Advancing PROTAC Characterization: Structural Insights through Adducts and Multimodal Tandem-MS Strategies.

Proteolysis targeting chimeras (PROTACs) are specialized molecules that bind to a target protein and a ubiquitin ligase to facilitate protein degradation. Despite their significance, native PROTACs have not undergone tandem mass spectrometry (MS) analysis. To address this gap, we conducted a pioneering investigation on the fragmentation patterns of two PROTACs in development, dBET1 and VZ185. Employing diverse cations (sodium, lithium, and silver) and multiple tandem-MS techniques, we enhanced their structural characterization. Notably, lithium cations facilitated comprehensive positive-mode coverage for dBET1, while negative polarity mode offered richer insights. Employing de novo structure determination on 2DMS data from degradation studies yielded crucial insights. In the case of VZ185, various charge states were observed, with [M + 2H]2+ revealing fewer moieties than [M + H]+ due to charge-related factors. Augmenting structural details through silver adducts suggested both charge-directed and charge-remote fragmentation. This comprehensive investigation identifies frequently dissociated bonds across multiple fragmentation techniques, pinpointing optimal approaches for elucidating PROTAC structures. The findings contribute to advancing our understanding of PROTACs, pivotal for their continued development as promising therapeutic agents.

Review of fragmentation of synthetic single-stranded oligonucleotides by tandem mass spectrometry from 2014 to 2022.

The fragmentation of oligonucleotides by mass spectrometry allows for the determination of their sequences. It is necessary to understand how oligonucleotides dissociate in the gas phase, which allows interpretation of data to obtain sequence information. Since 2014, a range of fragmentation mechanisms, including a novel internal rearrangement, have been proposed using different ion dissociation techniques. The recent publications have focused on the fragmentation of modified oligonucleotides such as locked nucleic acids, modified nucleobases (methylated, spacer, nebularine and aminopurine) and modification to the carbon 2'-position on the sugar ring; these modified oligonucleotides are of great interest as therapeutics. Comparisons of different dissociation techniques have been reported, including novel approaches such as plasma electron detachment dissociation and radical transfer dissociation. This review covers the period 2014-2022 and details the new knowledge gained with respect to oligonucleotide dissociation using tandem mass spectrometry (without priori sample digestion) during that time, with a specific focus on synthetic single-stranded oligonucleotides.

Dissection of schistosome tissues under LC-MS compatible preservative conditions for quantitative proteomics.

Schistosomes are blood flukes with specialised tissues and organs, each one playing a pivotal role in perpetuating the parasite life cycle. Herein, we describe a detailed methodology for preserving the proteome of adult Schistosoma mansoni worms during manual dissection for enrichment of tissues associated with the parasite's alimentary tract. We provide step-by-step directions for specimen storage and dissection while in preservative solution, tissue homogenisation, protein extraction and digestion using a methodology fully compatible with downstream quantitative liquid chromatography-mass spectrometry analysis. Our methodology uses label-free and QconCAT-based absolute quantification for detection of S. mansoni oesophageal gland products proposed as vaccine candidates. Through stabilisation of the proteome and minimising sample degradation during dissection our approach has allowed us to access the hidden proteome of target tissues not readily available from total lysates because of their small volume. This protocol can be replicated or adapted to other Schistosoma species lacking quantitative proteomics characterisation of specialised tissues for discovery of proteins with potential diagnostic and therapeutic utility.

Dispersant-First Dispersive Liquid-Liquid Microextraction (DF-DLLME), a Novel Sample Preparation Procedure for NDMA Determination in Metformin Products.

Since December 2019, global batch recalls of metformin pharmaceutical products have highlighted an urgent need to control N-nitrosodimethylamine (NDMA) contamination to demonstrate patient safety and maintain supply of this essential medicine. Due to their formulation, the metformin extended-release products present difficult analytical challenges for conventional sample preparation procedures, such as artefactual (in-situ) NDMA formation, gelling, and precipitation. To overcome these challenges, a new version of dispersive liquid-liquid microextraction (DLLME) termed dispersant-first DLLME (DF-DLLME) was developed and optimized for the analysis of NDMA in metformin extended-release products using a detailed Design of Experiments (DoE) to optimize sample preparation. Gas chromatography-high resolution accurate mass-mass spectrometry (GC-HRAM-MS) combined with automated DF-DLLME were successfully applied to monitor the NDMA levels of two different metformin extended-release AstraZeneca products to ultra-trace levels (parts per billion). The additional benefits associated with DF-DLLME, which include automation, time/costs saving, and greener sample preparation, make this novel technique easier to transfer from a development to Quality Control (QC) environment. In addition, this also offers an attractive candidate for the wider platform analysis of N-nitrosamines in pharmaceutical drug products.

