Gábor Transform-Based Signal Isolation, Rapid Deconvolution, and Quantitation of Intact Protein Ions with Mass Spectrometry.

High-resolution mass spectrometry (HRMS) is a powerful technique for the characterization and quantitation of complex biological mixtures, with several applications including clinical monitoring and tissue imaging. However, these medical and pharmaceutical applications are pushing the analytical limits of modern HRMS techniques, requiring either further development in instrumentation or data processing methods. Here, we demonstrate new developments in the interactive Fourier-transform analysis for mass spectrometry (iFAMS) software including the first application of Gábor transform (GT) to protein quantitation. Newly added automation tools detect signals from minimal user input and apply thresholds for signal selection, deconvolution, and baseline correction to improve the objectivity and reproducibility of deconvolution. Additional tools were added to improve the deconvolution of highly complex or congested mass spectra and are demonstrated here for the first time. The "Gábor Slicer" enables the user to explore trends in the Gábor spectrogram with instantaneous ion mass estimates accurate to 10 Da. The charge adjuster allows for easy visual confirmation of accurate charge state assignments and quick adjustment if necessary. Deconvolution refinement utilizes a second GT of isotopically resolved data to remove common deconvolution artifacts. To assess the quality of deconvolution from iFAMS, several comparisons are made to deconvolutions using other algorithms such as UniDec and an implementation of MaxEnt in Agilent MassHunter BioConfirm. Lastly, the newly added batch processing and quantitation capabilities of iFAMS are demonstrated and compared to a common extracted ion chromatogram approach.

The complete assembly of human LAT1-4F2hc complex provides insights into its regulation, function and localisation.

The LAT1-4F2hc complex (SLC7A5-SLC3A2) facilitates uptake of essential amino acids, hormones and drugs. Its dysfunction is associated with many cancers and immune/neurological disorders. Here, we apply native mass spectrometry (MS)-based approaches to provide evidence of super-dimer formation (LAT1-4F2hc)2. When combined with lipidomics, and site-directed mutagenesis, we discover four endogenous phosphatidylethanolamine (PE) molecules at the interface and C-terminus of both LAT1 subunits. We find that interfacial PE binding is regulated by 4F2hc-R183 and is critical for regulation of palmitoylation on neighbouring LAT1-C187. Combining native MS with mass photometry (MP), we reveal that super-dimerization is sensitive to pH, and modulated by complex N-glycans on the 4F2hc subunit. We further validate the dynamic assemblies of LAT1-4F2hc on plasma membrane and in the lysosome. Together our results link PTM and lipid binding with regulation and localisation of the LAT1-4F2hc super-dimer.

Label-Free Composition Analysis of Supramolecular Polymer-Nanoparticle Hydrogels by Reversed-Phase Liquid Chromatography Coupled with a Charged Aerosol Detector.

Supramolecular hydrogels formed through polymer-nanoparticle interactions are promising biocompatible materials for translational medicines. This class of hydrogels exhibits shear-thinning behavior and rapid recovery of mechanical properties, providing desirable attributes for formulating sprayable and injectable therapeutics. Characterization of hydrogel composition and loading of encapsulated drugs is critical to achieving the desired rheological behavior as well as tunable in vitro and in vivo payload release kinetics. However, quantitation of hydrogel composition is challenging due to material complexity, heterogeneity, high molecular weight, and the lack of chromophores. Here, we present a label-free approach to simultaneously determine hydrogel polymeric components and encapsulated payloads by coupling a reversed phase liquid chromatographic method with a charged aerosol detector (RPLC-CAD). The hydrogel studied consists of modified hydroxypropylmethylcellulose, self-assembled PEG-b-PLA nanoparticles, and a therapeutic compound, bimatoprost. The three components were resolved and quantitated using the RPLC-CAD method with a C4 stationary phase. The method demonstrated robust performance, applicability to alternative cargos (i.e., proteins) and was suitable for composition analysis as well as for evaluating in vitro release of cargos from the hydrogel. Moreover, this method can be used to monitor polymer degradation and material stability, which can be further elucidated by coupling the RPLC method with (1) a multi-angle light scattering detector (RPLC-MALS) or (2) high resolution mass spectrometry (RPLC-MS) and a Fourier-transform based deconvolution algorithm. We envision that this analytical strategy could be generalized to characterize critical quality attributes of other classes of supramolecular hydrogels, establish structure-property relationships, and provide rational design guidance in hydrogel drug product development.

Label-Free Composition Analysis of Supramolecular Polymer - Nanoparticle Hydrogels by Reversed-Phase Liquid Chromatography Coupled with a Charged Aerosol Detector.

