Top-down mass spectrometry of native proteoforms and their complexes: a community study.

The combination of native electrospray ionization with top-down fragmentation in mass spectrometry (MS) allows simultaneous determination of the stoichiometry of noncovalent complexes and identification of their component proteoforms and cofactors. Although this approach is powerful, both native MS and top-down MS are not yet well standardized, and only a limited number of laboratories regularly carry out this type of research. To address this challenge, the Consortium for Top-Down Proteomics initiated a study to develop and test protocols for native MS combined with top-down fragmentation of proteins and protein complexes across 11 instruments in nine laboratories. Here we report the summary of the outcomes to provide robust benchmarks and a valuable entry point for the scientific community.

Deletion of a previously uncharacterized lipoprotein lirL confers resistance to an inhibitor of type II signal peptidase in Acinetobacter baumannii.

Acinetobacter baumannii is a clinically important, predominantly health care-associated gram-negative bacterium with high rates of emerging resistance worldwide. Given the urgent need for novel antibacterial therapies against A. baumannii, we focused on inhibiting lipoprotein biosynthesis, a pathway that is essential for envelope biogenesis in gram-negative bacteria. The natural product globomycin, which inhibits the essential type II signal peptidase prolipoprotein signal peptidase (LspA), is ineffective against wild-type A. baumannii clinical isolates due to its poor penetration through the outer membrane. Here, we describe a globomycin analog, G5132, that is more potent against wild-type and clinical A. baumannii isolates. Mutations leading to G5132 resistance in A. baumannii map to the signal peptide of a single hypothetical gene, which we confirm encodes an alanine-rich lipoprotein and have renamed lirL (prolipoprotein signal peptidase inhibitor resistance lipoprotein). LirL is a highly abundant lipoprotein primarily localized to the inner membrane. Deletion of lirL leads to G5132 resistance, inefficient cell division, increased sensitivity to serum, and attenuated virulence. Signal peptide mutations that confer resistance to G5132 lead to the accumulation of diacylglyceryl-modified LirL prolipoprotein in untreated cells without significant loss in cell viability, suggesting that these mutations overcome a block in lipoprotein biosynthetic flux by decreasing LirL prolipoprotein substrate sensitivity to processing by LspA. This study characterizes a lipoprotein that plays a critical role in resistance to LspA inhibitors and validates lipoprotein biosynthesis as a antibacterial target in A. baumannii.

The rapid detection of procalcitonin in septic serum using immunoaffinity MALDI chips.

BACKGROUND: Sepsis is a common worldwide health condition with high mortality. It is caused by a dysregulated immune response to the pathogen. Severe infections resulting in sepsis can be also determined by monitoring several bloodstream biomarkers, one of them being pro-hormone procalcitonin (PCT). PCT concentration in the bloodstream correlates well with sepsis and in severe cases increases up to a thousand times from the healthy physiological values in a short time. In this study, we developed a rapid technique for PCT detection by MALDI-TOF mass spectrometry, that uses in-situ enrichment directly on the specialized immuno MALDI chips that are utilized as MALDI plates. The method's ability to detect PCT was confirmed by comparing the results with LC-MS bottom-up workflow. The new method detects intact PCT by its m/z and uncovers its alternations in septic serum. METHODS: The MALDI chips used for the detection of PCT were prepared by ambient ion soft landing of anti-PCT antibody on an ITO glass slide. The chips were used for the development of the rapid MALDI-TOF MS method. A parallel method based on affinity enrichment on magnetic beads followed by LC-MS/MS data-dependent peptide microsequencing was used to prove PCT presence in the sample. All samples were also tested by ELISA to determine PCT concentration prior to analyzing them by mass spectrometry methods. RESULTS: The MALDI chip method was optimized using recombinant PCT spiked into the human serum. The PCT detection limit was 10 ng/mL. The optimized method was used to analyze 13 sera from patients suffering sepsis. The PCT results were confirmed by LC-MS/MS. The measurement of the intact PCT by the MALDI chip method revealed that sera of patients with severe sepsis have other forms of PCT present, which show post-processing of the primary sequence by cleavage of PCT, resulting in the formation of N and C termini fragments. CONCLUSIONS: Procalcitonin from human serum was successfully enriched and detected using immunoaffinity MALDI chips. The intact PCT was characterized in 13 septic patients. The method is more specific compared to non-MS-based immunoaffinity techniques and allows observation of different variants of PCT in septic patients.

