Combining Fourier Transform Ion Mobility with Charge Detection Mass Spectrometry for the Analysis of Multimeric Protein Complexes.

Charge detection mass spectrometry (CDMS) allows direct mass measurement of heterogeneous samples by simultaneously determining the charge state and the mass-to-charge ratio (/) of individual ions, unlike conventional MS methods that use large ensembles of ions. CDMS typically requires long acquisition times and the collection of thousands of spectra, each containing tens to hundreds of ions, to generate sufficient ion statistics, making it difficult to interface with the time scales of online separation techniques such as ion mobility. Here, we demonstrate the application of Fourier transform multiplexing and drift tube ion mobility joined with Orbitrap-based CDMS for the analysis of multimeric protein complexes.

Progress in Tandem Mass Spectrometry Data Analysis for Nucleic Acids.

Mass spectrometry (MS) has become a critical tool in the characterization of covalently modified nucleic acids. Well-developed bottom-up approaches, where nucleic acids are digested with an endonuclease and the resulting oligonucleotides are separated before MS and MS/MS analysis, provide substantial insight into modified nucleotides in biological and synthetic nucleic. Top-down MS presents an alternative approach where the entire nucleic acid molecule is introduced to the mass spectrometer intact and then fragmented by MS/MS.

Proteo-SAFARI: An R Application for Identification of Fragment Ions in Top-Down MS/MS Spectra of Proteins.

Proteo-SAFARI is a shiny application for fragment assignment by relative isotopes, an R-based software application designed for identification of protein fragment ions directly in the / domain. This program provides an open-source, user-friendly application for identification of fragment ions from a candidate protein sequence with support for custom covalent modifications and various visualizations of identified fragments. Additionally, Proteo-SAFARI includes a nonnegative least-squares fitting approach to determine the contributions of various hydrogen shifted fragment ions ( + 1, + 1, - 1, - 2) observed in UVPD mass spectra which exhibit overlapping isotopic distributions.

Structural Analysis of the 20S Proteasome Using Native Mass Spectrometry and Ultraviolet Photodissociation.

Owing to the role of the 20S proteasome in a wide spectrum of pathologies, including neurodegenerative disorders, proteasome-associated autoinflammatory syndromes (PRAAS), and cardiovascular diseases, understanding how its structure and composition contribute to dysfunction is crucial. As a 735 kDa protein assembly, the 20S proteasome facilitates normal cellular proteostasis by degrading oxidized and misfolded proteins. Declined proteasomal activity, which can be attributed to perturbations in the structural integrity of the 20S proteasome, is considered one of the main contributors to multiple proteasome-related diseases.

Nucleo-SAFARI: Automated Identification of Fragment Ions in Top-Down MS/MS Spectra of Nucleic Acids.

Recent progress in top-down mass spectrometry analysis of progressively larger nucleic acids has enabled in-depth characterization of intact, modified RNA molecules. Development of methods for desalting and MS/MS fragmentation allows rapid acquisition of high-quality top-down MS/MS spectra of nucleic acids up to 100 nt, which has spurred the need for development of software approaches to identify and validate nucleic acid fragment ions. We have implemented an R-based approach to aid in analysis of MS/MS spectra of nucleic acids based on fragment ions observed directly in the / domain.

Conformational Characterization of Peptides and Proteins by 193 nm Ultraviolet Photodissociation in the Collision Cell of a Trapped Ion Mobility Spectrometry-Time-of-Flight Mass Spectrometer.

Ultraviolet photodissociation (UVPD) has been shown to be a versatile ion activation strategy for the characterization of peptides and intact proteins among other classes of biological molecules. Combining the high-performance mass spectrometry (MS/MS) capabilities of UVPD with the high-resolution separation of trapped ion mobility spectrometry (TIMS) presents an opportunity for enhanced structural elucidation of biological molecules. In the present work, we integrate a 193 nm excimer laser in a TIMS-time-of-flight (TIMS-TOF) mass spectrometer for UVPD in the collision cell and use it for the analysis of several mass-mobility-selected species of ubiquitin and myoglobin.

Alternative lipid synthesis in response to phosphate limitation promotes antibiotic tolerance in Gram-negative ESKAPE pathogens.

The Gram-negative outer membrane protects bacterial cells from environmental toxins such as antibiotics. The outer membrane lipid bilayer is asymmetric; while glycerophospholipids compose the periplasmic facing leaflet, the surface layer is enriched with phosphate-containing lipopolysaccharides. The anionic phosphates that decorate the cell surface promote electrostatic interactions with cationic antimicrobial peptides such as colistin, allowing them to penetrate the bilayer, form pores, and lyse the cell.

Hydrophilic Interaction Chromatography Coupled to Ultraviolet Photodissociation Affords Identification, Localization, and Relative Quantitation of Glycans on Intact Glycoproteins.

Protein glycosylation is implicated in a wide array of diseases, yet glycoprotein analysis remains elusive owing to the extreme heterogeneity of glycans, including microheterogeneity of some of the glycosites (amino acid residues). Various mass spectrometry (MS) strategies have proven tremendously successful for localizing and identifying glycans, typically utilizing a bottom-up workflow in which glycoproteins are digested to create glycopeptides to facilitate analysis. An emerging alternative is top-down MS that aims to characterize intact glycoproteins to allow precise identification and localization of glycans.

Native Mass Spectrometry Reveals Binding Interactions of SARS-CoV-2 PLpro with Inhibitors and Cellular Targets.

