Characterization of antisense oligonucleotide and guide ribonucleic acid diastereomers by hydrophilic interaction liquid chromatography coupled to mass spectrometry.

Oligonucleotides have become an essential modality for a variety of therapeutic approaches, including cell and gene therapies. Rapid progress in the field has attracted significant research in designing novel oligonucleotide chemistries and structures. Beyond their polar nature, the length of large RNAs and presence of numerous diastereomers for phosphorothioate (PS)-modified RNAs pose heightened challenges for their characterization. In this study, the stereochemistry of a fully-modified antisense oligonucleotide (ASO) and partially-modified guide RNAs (gRNAs) was investigated using HILIC and orthogonal techniques. The profiles of three lots of a fully-modified ASO with PS linkages were compared using ion-pairing RPLC (IPRP) and HILIC. Interestingly, three isomer peaks were partially resolved by HILIC for two lots while only one peak was observed on the IPRP profile. Model oligonucleotides having the same sequence of the five nucleotides incorporated to the 3'-end of the gRNA but differing in their number and position of PS linkages were investigated by HILIC, IPRP, ion mobility spectrometry-mass spectrometry (IM-MS) and nuclear magnetic resonance (NMR). An strategy was ultimately designed to aid in the characterization of gRNA stereochemistry. Ribonuclease (RNase) T1 digestion enabled the characterization of gRNA diastereomers by reducing their number from 32 at the gRNA intact level to 4 or 8 at the fragment level. To our knowledge, this is the first time that HILIC has successfully been utilized for the profiling of diastereomers for various oligonucleotide formats and chemical modifications.

Deeper Understanding of Solvent-Based Ambient Ionization Mass Spectrometry: Are Molecular Profiles Primarily Dictated by Extraction Mechanisms?

Solvent-based ambient ionization mass spectrometry (MS) techniques provide a powerful approach for direct chemical analysis and molecular profiling of biological tissues. While molecular profiling of tissues has been widely used for disease diagnosis, little is understood about how the interplay among solvent properties, matrix effects, and ion suppression can influence the detection of biological molecules. Here, we perform a systematic investigation of the extraction processes of lipids using an ambient ionization droplet microsampling platform to investigate how the physicochemical properties of the solvent systems and extraction time influence molecular extraction and detection. Direct molecular profiling and quantitative liquid chromatography-mass spectrometry (LC-MS) of discrete solvent droplets after surface sampling were investigated to provide insights into extraction and ionization mechanisms. The results of this study suggest that intermolecular interactions such as hydrogen bonding play a major role in extraction and detection of lipids using solvent-based ambient ionization techniques. In addition, extraction time was observed to impact the molecular profiles obtained, suggesting optimization of this parameter can be performed to favor detection of specific analytes.

Structural Characterization and Quantitation of Ether-Linked Glycerophospholipids in Peroxisome Biogenesis Disorder Tissue by Ultraviolet Photodissociation Mass Spectrometry.

The biological impact of ether glycerophospholipids (GP) in peroxisomal disorders and other diseases makes them significant targets as biomarkers for diagnostic assays or deciphering pathology of the disorders. Ether lipids include both plasmanyl and plasmenyl lipids, which each contain an ether or a vinyl ether bond at the sn-1 linkage position, respectively. This linkage, in contrast to traditional diacyl GPs, precludes their detailed characterization by mass spectrometry via traditional collisional-based MS/MS techniques. Additionally, the isomeric nature of plasmanyl and plasmenyl pairs of ether lipids introduces a further level of complexity that impedes analysis of these species. Here, we utilize 213 nm ultraviolet photodissociation mass spectrometry (UVPD-MS) for detailed characterization of phosphatidylethanolamine (PE) and phosphatidylcholine (PC) plasmenyl and plasmanyl lipids in mouse brain tissue. 213 nm UVPD-MS enables the successful differentiation of these four ether lipid subtypes for the first time. We couple this UVPD-MS methodology to reversed-phase liquid chromatography (RPLC) for characterization and relative quantitation of ether lipids from normal and diseased (Pex7 deficiency modeling the peroxisome biogenesis disorder, RCDP) mouse brain tissue, highlighting the ability to pinpoint specific structural features of ether lipids that are important for monitoring aberrant lipid metabolism in peroxisomal disorders.