The Global Characterisation of a Drug-Dendrimer Conjugate - PEGylated poly-lysine Dendrimer.

The recent emergence of drug-dendrimer conjugates within pharmaceutical industry research and development introduces a range of challenges for analytical and measurement science. These molecules are very high molecular weight (100-200kDa) with a significant degree of structural complexity. The characteristics and quality attributes that require understanding and definition, and impact efficacy and safety, are diverse. They relate to the intact conjugate, the various building blocks of these complex systems and the level of the free and bound active pharmaceutical ingredient (API). From an analytical and measurement science perspective, this necessitates the measurement of the molecular weight, impurity characterisation, the quantitation of the number of conjugated versus free API molecules, the determination of the impurity profiles of the building blocks, primary structure and both particle size and morphology. Here we report the first example of a global characterisation of a drug-dendrimer conjugate - PEGylated poly-lysine dendrimer currently under development (AZD0466). The impact of the wide variety of analytical and measurement techniques on the overall understanding of this complex molecular entity is discussed, with the relative capabilities of the various approaches compared. The results of this study are an essential platform for the research and development of the future generations of related dendrimer-based medicines.

Whole-cell modeling in yeast predicts compartment-specific proteome constraints that drive metabolic strategies.

When conditions change, unicellular organisms rewire their metabolism to sustain cell maintenance and cellular growth. Such rewiring may be understood as resource re-allocation under cellular constraints. Eukaryal cells contain metabolically active organelles such as mitochondria, competing for cytosolic space and resources, and the nature of the relevant cellular constraints remain to be determined for such cells. Here, we present a comprehensive metabolic model of the yeast cell, based on its full metabolic reaction network extended with protein synthesis and degradation reactions. The model predicts metabolic fluxes and corresponding protein expression by constraining compartment-specific protein pools and maximising growth rate. Comparing model predictions with quantitative experimental data suggests that under glucose limitation, a mitochondrial constraint limits growth at the onset of ethanol formation-known as the Crabtree effect. Under sugar excess, however, a constraint on total cytosolic volume dictates overflow metabolism. Our comprehensive model thus identifies condition-dependent and compartment-specific constraints that can explain metabolic strategies and protein expression profiles from growth rate optimisation, providing a framework to understand metabolic adaptation in eukaryal cells.

Advancements in the characterisation of oligonucleotides by high performance liquid chromatography-mass spectrometry in 2021: A short review.

The first oligonucleotide therapeutic was approved by the Food and Drug Administration in 1998, and since then, 12 nucleic acids have been commercialised as medicines. To be approved, the oligonucleotides need to be identified and characterised as well as its related impurities. Different methods exist, but the most commonly used is ion-pairing reversed-phase liquid chromatography with tandem mass spectrometry. The separation obtained depends on the mobile phase and column used. Other methods have been developed, notably by using hydrophilic interaction chromatography and two-dimensional high performance liquid chromatography. Furthermore, ion-pairing reversed-phase high performance liquid chromatography ultra-violet spectroscopy detection and mass spectrometry has been optimised for the analysis of methylated nucleobases due to the utilisation of this modification in the drugs. This review covers the recent advancements in the analysis and characterisation of oligonucleotides in 2021 by high performance liquid chromatography mass spectrometry, notably by hydrophilic interaction chromatography and two-dimensional liquid chromatography but also the different parameters that influence the analysis by ion-pairing reversed-phase high performance liquid chromatography, the characterisation of methylated nucleobases, and the recent software developed for oligonucleotides.

Application of open port sampling interface mass spectrometry (OPSI-MS) to deuterium exchange as an aid for structural elucidation.