[A case of botulism in the Czech Republic and current possibilities for detecting the neurotoxin produced by Clostridium botulinum].

In the Czech Republic, botulism is a rare life-threatening disease. A total of 155 cases have been reported since 1960; according to the ISIN (formerly EPIDAT) database, there have been only three isolated cases since 2013, with the exception of a single occurrence of familial botulism in 2013. In our work, we present the occurrence of botulism after ingestion of pâté of untraceable origin by a couple who were hospitalized for botulotoxin food poisoning in July 2022. Their neurological symptoms were dominated by dysarthria. After administration of antibotulinum serum, their condition improved significantly. Patient samples were analyzed using affinity carriers and MALDI mass spectrometry, a modern highly sensitive technique for detecting the presence of botulinum neurotoxins. Unlike traditional detection by a difficult and costly biological experiment on mice, the above analysis does not require the killing of laboratory animals.

Top-Down Detection of Oxidative Protein Footprinting by Collision-Induced Dissociation, Electron-Transfer Dissociation, and Electron-Capture Dissociation.

Fast photochemical oxidation of proteins (FPOP) footprinting is a structural mass spectrometry method that maps proteins by fast and irreversible chemical reactions. The position of oxidative modification reflects solvent accessibility and site reactivity and thus provides information about protein conformation, structural dynamics, and interactions. Bottom-up mass spectrometry is an established standard method to analyze FPOP samples. In the bottom-up approach, all forms of the protein are digested together by a protease of choice, which results in a mixture of peptides from various subpopulations of proteins with varying degrees of photochemical oxidation. Here, we investigate the possibility to analyze a specifically selected population of only singly oxidized proteins. This requires utilization of more specific top-down mass spectrometry approaches. The key element of any top-down experiment is the selection of a suitable method of ion isolation, excitation, and fragmentation. Here, we employ and compare collision-induced dissociation, electron-transfer dissociation, and electron-capture dissociation combined with multi-continuous accumulation of selected ions. A singly oxidized subpopulation of FPOP-labeled ubiquitin was used to optimize the method. The top-down approach in FPOP is limited to smaller proteins, but its usefulness was demonstrated by using it to visualize structural changes induced by co-factor removal from the holo/apo myoglobin system. The top-down data were compared with the literature and with the bottom-up data set obtained on the same samples. The top-down results were found to be in good agreement, which indicates that monitoring a singly oxidized FPOP ion population by the top-down approach is a functional workflow for oxidative protein footprinting.

Inhibition of Escherichia coli Lipoprotein Diacylglyceryl Transferase Is Insensitive to Resistance Caused by Deletion of Braun's Lipoprotein.

Lipoprotein diacylglyceryl transferase (Lgt) catalyzes the first step in the biogenesis of Gram-negative bacterial lipoproteins which play crucial roles in bacterial growth and pathogenesis. We demonstrate that Lgt depletion in a clinical uropathogenic Escherichia coli strain leads to permeabilization of the outer membrane and increased sensitivity to serum killing and antibiotics. Importantly, we identify G2824 as the first-described Lgt inhibitor that potently inhibits Lgt biochemical activity in vitro and is bactericidal against wild-type Acinetobacter baumannii and E. coli strains. While deletion of a gene encoding a major outer membrane lipoprotein, lpp, leads to rescue of bacterial growth after genetic depletion or pharmacologic inhibition of the downstream type II signal peptidase, LspA, no such rescue of growth is detected after Lgt depletion or treatment with G2824. Inhibition of Lgt does not lead to significant accumulation of peptidoglycan-linked Lpp in the inner membrane. Our data validate Lgt as a novel antibacterial target and suggest that, unlike downstream steps in lipoprotein biosynthesis and transport, inhibition of Lgt may not be sensitive to one of the most common resistance mechanisms that invalidate inhibitors of bacterial lipoprotein biosynthesis and transport. IMPORTANCE As the emerging threat of multidrug-resistant (MDR) bacteria continues to increase, no new classes of antibiotics have been discovered in the last 50 years. While previous attempts to inhibit the lipoprotein biosynthetic (LspA) or transport (LolCDE) pathways have been made, most efforts have been hindered by the emergence of a common mechanism leading to resistance, namely, the deletion of the gene encoding a major Gram-negative outer membrane lipoprotein lpp. Our unexpected finding that inhibition of Lgt is not susceptible to lpp deletion-mediated resistance uncovers the complexity of bacterial lipoprotein biogenesis and the corresponding enzymes involved in this essential outer membrane biogenesis pathway and potentially points to new antibacterial targets in this pathway.