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.

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%.

Characterization of the T4 gp32-ssDNA complex by native, cross-linking, and ultraviolet photodissociation mass spectrometry.

Protein-DNA interactions play crucial roles in DNA replication across all living organisms. Here, we apply a suite of mass spectrometry (MS) tools to characterize a protein-ssDNA complex, T4 gp32·ssDNA, with results that both support previous studies and simultaneously uncover novel insight into this non-covalent biological complex. Native mass spectrometry of the protein reveals the co-occurrence of Zn-bound monomers and homodimers, while addition of differing lengths of ssDNA generates a variety of protein:ssDNA complex stoichiometries (1 : 1, 2 : 1, 3 : 1), indicating sequential association of gp32 monomers with ssDNA. Ultraviolet photodissociation (UVPD) mass spectrometry allows characterization of the binding site of the ssDNA within the protein monomer via analysis of holo ions, i.e. ssDNA-containing protein fragments, enabling interrogation of disordered regions of the protein which are inaccessible via traditional crystallographic techniques. Finally, two complementary cross-linking (XL) approaches, bottom-up analysis of the crosslinked complexes as well as MS1 analysis of the intact complexes, are used to showcase the absence of ssDNA binding with the intact cross-linked homodimer and to generate two homodimer gp32 model structures which highlight that the homodimer interface overlaps with the monomer ssDNA-binding site. These models suggest that the homodimer may function in a regulatory capacity by controlling the extent of ssDNA binding of the protein monomer. In sum, this work underscores the utility of a multi-faceted mass spectrometry approach for detailed investigation of non-covalent protein-DNA complexes.

Directed Evolution of an Improved Aminoacyl-tRNA Synthetase for Incorporation of L-3,4-Dihydroxyphenylalanine (L-DOPA).

The catechol group of 3,4-dihydroxyphenylalanine (L-DOPA) derived from L-tyrosine oxidation is a key post-translational modification (PTM) in many protein biomaterials and has potential as a bioorthogonal handle for precision protein conjugation applications such as antibody-drug conjugates. Despite this potential, indiscriminate enzymatic modification of exposed tyrosine residues or complete replacement of tyrosine using auxotrophic hosts remains the preferred method of introducing the catechol moiety into proteins, which precludes many protein engineering applications. We have developed new orthogonal translation machinery to site-specifically incorporate L-DOPA into recombinant proteins and a new fluorescent biosensor to selectively monitor L-DOPA incorporation in vivo. We show simultaneous biosynthesis and incorporation of L-DOPA and apply this translation machinery to engineer a novel metalloprotein containing a DOPA-Fe chromophore.

Unsaturation Elements and Other Modifications of Phospholipids in Bacteria: New Insight from Ultraviolet Photodissociation Mass Spectrometry.

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.

Localization of Double Bonds in Bacterial Glycerophospholipids Using 193 nm Ultraviolet Photodissociation in the Negative Mode.

The need for detailed structural characterization of glycerophospholipids (GPLs) for many types of biologically motivated applications has led to the development of novel mass spectrometry-based methodologies that utilize alternative ion activation methods. Ultraviolet photodissociation (UVPD) has shown great utility for localizing sites of unsaturation within acyl chains and to date has predominantly been used for positive mode analysis of GPLs. In the present work, UVPD is used to localize sites of unsaturation in GPL anions. Similar to UVPD mass spectra of GPL cations, UVPD of deprotonated or formate-adducted GPLs yields diagnostic fragment ions spaced 24 Da apart. This method was integrated into a liquid chromatography workflow and used to evaluate profiles of sites of unsaturation of lipids in Escherichia coli (E. coli) and Acinetobacter baumannii (A. baumannii). When assigning sites of unsaturation, E. coli was found to contain all unsaturation elements at the same position relative to the terminal methyl carbon of the acyl chain; the first carbon participating in a site of unsaturation was consistently seven carbons along the acyl chain when counting carbons from the terminal methyl carbon. GPLs from A. baumannii exhibited more variability in locations of unsaturation. For GPLs containing sites of unsaturation in both acyl chains, an MS3 method was devised to assign sites to specific acyl chains.