RATIONALE: Deuterium exchange has been demonstrated to provide additional information to accurate mass measurement and collision-induced dissociation on unknown chemical structures. An enhanced method for rapid deuterium exchange could make this technique more routine for structural elucidation. Open port sampling interface mass spectrometry (OPSI-MS) with an aprotic solvent offers a rapid method for performing deuterium incorporation. METHODS: Samples of standard drug molecules have been analysed by OPSI-MS directly from solids using a make-up flow of acetonitrile + 0.1% trifluoroacetic acid. The resultant spectra were compared with those obtained by OPSI-MS analysis of the samples dissolved in deuterium oxide (D2 O). Solutions of these molecules in acetonitrile/D2 O were analysed using an Atmospheric Solids Analysis Probe (ASAP) at different temperatures to compare the suitability of this technique. RESULTS: The number of exchangeable hydrogens was obtained through deuterium exchange using the OPSI source, although there was some incomplete exchange or back-exchange observed. Molecules with one to five exchangeable hydrogens were successfully analysed. ASAP analysis produced more complicated spectra with higher levels of incomplete or back-exchanged ions; this was more pronounced at higher temperatures. CONCLUSIONS: The use of OPSI provides a method for the rapid determination of the number of exchangeable hydrogens within a molecule. This yields useful information as an aid to the structural elucidation of unknowns. ASAP produces incomplete exchange and cannot be used for incorporation studies.

Advances in high-resolution mass spectrometry applied to pharmaceuticals in 2020: A whole new age of information.

Continuous improvements in mass spectrometry (MS) have resulted in the widespread availability and adoption of high-resolution mass spectrometry (HRMS) across laboratories worldwide. The capabilities and the associated advantages of HRMS make it an invaluable analytical tool for analyte characterization, screening, and quantification methodologies for a wide scope of applications across pharmaceutical development. These applications include drug discovery, product characterizations of both small molecules and novel drug modalities, in vitro and in vivo metabolism studies, post-approval quality control, and pharmacovigilance. This review gives an overview of the current capabilities of HRMS and its pharmaceutical applications in 2020, and provides a perspective on the future of HRMS within the pharmaceutical industry.

Quantitative Proteomics of Enriched Esophageal and Gut Tissues from the Human Blood Fluke Schistosoma mansoni Pinpoints Secreted Proteins for Vaccine Development.

Schistosomes are blood-dwelling helminth parasites that cause schistosomiasis, a debilitating disease resulting in inflammation and, in extreme cases, multiple organ damage. Major challenges to control the transmission persist, and the discovery of protective antigens remains of critical importance for vaccine development. Rhesus macaques can self-cure following schistosome infection, generating antibodies that target proteins from the tegument, gut, and esophagus, the last of which is the least investigated. We developed a dissection technique that permitted increased sensitivity in a comparative proteomics profiling of schistosome esophagus and gut. Proteome analysis of the male schistosome esophagus identified 13 proteins encoded by microexon genes (MEGs), 11 of which were uniquely located in the esophageal glands. Based on this and transcriptome information, a QconCAT was designed for the absolute quantification of selected targets. MEGs 12, 4.2, and 4.1 and venom allergen-like protein 7 were the most abundant, spanning over 245 million to 6 million copies per cell, while aspartyl protease, palmitoyl thioesterase, and galactosyl transferase were present at <1 million copies. Antigenic variation by alternative splicing of MEG proteins was confirmed together with a specialized machinery for protein glycosylation/secretion in the esophagus. Moreover, some gastrodermal secretions were highly enriched in the gut, while others were more uniformly distributed throughout the parasite, potentially indicating lysosomal activity. Collectively, our findings provide a more rational, better-oriented selection of schistosome vaccine candidates in the context of a proven model of protective immunity.

Towards a generic method for ion chromatography/mass spectrometry of low-molecular-weight amines in pharmaceutical drug discovery and development.

