Discriminative Identification of SARS-CoV-2 Variants Based on Mass-Spectrometry Analysis.

The spread of SARS-CoV-2 variants of concern (VOCs) is of great importance since genetic changes may increase transmissibility, disease severity and reduce vaccine effectiveness. Moreover, these changes may lead to failure of diagnostic measures. Therefore, variant-specific diagnostic methods are essential. To date, genetic sequencing is the gold-standard method to discriminate between variants. However, it is time-consuming (taking several days) and expensive. Therefore, the development of rapid diagnostic methods for SARS-CoV-2 in accordance with its genetic modification is of great importance. In this study we introduce a Mass Spectrometry (MS)-based methodology for the diagnosis of SARS-CoV-2 in propagated in cell-culture. This methodology enables the universal identification of SARS-CoV-2, as well as variant-specific discrimination. The universal identification of SARS-CoV-2 is based on conserved markers shared by all variants, while the identification of specific variants relies on variant-specific markers. Determining a specific set of peptides for a given variant consists of a multistep procedure, starting with an in-silico search for variant-specific tryptic peptides, followed by a tryptic digest of a cell-cultured SARS-CoV-2 variant, and identification of these markers by HR-LC-MS/MS analysis. As a proof of concept, this approach was demonstrated for four representative VOCs compared to the wild-type Wuhan reference strain. For each variant, at least two unique markers, derived mainly from the spike (S) and nucleocapsid (N) viral proteins, were identified. This methodology is specific, rapid, easy to perform and inexpensive. Therefore, it can be applied as a diagnostic tool for pathogenic variants.

PICA: Pixel Intensity Correlation Analysis for Deconvolution and Metabolite Identification in Mass Spectrometry Imaging.

In-source fragmentation (ISF) is a naturally occurring phenomenon in various ion sources including soft ionization techniques such as matrix-assisted laser desorption/ionization (MALDI). It has traditionally been minimized as it makes the dataset more complex and often leads to mis-annotation of metabolites. Here, we introduce an approach termed PICA (for pixel intensity correlation analysis) that takes advantage of ISF in MALDI imaging to increase confidence in metabolite identification. In PICA, the extraction and association of in-source fragments to their precursor ion results in "pseudo-MS/MS spectra" that can be used for identification. We examined PICA using three different datasets, two of which were published previously and included validated metabolites annotation. We show that highly colocalized ions possessing Pearson correlation coefficient (PCC) ≥ 0.9 for a given precursor ion are mainly its in-source fragments, natural isotopes, adduct ions, or multimers. These ions provide rich information for their precursor ion identification. In addition, our results show that moderately colocalized ions (PCC < 0.9) may be structurally related to the precursor ion, which allows for the identification of unknown metabolites through known ones. Finally, we propose three strategies to reduce the total computation time for PICA in MALDI imaging. To conclude, PICA provides an efficient approach to extract and group ions stemming from the same metabolites in MALDI imaging and thus allows for high-confidence metabolite identification.

SARS-CoV-2 spike antigen quantification by targeted mass spectrometry of a virus-based vaccine.

The spike glycoprotein mediates virus binding to the host cells and is a key target for vaccines development. One SARS-CoV-2 vaccine is based on vesicular stomatitis virus (VSV), in which the native surface glycoprotein has been replaced by the SARS-CoV-2 spike protein (VSV-ΔG-spike). The titer of the virus is quantified by the plaque forming unit (PFU) assay, but there is no method for spike protein quantitation as an antigen in a VSV-based vaccine. Here, we describe a mass spectrometric (MS) spike protein quantification method, applied to VSV-ΔG-spike based vaccine. Proof of concept of this method, combining two different sample preparations, is shown for complex matrix samples, produced during the vaccine manufacturing processes. Total spike levels were correlated with results from activity assays, and ranged between 0.3-0.5 µg of spike protein per 107 PFU virus-based vaccine. This method is simple, linear over a wide range, allows quantification of antigen within a sample and can be easily implemented for any vaccine or therapeutic sample.

Coupling immuno-magnetic capture with LC-MS/MS(MRM) as a sensitive, reliable, and specific assay for SARS-CoV-2 identification from clinical samples.

