cross sections (CCS, σ) offers supplemental information about sizes and conformations of ions beyond mass analysis alone. We have previously shown that CCSs can be determined directly from the time-domain transient decay of ions in an Orbitrap mass analyzer as ions oscillate around the central electrode and collide with neutral gas, thus removing them from the ion packet. Herein, we develop the modified hard collision model, thus deviating from the prior FT-MS hard sphere model, to determine CCSs as a function of center-of-mass collision energy in the Orbitrap analyzer. With this model, we aim to increase the upper mass limit of CCS measurement for native-like proteins, characterized by low charge states and presumed to be in more compact conformations. We also combine CCS measurements with collision induced unfolding and tandem mass spectrometry experiments to monitor protein unfolding and disassembly of protein complexes and measure CCSs of ejected monomers from protein complexes.