RATIONALE: Low-molecular-weight amines are encountered in pharmaceutical analysis, e.g. as reactants in chemical syntheses, but are challenging to analyse using ultrahigh-performance liquid chromatography/mass spectrometry (UHPLC/MS) due to their high polarity causing poor retention. Ion chromatography/mass spectrometry (IC/MS) is an emerging technique for polar molecule analysis that offers better separation. A generic IC/MS method would overcome problems associated with using UHPLC/MS in drug discovery and development environments. METHODS: Amine standards were analysed using IC/MS with gradient elution (variety of column temperatures evaluated). An electrospray ionisation (ESI) quadrupole mass spectrometer was operated in positive ion polarity in scanning mode. The make-up flow composition was evaluated by assessing the performance of a range of organic modifiers (acetonitrile, ethanol, methanol) and additives (acetic acid, formic acid, methanesulfonic acid). The ESI conditions were optimised to minimise adduct formation and promote generation of protonated molecules. RESULTS: The performance attributes were investigated and optimised for low-molecular-weight amine analysis. Organic solvents and acidic additives were evaluated as make-up flow components to promote ESI, with 0.05% acetic acid in ethanol optimal for producing protonated molecules. The hydrogen bonding capability of amines led to abundant protonated molecule-solvent complexes; optimisation of source conditions reduced these, with collision-induced dissociation voltage having a strong effect. The detection limit was ≤1.78 ng for the amines analysed, which is fit-for-purpose for an open-access chemistry environment. CONCLUSIONS: This study demonstrates the value of IC/MS for analysing low-molecular-weight amines. Good chromatographic separation of mixtures was possible without derivatisation. Ionisation efficiency was greatest using a make-up flow of 0.05% acetic acid in ethanol, and optimisation of ESI source conditions promoted protonated molecule generation for easy determination of molecular weight.

Glycolytic flux in Saccharomyces cerevisiae is dependent on RNA polymerase III and its negative regulator Maf1.

Protein biosynthesis is energetically costly, is tightly regulated and is coupled to stress conditions including glucose deprivation. RNA polymerase III (RNAP III)-driven transcription of tDNA genes for production of tRNAs is a key element in efficient protein biosynthesis. Here we present an analysis of the effects of altered RNAP III activity on the Saccharomyces cerevisiae proteome and metabolism under glucose-rich conditions. We show for the first time that RNAP III is tightly coupled to the glycolytic system at the molecular systems level. Decreased RNAP III activity or the absence of the RNAP III negative regulator, Maf1 elicit broad changes in the abundance profiles of enzymes engaged in fundamental metabolism in S. cerevisiae In a mutant compromised in RNAP III activity, there is a repartitioning towards amino acids synthesis de novo at the expense of glycolytic throughput. Conversely, cells lacking Maf1 protein have greater potential for glycolytic flux.

DOSCATs: Double standards for protein quantification.

The two most common techniques for absolute protein quantification are based on either mass spectrometry (MS) or on immunochemical techniques, such as western blotting (WB). Western blotting is most often used for protein identification or relative quantification, but can also be deployed for absolute quantification if appropriate calibration standards are used. MS based techniques offer superior data quality and reproducibility, but WB offers greater sensitivity and accessibility to most researchers. It would be advantageous to apply both techniques for orthogonal quantification, but workflows rarely overlap. We describe DOSCATs (DOuble Standard conCATamers), novel calibration standards based on QconCAT technology, to unite these platforms. DOSCATs combine a series of epitope sequences concatenated with tryptic peptides in a single artificial protein to create internal tryptic peptide standards for MS as well as an intact protein bearing multiple linear epitopes. A DOSCAT protein was designed and constructed to quantify five proteins of the NF-κB pathway. For three target proteins, protein fold change and absolute copy per cell values measured by MS and WB were in excellent agreement. This demonstrates that DOSCATs can be used as multiplexed, dual purpose standards, readily deployed in a single workflow, supporting seamless quantitative transition from MS to WB.

Protein turnover measurement using selected reaction monitoring-mass spectrometry (SRM-MS).

Protein turnover represents an important mechanism in the functioning of cells, with deregulated synthesis and degradation of proteins implicated in many diseased states. Therefore, proteomics strategies to measure turnover rates with high confidence are of vital importance to understanding many biological processes. In this study, the more widely used approach of non-targeted precursor ion signal intensity (MS1) quantification is compared with selected reaction monitoring (SRM), a data acquisition strategy that records data for specific peptides, to determine if improved quantitative data would be obtained using a targeted quantification approach. Using mouse liver as a model system, turnover measurement of four tricarboxylic acid cycle proteins was performed using both MS1 and SRM quantification strategies. SRM outperformed MS1 in terms of sensitivity and selectivity of measurement, allowing more confident determination of protein turnover rates. SRM data are acquired using cheaper and more widely available tandem quadrupole mass spectrometers, making the approach accessible to a larger number of researchers than MS1 quantification, which is best performed on high mass resolution instruments. SRM acquisition is ideally suited to focused studies where the turnover of tens of proteins is measured, making it applicable in determining the dynamics of proteins complexes and complete metabolic pathways.This article is part of the themed issue 'Quantitative mass spectrometry'.