Recently, numerous diagnostic approaches from different disciplines have been developed for SARS-CoV-2 diagnosis to monitor and control the COVID-19 pandemic. These include MS-based assays, which provide analytical information on viral proteins. However, their sensitivity is limited, estimated to be 5 × 104 PFU/ml in clinical samples. Here, we present a reliable, specific, and rapid method for the identification of SARS-CoV-2 from nasopharyngeal (NP) specimens, which combines virus capture followed by LC-MS/MS(MRM) analysis of unique peptide markers. The capture of SARS-CoV-2 from the challenging matrix, prior to its tryptic digestion, was accomplished by magnetic beads coated with polyclonal IgG-α-SARS-CoV-2 antibodies, enabling sample concentration while significantly reducing background noise interrupting with LC-MS analysis. A sensitive and specific LC-MS/MS(MRM) analysis method was developed for the identification of selected tryptic peptide markers. The combined assay, which resulted in S/N ratio enhancement, achieved an improved sensitivity of more than 10-fold compared with previously described MS methods. The assay was validated in 29 naive NP specimens, 19 samples were spiked with SARS-CoV-2 and 10 were used as negative controls. Finally, the assay was successfully applied to clinical NP samples (n = 26) pre-determined as either positive or negative by RT-qPCR. This work describes for the first time a combined approach for immuno-magnetic viral isolation coupled with MS analysis. This method is highly reliable, specific, and sensitive; thus, it may potentially serve as a complementary assay to RT-qPCR, the gold standard test. This methodology can be applied to other viruses as well.

Characterization of the PRODUCTION of ANTHOCYANIN PIGMENT 1 Arabidopsis dominant mutant using DLEMMA dual isotope labeling approach.

Stable isotope labeling has emerged as a valuable tool for metabolite identification and quantification. In this study, we employed DLEMMA, a dual stable isotope labeling approach to identify and track phenylpropanoid pathway in Arabidopsis thaliana. Three forms of phenylalanine (Phe), including unlabeled, Phe13C6 and Phe13C62H5, were used as feeding precursors. The unique isotopic pattern obtained from MS spectra significantly simplified data processing and facilitated global mining of Phe-derived metabolites. Following this approach, we have identified 35 phenylalanine-derived metabolites with high confidence. We next compared phenylpropanoids contents between leaves of wild type (WT) and the dominant PRODUCTION OF ANTHOCYANIN PIGMENT 1 (pap1-D) Arabidopsis thaliana mutant using a combined sample matrices and label-swap approach. This approach was designed to correct any unequal matrix effects between the two divergent samples, and any possible uneven label incorporation efficiency between the two differently labeled Phe precursors. Thirty of the 35 identified metabolites were found differential between WT and pap1-D leaves. Our results shown that the ectopic PAP1 expression led to significant accumulation of cyanidin-type anthocyanins, quercetin-type flavonols and hydroxycinnamic acids and their glycosylated derivatives. While levels of kaempferol glycosides and a hydroxycinnamic acid amide were reduced in the pap1-D leaves.

Specific and Rapid SARS-CoV-2 Identification Based on LC-MS/MS Analysis.

SARS-CoV-2, the etiologic agent of the COVID-19 pandemic, emerged as the cause of a global crisis. Rapid and reliable clinical diagnosis is essential for effectively controlling transmission. The gold standard assay for SARS-CoV-2 identification is the highly sensitive real-time quantitative polymerase chain reaction (RT-qPCR); however, this assay depends on specialized reagents and may suffer from false results. Thus, additional assays based on different approaches could be beneficial. Here, we present a novel method for SARS-CoV-2 identification based on mass spectrometry. The approach we implemented combines a multistep procedure for the rational down-selection of a set of reliable markers out of all optional in silico derived tryptic peptides in viral proteins, followed by monitoring of peptides derived from tryptic digests of purified proteins, cell-cultured SARS-CoV-2, and nasopharyngeal (NP) swab matrix spiked with the virus. The marker selection was based on specificity to SARS-CoV-2 and on analytical parameters including sensitivity, linearity, and reproducibility. The final assay is based on six unique and specific peptide markers for SARS-CoV-2 identification. The simple and rapid (2.5 h) protocol we developed consists of virus heat inactivation and denaturation, tryptic digestion, and identification of the selected markers by liquid chromatography coupled to high-resolution mass spectrometry (LC-MS/MS). The developed assay enabled the identification of 104 PFU/mL SARS-CoV-2 spiked into buffer. Finally, the assay was successfully applied to 16 clinical samples diagnosed by RT-qPCR, achieving 94% concordance with the current gold standard assay. To conclude, the novel MS-based assay described here is specific, rapid, simple, and is believed to provide a complementary assay to the RT-qPCR method.

