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 m/z domain. This program, entitled Shiny Application for Fragment Assignment by Relative Isotopes (Nucleo-SAFARI), utilizes the Shiny HTML framework for deployment of a user-friendly application for automated annotation of top-down MS/MS spectra of nucleic acids recorded on Orbitrap mass spectrometer platforms. This approach proceeds through in silico generation of fragment ions and their isotopic distributions, followed by algorithmic assessment of the experimental isotopic distributions. Nucleo-SAFARI is available for download at https://github.com/mblanzillotti/Nucleo-SAFARI.

Tracking Inhibition of Human Small C-Terminal Domain Phosphatase 1 Using 193 nm Ultraviolet Photodissociation Mass Spectrometry.

Working in tandem with kinases via a dynamic interplay of phosphorylation and dephosphorylation of proteins, phosphatases regulate many cellular processes and thus represent compelling therapeutic targets. Here we leverage ultraviolet photodissociation to shed light on the binding characteristics of two covalent phosphatase inhibitors, T65 and rabeprazole, and their respective interactions with the human small C-terminal domain phosphatase 1 (SCP1) and its single-point mutant C181A, in which a nonreactive alanine replaces one key reactive cysteine. Top-down MS/MS analysis is used to localize the binding of T65 and rabeprazole on the two proteins and estimate the relative reactivities of each cysteine residue.

A High-Throughput Workflow for Mass Spectrometry Analysis of Nucleic Acids by Nanoflow Desalting.

Broad interest in nucleic acids, both their therapeutic capabilities and understanding the nuances of their structure and resulting function, has increased in recent years. Post-transcriptional modifications, in particular, have become an important analysis target, as these covalent modifications to the sugars, nitrogenous bases, and phosphate backbone impart differential functionality to synthetic and biological nucleic acids. Characterizing these post-transcriptional modifications can be difficult with traditional sequencing workflows; however, advancements in top-down mass spectrometry address these challenges. Online desalting platforms have enabled facile sample cleanup and reliable ionization of increasingly large (100 nt) oligonucleotides, and application of existing tandem mass spectrometry techniques has yielded information-rich spectra which can be used to interrogate primary sequences. To extend the capabilities of top-down MS and its analysis of nucleic acids, we have developed a nanoflow desalting platform for high-throughput and low sample-use desalting coupled with collision-induced dissociation (CID), 213 nm ultraviolet photodissociation (UVPD), and activated-ion electron photodetachment dissociation (a-EPD) to yield high-quality MS/MS spectra. Fragments identified using an m/z-domain isotope matching strategy yielded high sequence coverage (>70%) of a yeast phenylalanine tRNA.

Top-Down Mass Spectrometry of Synthetic Single Guide Ribonucleic Acids Enabled by Facile Sample Clean-Up.

In recent years, CRISPR-Cas9 genome editing has become an important technology in biomedical research and has demonstrated tremendous therapeutic potential. With Cas9 endonuclease, the use of single guide ribonucleic acids (sgRNAs) allows for sequence-specific cutting on target double-stranded deoxyribonucleic acids. Therefore, the design and quality of sgRNAs can greatly affect the efficiency and specificity of genome editing. Mass spectrometry (MS) has been a powerful tool to detect molecular features and sequence a variety of biomolecules; however, as the sizes of oligonucleotides get larger, it becomes more challenging to desalt samples and achieve high-quality intact spectra with effective fragmentation. Here, we develop a simple but effective online column-based clean-up method (reversed-phase column in a size exclusion mode) that removes formulation salts and metal adducts from larger oligonucleotides upon entering the mass spectrometer in a consistent manner. Using the top-down approach without any nuclease digestion, we characterized and sequenced 100-nucleotide-long sgRNAs by higher-energy collision dissociation (HCD), collision-induced dissociation (CID), ultraviolet photodissociation (UVPD), and activated electron photodetachment (a-EPD). In a single 10 min liquid chromatography-tandem MS (LC-MS/MS) run, CID yielded the best sequence coverage, of 67%. When adding complementary UVPD and a-EPD runs, we achieved 80% overall sequence coverage and 100% cleavages for the variable sequence, the first 20 nucleotides from the 5' end. This LC-MS/MS platform provides a facile top-down workflow to analyze and sequence larger chemically modified oligonucleotides with no sample treatment.

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

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.

Analysis of Viral Spike Protein N-Glycosylation Using Ultraviolet Photodissociation Mass Spectrometry.

Characterization of protein glycosylation by tandem mass spectrometry remains challenging owing to the vast diversity of oligosaccharides bound to proteins, the variation in monosaccharide linkage patterns, and the lability of the linkage between the glycan and protein. Here, we have adapted an HCD-triggered-ultraviolet photodissociation (UVPD) approach for the simultaneous localization of glycosites and full characterization of both glycan compositions and intersaccharide linkages, the latter provided by extensive cross-ring cleavages enabled by UVPD. The method is applied to study glycan compositions based on analysis of glycopeptides from proteolytic digestion of recombinant human coronaviruse spike proteins from SARS-CoV-2 and HKU1. UVPD reveals unique intersaccharide linkage information and is leveraged to localize N-linked glycoforms with confidence.

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

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%.

Mapping a Conformational Epitope of Hemagglutinin A Using Native Mass Spectrometry and Ultraviolet Photodissociation.

As the importance of effective vaccines and the role of protein therapeutics in the drug industry continue to expand, alternative strategies to characterize protein complexes are needed. Mass spectrometry (MS) in conjunction with enzymatic digestion or chemical probes has been widely used for mapping binding epitopes at the molecular level. However, advances in instrumentation and application of activation methods capable of accessing higher energy dissociation pathways have recently allowed direct analysis of protein complexes. Here we demonstrate a workflow utilizing native MS and ultraviolet photodissociation (UVPD) to map the antigenic determinants of a model antibody-antigen complex involving hemagglutinin (HA), the primary immunogenic antigen of the influenza virus, and the D1 H1-17/H3-14 antibody which has been shown to confer potent protection to lethal infection in mice despite lacking neutralization activity. Comparison of sequence coverages upon UV photoactivation of HA and of the HA·antibody complex indicates the elimination of some sequence ions that originate from backbone cleavages exclusively along the putative epitope regions of HA in the presence of the antibody. Mapping the number of sequence ions covering the HA antigen versus the HA·antibody complex highlights regions with suppressed backbone cleavage and allows elucidation of unknown epitopes. Moreover, examining the observed fragment ion types generated by UVPD demonstrates a loss in diversity exclusively along the antigenic determinants upon MS/MS of the antibody-antigen complex. UVPD-MS shows promise as a method to rapidly map epitope regions along antibody-antigen complexes as novel antibodies are discovered or developed.