Footprints of Nanoscale DNA-Silver Cluster Chromophores via Activated-Electron Photodetachment Mass Spectrometry.

DNA-templated silver clusters (AgC) are fluorescent probes and biosensors whose electronic spectra can be tuned by their DNA hosts. However, the underlying rules that relate DNA sequence and structure to DNA-AgC fluorescence and photophysics are largely empirical. Here, we employ 193 nm activated electron photodetachment (a-EPD) mass spectrometry as a hybrid MS3 approach to gain structural insight into these nanoscale chromophores. Two DNA-AgC systems are investigated with a 20 nt single-stranded DNA (ssDNA) and a 28 nt hybrid hairpin/single-stranded DNA (hpDNA). Both oligonucleotides template Ag10 clusters, but the two complexes are distinct chromophores: the former has a violet absorption at 400 nm with no observable emission, while the latter has a blue-green absorption at 490 nm with strong green emission at 550 nm. Via identification of both apo and holo (AgC-containing) sequence ions generated upon a-EPD and mapping areas of sequence dropout, specific DNA regions that encapsulate the AgC are assigned and attributed to the coordination with the DNA nucleobases. These a-EPD footprints are distinct for the two complexes. The ssDNA contacts the cluster via four nucleobases (CCTT) in the central region of the strand, whereas the hpDNA coordinates the cluster via 13 nucleobases (TTCCCGCCTTTTG) in the double-stranded region of the hairpin. This difference is consistent with prior X-ray scattering spectra and suggests that the clusters can adapt to different DNA hosts. More importantly, the a-EPD footprints directly identify the nucleobases that are in direct contact with the AgC. As these contacting nucleobases can tune the electronic structures of the Ag core and protect the AgC from collisional quenching in solution, understanding the DNA-silver contacts within these complexes will facilitate future biosensor designs.

Total Synthesis of an Immunogenic Trehalose Phospholipid from Salmonella Typhi and Elucidation of Its sn-Regiochemistry by Mass Spectrometry.

Diphosphatidyltrehalose (diPT) is an immunogenic glycolipid, recently isolated from Salmonella Typhi. Despite rigorous structure elucidation, the sn-position of the acyl chains on the glycerol backbone had not been unequivocally established. A stereoselective synthesis of diPT and its regioisomer is reported herein. Using a hybrid MS3 approach combining collisional dissociation and ultraviolet photodissociation mass spectrometry for analysis of the regioisomers and natural diPT, the regiochemistry of the acyl chains of this abundant immunostimulatory glycolipid was established.

Localization of Cyclopropane Modifications in Bacterial Lipids via 213 nm Ultraviolet Photodissociation Mass Spectrometry.

Subtle structural features in bacterial lipids such as unsaturation elements can have vast biological implications. Cyclopropane rings have been correlated with tolerance to a number of adverse conditions in bacterial phospholipids. They have also been shown to play a major role in Mycobacterium tuberculosis ( M. tuberculosis or Mtb) pathogenesis as they occur in mycolic acids (MAs) in the mycobacterial cell. Traditional collisional activation methods allow elucidation of basic structural features of lipids but fail to reveal the presence and position of cyclopropane rings. Here, we employ 213 nm ultraviolet photodissociation mass spectrometry (UVPD-MS) for structural characterization of cyclopropane rings in bacterial phospholipids and MAs. Upon UVPD, dual cross-ring C-C cleavages on both sides of the cyclopropane ring are observed for cyclopropyl lipids, resulting in diagnostic pairs of fragment ions spaced 14 Da apart, thus enabling cyclopropane localization. These diagnostic pairs of ions corresponding to dual cross-ring cleavage are observed in both negative and positive ion modes and afford localization of multiple cyclopropane rings within a single lipid. This method was integrated with liquid chromatography (LC) for LC/UVPD-MS analysis of cyclopropyl glycerophospholipids in Escherichia coli ( E. coli) and for analysis of MAs in Mycobacterium bovis ( M. bovis) and M. tuberculosis lipid extracts.