Supramolecular hydrogels formed through polymer-nanoparticle interactions are promising biocompatible materials for translational medicines. This class of hydrogels exhibits shear-thinning behavior and rapid recovery of mechanical properties following applied stresses, providing desirable attributes for formulating sprayable and injectable therapeutics. Characterization of hydrogel composition and loading of encapsulated drugs is critical to achieving desired rheological behavior as well as tunable in vitro and in vivo payload release kinetics. However, quantitation of hydrogel compositions is challenging due to material complexity, heterogeneity, high molecular weight, and the lack of chromophores. Here, we present a label-free approach to simultaneously determine hydrogel polymeric components and encapsulated payloads by coupling a reversed phase liquid chromatographic method with a charged aerosol detector (RPLC-CAD). The hydrogel studied consists of modified hydroxypropylmethylcellulose, self-assembled PEG-b-PLA nanoparticles, and a therapeutic compound, Bimatoprost. The three components were resolved and quantitated using the RPLC-CAD method with a C4 stationary phase. The method demonstrated robust performance, applicability to alternative cargos (i.e. proteins), and was suitable for composition analysis as well as for evaluating in vitro release of cargos from the hydrogel. Moreover, this method can be used to monitor polymer degradation and material stability, which can be further elucidated by coupling the RPLC method with high resolution mass spectrometry and a Fourier-transform based deconvolution algorithm. To our knowledge, this is the first RPLC-CAD method for characterizing the critical quality attributes of supramolecular hydrogels. We envision this analytical strategy could be generalized to characterize other classes of supramolecular hydrogels, establish structure-property relationships, and provide rational design guidance in hydrogel drug product development.

Linker Length Drives Heterogeneity of Multivalent Complexes of Hub Protein LC8 and Transcription Factor ASCIZ.

LC8, a ubiquitous and highly conserved hub protein, binds over 100 proteins involved in numerous cellular functions, including cell death, signaling, tumor suppression, and viral infection. LC8 binds intrinsically disordered proteins (IDPs), and although several of these contain multiple LC8 binding motifs, the effects of multivalency on complex formation are unclear. Drosophila ASCIZ has seven motifs that vary in sequence and inter-motif linker lengths, especially within subdomain QT2-4 containing the second, third, and fourth LC8 motifs. Using isothermal-titration calorimetry, analytical-ultracentrifugation, and native mass-spectrometry of QT2-4 variants, with methodically deactivated motifs, we show that inter-motif spacing and specific motif sequences combine to control binding affinity and compositional heterogeneity of multivalent duplexes. A short linker separating strong and weak motifs results in stable duplexes but forms off-register structures at high LC8 concentrations. Contrastingly, long linkers engender lower cooperativity and heterogeneous complexation at low LC8 concentrations. Accordingly, two-mers, rather than the expected three-mers, dominate negative-stain electron-microscopy images of QT2-4. Comparing variants containing weak-strong and strong-strong motif combinations demonstrates sequence also regulates IDP/LC8 assembly. The observed trends persist for trivalent ASCIZ subdomains: QT2-4, with long and short linkers, forms heterogeneous complexes, whereas QT4-6, with similar mid-length linkers, forms homogeneous complexes. Implications of linker length variations for function are discussed.

Fourier-Transform Approach for Reconstructing Macromolecular Mass Defect Profiles.

State-of-the-art native mass spectrometry (MS) methods have been developed for analysis of highly heterogeneous intact complexes and have provided much insight into the structure and properties of noncovalent assemblies that can be difficult to study using denatured proteins. These native MS methods can often be used to study even highly polydisperse membrane proteins embedded in detergent micelles, nanodiscs, and other membrane mimics. However, characterizing highly polydisperse native complexes which are also heterogeneous presents additional challenges for native MS. Macromolecular mass defect (MMD) analysis aims to characterize heterogeneous ion populations obfuscated by adduct polydispersity and reveal the distribution of "base" masses, and was recently implemented in the Bayesian analysis software UniDec. Here, we illustrate an alternative, orthogonal MMD analysis method implemented in the deconvolution program iFAMS, which takes advantage of Fourier transform (FT) to deconvolve low-resolution data with few user-input parameters and which can provide high quality results even for mass spectra with a signal-to-noise ratio of ∼5:1. Agreement between this method, which is based on frequency-domain data, and the mass-domain algorithm of UniDec provides strong evidence that both methods can accurately characterize highly polydisperse and heterogeneous ion populations. The FT algorithm is expected to be very useful in characterizing many types of analytes ranging from membrane proteins to polymer-conjugated proteins, branched polymers, and other large analytes, as well as for reconstructing isotope profiles for highly complex but still isotope-resolved mass spectra.