Dynamic phosphorylation of RelA on Ser42 and Ser45 in response to TNFα stimulation regulates DNA binding and transcription.

The NF-κB signalling module controls transcription through a network of protein kinases such as the IKKs, as well as inhibitory proteins (IκBs) and transcription factors including RelA/p65. Phosphorylation of the NF-κB subunits is critical for dictating system dynamics. Using both non-targeted discovery and quantitative selected reaction monitoring-targeted proteomics, we show that the cytokine TNFα induces dynamic multisite phosphorylation of RelA at a number of previously unidentified residues. Putative roles for many of these phosphorylation sites on RelA were predicted by modelling of various crystal structures. Stoichiometry of phosphorylation determination of Ser45 and Ser42 revealed preferential early phosphorylation of Ser45 in response to TNFα. Quantitative analyses subsequently confirmed differential roles for pSer42 and pSer45 in promoter-specific DNA binding and a role for both of these phosphosites in regulating transcription from the IL-6 promoter. These temporal dynamics suggest that RelA-mediated transcription is likely to be controlled by functionally distinct NF-κB proteoforms carrying different combinations of modifications, rather than a simple 'one modification, one effect' system.

Analysis of Intrinsic Peptide Detectability via Integrated Label-Free and SRM-Based Absolute Quantitative Proteomics.

Quantitative mass spectrometry-based proteomics of complex biological samples remains challenging in part due to the variability and charge competition arising during electrospray ionization (ESI) of peptides and the subsequent transfer and detection of ions. These issues preclude direct quantification from signal intensity alone in the absence of a standard. A deeper understanding of the governing principles of peptide ionization and exploitation of the inherent ionization and detection parameters of individual peptides is thus of great value. Here, using the yeast proteome as a model system, we establish the concept of peptide F-factor as a measure of detectability, closely related to ionization efficiency. F-factor is calculated by normalizing peptide precursor ion intensity by absolute abundance of the parent protein. We investigated F-factor characteristics in different shotgun proteomics experiments, including across multiple ESI-based LC-MS platforms. We show that F-factors mirror previously observed physicochemical predictors as peptide detectability but demonstrate a nonlinear relationship between hydrophobicity and peptide detectability. Similarly, we use F-factors to show how peptide ion coelution adversely affects detectability and ionization. We suggest that F-factors have great utility for understanding peptide detectability and gas-phase ion chemistry in complex peptide mixtures, selection of surrogate peptides in targeted MS studies, and for calibration of peptide ion signal in label-free workflows. Data are available via ProteomeXchange with identifier PXD003472.

Absolute protein quantification of the yeast chaperome under conditions of heat shock.

Chaperones are fundamental to regulating the heat shock response, mediating protein recovery from thermal-induced misfolding and aggregation. Using the QconCAT strategy and selected reaction monitoring (SRM) for absolute protein quantification, we have determined copy per cell values for 49 key chaperones in Saccharomyces cerevisiae under conditions of normal growth and heat shock. This work extends a previous chemostat quantification study by including up to five Q-peptides per protein to improve confidence in protein quantification. In contrast to the global proteome profile of S. cerevisiae in response to heat shock, which remains largely unchanged as determined by label-free quantification, many of the chaperones are upregulated with an average two-fold increase in protein abundance. Interestingly, eight of the significantly upregulated chaperones are direct gene targets of heat shock transcription factor-1. By performing absolute quantification of chaperones under heat stress for the first time, we were able to evaluate the individual protein-level response. Furthermore, this SRM data was used to calibrate label-free quantification values for the proteome in absolute terms, thus improving relative quantification between the two conditions. This study significantly enhances the largely transcriptomic data available in the field and illustrates a more nuanced response at the protein level.