Detection and Quantification of Carbohydrate-Deficient Transferrin by MALDI-Compatible Protein Chips Prepared by Ambient Ion Soft Landing.

BACKGROUND: Transferrin is synthetized in the liver and is the most important iron-transport carrier in the human body. Severe alcohol consumption leads to alterations in glycosylation of transferrin. Mass spectrometry can provide fast detection and quantification of transferrin isoforms because they have different molecular masses. In this study, we used antibody chips in combination with MALDI-TOF MS for the detection and quantification of transferrin isoforms. METHODS: Protein chips were prepared by functionalization of indium tin oxide glass using ambient ion soft landing of electrosprayed antitransferrin antibody. Two microliters of patient serum was applied on the antibody-modified spots, and after incubation, washing, and matrix deposition, transferrin isoforms were detected by MALDI-TOF MS. Peak intensities of each transferrin form were used to calculate total carbohydrate-deficient transferrin (CDT). The CDT values obtained by the MALDI chip method were compared with the results obtained by a standard capillary electrophoresis (CE). RESULTS: The chip-based MALDI-TOF MS method was used for enrichment and detection of CDT from human serum. A sample cohort from 186 patients was analyzed. Of these samples, 44 were positively identified as belonging to alcoholic patients, whereas 142 were negative by the MALDI chip approach. The correlation of the data obtained by the CE and the chip-based MALDI was r = 0.986, 95% CI. CONCLUSIONS: Functionalized MALDI chips modified by antitransferrin antibody prepared by ambient ion soft landing were successfully used for detection and quantification of CDT from human sera.

Protein Chips Compatible with MALDI Mass Spectrometry Prepared by Ambient Ion Landing.

We present a technology that allows the preparation of matrix-assisted laser desorption/ionization (MALDI)-compatible protein chips by ambient ion landing of proteins and successive utilization of the resulting protein chips for the development of bioanalytical assays. These assays are based on the interaction between the immobilized protein and the sampled analyte directly on the protein chip and subsequent in situ analysis by MALDI mass spectrometry. The electrosprayed proteins are immobilized on dry metal and metal oxide surfaces, which are nonreactive under normal conditions. The ion landing of electrosprayed protein molecules is performed under atmospheric pressure by an automated ion landing apparatus that can manufacture protein chips with a predefined array of sample positions or any other geometry of choice. The protein chips prepared by this technique are fully compatible with MALDI ionization because the metal-based substrates are conductive and durable enough to be used directly as MALDI plates. Compared to other materials, the nonreactive surfaces show minimal nonspecific interactions with chemical species in the investigated sample and are thus an ideal substrate for selective protein chips. Three types of protein chips were used in this report to demonstrate the bioanalytical applications of ambient ion landing. The protein chips with immobilized proteolytic enzymes showed the usefulness for fast in situ peptide MALDI sequencing; the lectin-based protein chips showed the ability to enrich glycopeptides from complex mixtures with subsequent MALDI analysis, and the protein chips with immobilized antibodies were used for a novel immunoMALDI workflow that allowed the enrichment of antigens from the serum followed by highly specific MALDI detection.

Planar Functionalized Surfaces for Direct Immunoaffinity Desorption/Ionization Mass Spectrometry.