New approach for the rational selection of markers to identify botulinum toxins.

The application of mass spectrometry (MS) to detect unique peptide markers has been widely employed as a means of identifying bacterial proteins. Botulinum neurotoxins (BoNTs) are bacterial proteins that cause the life-threatening disease botulism. BoNTs are divided into several antigenically distinct serotypes and several dozen subtypes. The toxins' molecular heterogeneity makes their detection highly challenging. In this study, we describe a new LC-MS/MS-based platform for the direct identification of proteins derived from various species and subspecies in a single assay, as exemplified by BoNTs. The platform employs a rational down-selection process through several steps based on a combination of bioinformatics, tryptic digestion, and LC-MS, each leads to the final panel of markers. This approach has been demonstrated for all 8 subtypes of botulinum serotype A (BoNT/A). Ab-independent and Ab-dependent assays were developed based on the identification of 4 rationally selected markers or a combination of some of them, which enables full selectivity coverage. The Ab-independent assay, which is highly simple and rapid, has a sample-to-result turnaround time of approximately 40 min and enables the identification of 500 MsLD50/mL (5 ng/mL) BoNT/A in complex environmental matrices. The Ab-dependent assay, which is based on toxin's specific enrichment, has a turnaround time of 100 min, but enables improved sensitivity (50 MsLD50/mL, 0.5 ng/mL). Both assays were verified and validated using various environmental samples. This approach can easily be expanded to other botulinum serotypes and exhibits the potential for even further extension as a highly multiplexed assay for protein-based toxins, viruses, and organisms.

Rapid, Sensitive and Reliable Ricin Identification in Serum Samples Using LC-MS/MS.

Ricin, a protein derived from the seeds of the castor bean plant (Ricinus communis), is a highly lethal toxin that inhibits protein synthesis, resulting in cell death. The widespread availability of ricin, its ease of extraction and its extreme toxicity make it an ideal agent for bioterrorism and self-poisoning. Thus, a rapid, sensitive and reliable method for ricin identification in clinical samples is required for applying appropriate and timely medical intervention. However, this goal is challenging due to the low predicted toxin concentrations in bio-fluids, accompanied by significantly high matrix interferences. Here we report the applicability of a sensitive, selective, rapid, simple and antibody-independent assay for the identification of ricin in body fluids using mass spectrometry (MS). The assay involves lectin affinity capturing of ricin by easy-to-use commercial lactose-agarose (LA) beads, following by tryptic digestion and selected marker identification using targeted LC-MS/MS (Multiple Reaction Monitoring) analysis. This enables ricin identification down to 5 ng/mL in serum samples in 2.5 h. To validate the assay, twenty-four diverse naive- or ricin-spiked serum samples were evaluated, and both precision and accuracy were determined. A real-life test of the assay was successfully executed in a challenging clinical scenario, where the toxin was identified in an abdominal fluid sample taken 72 h post self-injection of castor beans extraction in an eventual suicide case. This demonstrates both the high sensitivity of this assay and the extended identification time window, compared to similar events that were previously documented. This method developed for ricin identification in clinical samples has the potential to be applied to the identification of other lectin toxins.

High mass resolution, spatial metabolite mapping enhances the current plant gene and pathway discovery toolbox.