Here we used native mass spectrometry (native MS) to probe a SARS-CoV protease, PLpro, which plays critical roles in coronavirus disease by affecting viral protein production and antagonizing host antiviral responses. Ultraviolet photodissociation (UVPD) and variable temperature electrospray ionization (vT ESI) were used to localize binding sites of PLpro inhibitors and revealed the stabilizing effects of inhibitors on protein tertiary structure. We compared PLpro from SARS-CoV-1 and SARS-CoV-2 in terms of inhibitor and ISG15 interactions to discern possible differences in protease function.

Advancing Protein Analysis: A Low-Pressure Drift Tube Orbitrap Mass Spectrometer for Ultraviolet Photodissociation-Based Structural Characterization.

Owing to its ability to generate extensive fragmentation of proteins, ultraviolet photodissociation (UVPD) mass spectrometry (MS) has emerged as a versatile ion activation technique for the structural characterization of native proteins and protein complexes. Interpreting these fragmentation patterns provides insight into the secondary and tertiary structures of protein ions. However, the inherent complexity and diversity of proteins often pose challenges in resolving their numerous conformations.

Investigating Lipid Transporter Protein and Lipid Interactions Using Variable Temperature Electrospray Ionization, Ultraviolet Photodissociation Mass Spectrometry, and Collision Cross Section Analysis.

Gram-negative bacteria develop and exhibit resistance to antibiotics, owing to their highly asymmetric outer membrane maintained by a group of six proteins comprising the Mla (maintenance of lipid asymmetry) pathway. Here, we investigate the lipid binding preferences of one Mla protein, MlaC, which transports lipids through the periplasm. We used ultraviolet photodissociation (UVPD) to identify and characterize modifications of lipids endogenously bound to MlaC expressed in three different bacteria strains.

Impact of Charge State on Characterization of Large Middle-Down Sized Peptides by Tandem Mass Spectrometry.

Fragmentation trends of large peptides were characterized by five activation methods, including HCD, ETD, EThcD, 213 nm UVPD, and 193 nm UVPD. Sequence coverages and scores were assessed based on charge site, peptide sequence, and peptide size. The effect of charge state and peptide size on sequence coverage was explored for a Glu-C digest of ribosomal proteins, and linear regression analysis of the collection of peptides indicated that HCD, ETD, and EThcD have a higher dependence charge state than 193 and 213 nm UV.

Top-Down Characterization of Bacterial Lipopolysaccharides and Lipooligosaccharides Using Activated-Electron Photodetachment Mass Spectrometry.

Lipopolysaccharides (LPS) and lipooligosaccharides (LOS) are located in the outer membrane of Gram-negative bacteria and are comprised of three distinctive parts: lipid A, core oligosaccharide (OS), and O-antigen. The structure of each region influences bacterial stability, toxicity, and pathogenesis. Here, we highlight the use of targeted activated-electron photodetachment (a-EPD) tandem mass spectrometry to characterize LPS and LOS from two crucial players in the human gut microbiota, Nissle and .

Enhanced Characterization of Lysine-Linked Antibody Drug Conjugates Enabled by Middle-Down Mass Spectrometry and Higher-Energy Collisional Dissociation-Triggered Electron-Transfer/Higher-Energy Collisional Dissociation and Ultraviolet Photodissociation.

As the development of new biotherapeutics advances, increasingly sophisticated tandem mass spectrometry methods are needed to characterize the most complex molecules, including antibody drug conjugates (ADCs). Lysine-linked ADCs, such as trastuzumab-emtansine (T-DM1), are among the most heterogeneous biotherapeutics. Here, we implement a workflow that combines limited proteolysis with HCD-triggered EThcD and UVPD mass spectrometry for the characterization of the resulting middle-down large-sized peptides of T-DM1.

RNA targeting and cleavage by the type III-Dv CRISPR effector complex.

CRISPR-Cas are adaptive immune systems in bacteria and archaea that utilize CRISPR RNA-guided surveillance complexes to target complementary RNA or DNA for destruction. Target RNA cleavage at regular intervals is characteristic of type III effector complexes. Here, we determine the structures of the Synechocystis type III-Dv complex, an apparent evolutionary intermediate from multi-protein to single-protein type III effectors, in pre- and post-cleavage states.

Characterization of Phosphorylated Peptides by Electron-Activated and Ultraviolet Dissociation Mass Spectrometry: A Comparative Study with Collision-Induced Dissociation.

Mass-spectrometry-based methods have made significant progress in the characterization of post-translational modifications (PTMs) in peptides and proteins; however, room remains to improve fragmentation methods. Ideal MS/MS methods are expected to simultaneously provide extensive sequence information and localization of PTM sites and retain labile PTM groups. This collection of criteria is difficult to meet, and the various activation methods available today offer different capabilities.

Mass Spectrometry Strategies for -Glycoproteomics.

Glycoproteomics has accelerated in recent decades owing to numerous innovations in the analytical workflow. In particular, new mass spectrometry strategies have contributed to inroads in -glycoproteomics, a field that lags behind -glycoproteomics due to several unique challenges associated with the complexity of -glycosylation. This review will focus on progress in sample preparation, enrichment strategies, and MS/MS techniques for the identification and characterization of -glycoproteins.

A non-FRET DNA reporter that changes fluorescence colour upon nuclease digestion.

Fluorescence resonance energy transfer (FRET) reporters are commonly used in the final stages of nucleic acid amplification tests to indicate the presence of nucleic acid targets, where fluorescence is restored by nucleases that cleave the FRET reporters. However, the need for dual labelling and purification during manufacturing contributes to the high cost of FRET reporters. Here we demonstrate a low-cost silver nanocluster reporter that does not rely on FRET as the on/off switching mechanism, but rather on a cluster transformation process that leads to fluorescence color change upon nuclease digestion.