BACKGROUND: Recent studies show that the haptoglobin phenotype in individuals with diabetes mellitus is an important factor for predicting the risk of myocardial infarction, cardiovascular death, and stroke. Current methods for haptoglobin phenotyping include PCR and gel electrophoresis. A need exists for a reliable method for high-throughput clinical applications. Mass spectrometry (MS) can in principle provide fast phenotyping because haptoglobin α 1 and α 2, which define the phenotype, have different molecular masses. Because of the complexity of the serum matrix, an efficient and fast enrichment technique is necessary for an MS-based assay. METHODS: MALDI plates were functionalized by ambient ion landing of electrosprayed antihaptoglobin antibody. The array was deposited on standard indium tin oxide slides. Fast immunoaffinity enrichment was performed in situ on the plate, which was further analyzed by MALDI-TOF MS. The haptoglobin phenotype was determined from the spectra by embedded software script. RESULTS: The MALDI mass spectra showed ion signals of haptoglobin α subunits at m/z 9192 and at m/z 15 945. A cohort of 116 sera was analyzed and the reliability of the method was confirmed by analyzing the identical samples by Western blot. One hundred percent overlap of results between the direct immunoaffinity desorption/ionization MS and Western Blot analysis was found. CONCLUSIONS: MALDI plates modified by antihaptoglobin antibody using ambient ion landing achieve low nonspecific interactions and efficient MALDI ionization and are usable for quick haptoglobin phenotyping.

Fabry disease: renal sphingolipid distribution in the α-Gal A knockout mouse model by mass spectrometric and immunohistochemical imaging.

Fabry disease is an X-linked lysosomal storage disease due to deficient α-galactosidase A (α-Gal A) activity and the resultant lysosomal accumulation of globotriaosylceramide (Gb3) and related lipids primarily in blood vessels, kidney, heart, and other organs. The renal distribution of stored glycolipid species in the α-Gal A knockout mouse model was compared to that in mice to assess relative distribution and absolute amounts of accumulated sphingolipid isoforms. Twenty isoforms of five sphingolipid groups were visualized by mass spectrometry imaging (MSI), and their distribution was compared with immunohistochemical (IHC) staining of Gb3, the major stored glycosphingolipid in consecutive tissue sections. Quantitative bulk lipid analysis of tissue sections was assessed by electrospray ionization with tandem mass spectrometry (ESI-MS/MS). In contrast to the findings in wild-type mice, all three analytical techniques (MSI, IHC, and ESI-MS/MS) revealed increases in Gb3 isoforms and ceramide dihexosides (composed mostly of galabiosylceramides), respectively. To our knowledge, this is the first report of the distribution of individual molecular species of Gb3 and galabiosylceramides in kidney sections in Fabry disease mouse. In addition, the spatial distribution of ceramides, ceramide monohexosides, and sphingomyelin forms in renal tissue is presented and discussed in the context of their biosynthesis.

Nanoliter segmented-flow sampling mass spectrometry with online compartmentalization.

We report a microfluidic device, using segmented flow in a two-phase system of immiscible liquids, which delivers aqueous droplets into a modified commercial mass spectrometer. The interface coupling the microfluidics to the mass spectrometer achieves up to 96% sample transfer efficiency to the vacuum chamber. Sample ionization is assisted by multipass infrared laser beam in the interface. The system achieves low femtomole detection limits of several analytes ranging from drugs to proteins. Sample ionization in this segmented-flow sampling was found to be remarkably insensitive to the presence of buffer salts and other matrices.

Ambient ionization mass spectrometry provides screening of selective androgen receptor modulators.

Ambient ionization mass spectrometry allows for analysis of samples in their natural state, i.e., with no sample pre-treatment. It can be viewed as a fast, simple, and economical analysis, but its main disadvantages include a lower analytical performance due to the presence of complex sample matrix and the lack of chromatographic separation prior to the introduction of the sample into the mass spectrometer. Here we present an application of two ambient ionization mass spectrometry techniques, i.e., Desorption Atmospheric Pressure Photoionization and Dielectric Barrier Discharge Ionization, for the analysis of known Selective Androgen Receptor Modulators, which represent common compounds of abuse in professional and semiprofessional sport. Eight real samples of illegal food supplements, seized by the local law enforcement, were used to test the performance of the ambient mass spectrometry and the results were validated against a newly developed targeted LC-UV-MS/MS method performed in multiple reaction monitoring mode with an external calibration for each analyte. In order to decide whether or not the compound can be declared as present, we proposed a system of rules for the interpretation of the obtained spectra. The criteria are based on mass spectrum matching (5-10 ppm accuracy from the theoretical exact mass and a correct isotopic pattern), duration of the mass signal (three or five consecutive scans, depending on the instrumentation used), and intensity above the background noise (threefold increase in intensity and absolute intensity above 5E4 or 1E5, depending on the instrumentation). When applying these criteria, good agreement was found between the tested methods. Ambient ionization techniques were effective at detecting SARMs at pharmacologically relevant doses, i.e., approximately above 1 mg per capsule, although they may fail to detect lower levels or isomeric species. It is demonstrated that when adhering to a set of clear and consistent rules, ambient mass spectrometry can be employed as a qualitative technique for the screening of illegal SARMs with sufficient confidence and without the necessity to perform a regular LC-MS analysis.

Controlled generation of double emulsions in air.

This Letter describes the controlled generation of double emulsions in the gas phase, which was carried out using an integrated emitter in a poly(dimethylsiloxane) (PDMS) microfluidic chip. The integrated emitter was formed using a molding approach, in which metal wires with desirable diameters were used as emitter molds. The generation of double emulsions in air was achieved with electrohydrodynamics actuation, which offers controllable force exerting on the double emulsions. We developed this capability for future integration of droplet microfluidics with mass spectrometry (MS), where each aqueous droplet in the microchannel is introduced into the gas phase as a double emulsion for subsequent ionization and MS analysis.

Probing Antibody Structures by Hydrogen/Deuterium Exchange Mass Spectrometry.

Hydrogen/deuterium exchange (HDX) followed by mass spectrometry detection (MS) provides a fast, reliable, and detailed solution for the assessment of a protein structure. It has been widely recognized as an indispensable tool and already approved by several regulatory agencies as a structural technique for the validation of protein biopharmaceuticals, including antibody-based drugs. Antibodies are of a key importance in life and medical sciences but considered to be challenging analytical targets because of their compact structure stabilized by disulfide bonds and due to the presence of glycosylation. Despite these difficulties, there are already numerous excellent studies describing MS-based antibody structure characterization. In this chapter, we describe a universal HDX-MS workflow. Deeper attention is paid to sample handling, optimization procedures, and feasibility stages, as these elements of the HDX experiment are crucial for obtaining reliable detailed and spatially well-resolved information.

Top-down mass spectrometry of native proteoforms and their complexes: A community study.

The combination of native electrospray ionisation with top-down fragmentation in mass spectrometry allows simultaneous determination of the stoichiometry of noncovalent complexes and identification of their component proteoforms and co-factors. While this approach is powerful, both native mass spectrometry and top-down mass spectrometry are not yet well standardised, and only a limited number of laboratories regularly carry out this type of research. To address this challenge, the Consortium for Top-Down Proteomics (CTDP) initiated a study to develop and test protocols for native mass spectrometry combined with top-down fragmentation of proteins and protein complexes across eleven instruments in nine laboratories. The outcomes are summarised in this report to provide robust benchmarks and a valuable entry point for the scientific community.

Preferred ß-lactone synthesis can explain high rate of false-negative results in the detection of OXA-48-like carbapenemases.

The resistance to carbapenems is usually mediated by enzymes hydrolyzing ß-lactam ring. Recently, an alternative way of the modification of the antibiotic, a ß-lactone formation by OXA-48-like enzymes, in some carbapenems was identified. We focused our study on a deep analysis of OXA-48-like-producing Enterobacterales, especially strains showing poor hydrolytic activity. In this study, well characterized 74 isolates of Enterobacterales resistant to carbapenems were used. Carbapenemase activity was determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), liquid chromatography/mass spectrometry (LC-MS), Carba-NP test and modified Carbapenem Inactivation Method (mCIM). As meropenem-derived ß-lactone possesses the same molecular weight as native meropenem (MW 383.46 g/mol), ß-lactonization cannot be directly detected by MALDI-TOF MS. In the spectra, however, the peaks of m/z = 340.5 and 362.5 representing decarboxylated ß-lactone and its sodium adduct were detected in 25 out of 35 OXA-48-like producers. In the rest 10 isolates, decarboxylated hydrolytic product (m/z = 358.5) and its sodium adduct (m/z = 380.5) have been detected. The peak of m/z = 362.5 was detected in 3 strains co-producing OXA-48-like and NDM-1 carbapenemases. The respective signal was identified in no strain producing class A or class B carbapenemase alone showing its specificity for OXA-48-like carbapenemases. Using LC-MS, we were able to identify meropenem-derived ß-lactone directly according to the different retention time. All strains with a predominant ß-lactone production showed negative results of Carba NP test. In this study, we have demonstrated that the strains producing OXA-48-like carbapenemases showing false-negative results using Carba NP test and MALDI-TOF MS preferentially produced meropenem-derived ß-lactone. We also identified ß-lactone-specific peak in MALDI-TOF MS spectra and demonstrated the ability of LC-MS to detect meropenem-derived ß-lactone.

Time-dependent oxidation during nano-assisted laser desorption ionization mass spectrometry: a useful tool for structure determination or a source of possible confusion?

This work reports on a new and extremely simple approach for determination of a double bond position by a laser desorption ionization mass spectrometry. It is solely based on the catalytic properties of nanostructured surfaces used in nanoassisted laser desorption ionization experiments. These surfaces can induce oxidation of analytes, which results in a mass shift that can be detected by mass spectrometry. If a site of unsaturation is oxidized and cleaved, the m/z difference is diagnostic of the position of a double bond. By demonstrating that the oxidation depends on the analyte surface dwell time, it was proven that it is caused by the surface activity and not by the laser desorption ionization process itself. Control matrix-assisted laser desorption/ionization (MALDI) experiment showed only a limited partial oxidation and no time dependency of the process. The ability to determine a position of a double bond was demonstrated on polyunsaturated phospholipids and cyclosporine A. In some other cases, however, the unexpected oxidation could cause confusion, as demonstrated for a glycosphingolipid from a porcine brain extract.

Poly[N-(2-hydroxypropyl)methacrylamide]-based tissue-embedding medium compatible with MALDI mass spectrometry imaging experiments.

Traditional tissue-sectioning techniques for histological samples utilize various embedding media to stabilize the tissue on a sectioning target and to provide a smooth cutting surface. Due to the ion suppression effect in MALDI ionization and number of background peaks in the low-mass region, these media are not suitable for mass spectrometry imaging (MSI) experiments. To overcome this, droplets of water are often used to mount the tissue on a sectioning target, but the ice block formed around the tissue does not provide a good support for sectioning of fragile samples. In this work, we propose a novel embedding media, compatible with MALDI ionization and MSI experiments, based on poly[N-(2-hydroxypropyl)methacrylamide] (pHPMA). Using a reversible addition-fragmentation chain transfer polymerization technique, well-defined pHPMA polymer with narrow mass distribution was prepared. Benefits of the resulted pHPMA-based embedding media were tested on different tissue samples.

Visualizing spatial lipid distribution in porcine lens by MALDI imaging high-resolution mass spectrometry.

The intraocular lens contains high levels of both cholesterol and sphingolipids, which are believed to be functionally important for normal lens physiology. The aim of this study was to explore the spatial distribution of sphingolipids in the ocular lens using mass spectrometry imaging (MSI). Matrix-assisted laser desorption/ionization (MALDI) imaging with ultra high resolution Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) was used to visualize the lipid spatial distribution. Equatorially-cryosectioned, 12 microm thick slices of tissue were thaw-mounted to an indium-tin oxide (ITO) glass slide by soft-landing to an ethanol layer. This procedure maintained the tissue integrity. After the automated MALDI matrix deposition, the entire lens section was examined by MALDI MSI in a 150 microm raster. We obtained spatial- and concentration-dependent distributions of seven lens sphingomyelins (SM) and two ceramide-1-phosphates (CerP), which are important lipid second messengers. Glycosylated sphingolipids or sphingolipid breakdown products were not observed. Owing to ultra high resolution MS, all lipids were identified with high confidence, and distinct distribution patterns for each of them are presented. The distribution patterns of SMs provide an understanding of the physiological functioning of these lipids in clear lenses and offer a novel pathophysiological means for understanding diseases of the lens.