Understanding when and where metabolites accumulate provides important cues to the gene function. Mass spectrometry imaging (MSI) enables in situ temporal and spatial measurement of a large assortment of metabolites, providing mapping information regarding their cellular distribution. To describe the current state and technical advances using MSI in plant sciences, we employed MSI to demonstrate its significant contribution to the study of plant specialised metabolism. We show that coupling MSI with: (1) RNA interference (RNAi), (2) virus induced gene silencing (VIGS), (3) agroinfiltration or (4) samples derived from plant natural variation provides great opportunities to understand the accurate gene-metabolite relationship and discover novel gene-associated metabolites. This was exemplified in three plant species (i.e. tomato, tobacco and wheat) by mapping the distribution of metabolites possessing a range of polarities. In particular, we demonstrated that MSI is able to spatially map an entire metabolic pathway, including intermediates and final products, in the intricate biosynthetic route to tomato fruit steroidal glycoalkaloids. We therefore envisage MSI as a key component of the metabolome analysis arsenal employed in plant gene discovery strategies.

Rapid and sensitive identification of ricin in environmental samples based on lactamyl agarose beads using LC-MS/MS (MRM).

Ricin, a plant-derived toxin extracted from the seeds of Ricinus communis (castor bean plant), is one of the most toxic proteins known. Ricin's high toxicity, widespread availability, and ease of its extraction make it a potential agent for bioterrorist attacks. Most ricin detection methods are based on immunoassays. These methods may suffer from low efficiency in matrices containing interfering substances, or from false positive results due to antibody cross reactivity, with highly homologous proteins. In this study, we have developed a simple, rapid, sensitive, and selective mass spectrometry assay, for the identification of ricin in complex environmental samples. This assay involves three main stages: (a) Ricin affinity capture by commercial lactamyl-agarose (LA) beads. (b) Tryptic digestion. (c) LC-MS/MS (MRM) analysis of tryptic fragments. The assay was validated using 60 diverse environmental samples such as soil, asphalt, and vegetation, taken from various geographic regions. The assay's selectivity was established in the presence of high concentrations of competing lectin interferences. Based on our findings, we have defined strict criteria for unambiguous identification of ricin. Our novel method, which combines affinity capture beads followed by MRM-based analysis, enabled the identification of 1 ppb ricin spiked into complex environmental matrices. This methodology has the potential to be extended for the identification of ricin in body fluids from individuals exposed (deliberately or accidentally) to the toxin, contaminated food or for the detection of the entire family of RIP-II toxins, by applying multiplex format.

Miso: an R package for multiple isotope labeling assisted metabolomics data analysis.

MOTIVATION: The use of stable isotope labeling is highly advantageous for structure elucidation in metabolomics studies. However, computational tools dealing with multiple-precursor-based labeling studies are still missing. Hence, we developed Miso, an R package providing automated and efficient data analysis workflow to detect the complete repertoire of labeled molecules from multiple-precursor-based labeling experiments. RESULTS: The capability of Miso is demonstrated by the analysis of liquid chromatography-mass spectrometry data obtained from duckweed plants fed with one unlabeled and two differently labeled tyrosine (unlabeled tyrosine, tyrosine-2H4 and tyrosine-13C915N1). The resulting data matrix generated by Miso contains sets of unlabeled and labeled ions with their retention time, m/z values and number of labeled atoms that can be directly utilized for database query and biological studies. AVAILABILITY AND IMPLEMENTATION: Miso is publicly available on the CRAN repository (https://cran.r-project.org/web/packages/Miso). A reproducible case study and a detailed tutorial are available from GitHub (https://github.com/YonghuiDong/Miso_example). SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

DLEMMA-MS-Imaging for Identification of Spatially Localized Metabolites and Metabolic Network Map Reconstruction.

Regardless of major advances in mass spectrometry imaging (MSI), there are three intrinsic limitations associated with MSI, including intricate molecular identification, low molecular coverage, and incapability to obtain "true" spatial distribution due to isobaric and particularly isomeric ions interference. We developed a novel approach that integrates in vivo dual isotope labeling of precursor metabolites with MSI (DLEMMA-MS-Imaging) for identification of spatially localized metabolite and metabolic network map reconstruction. In a proof-of-concept study, we identified 59 and 6 novel metabolites in lemna and tomato fruit, respectively. Significantly, 20-30% of the identified metabolites were found to contain at least one structural isomer that displays a different distribution pattern. The notable feature of this approach is the ability to differentiate localization of structural isomers, hence, providing the "true" distribution of molecules of interest.

Development of a multiplex Endopep-MS assay for simultaneous detection of botulinum toxins A, B and E.

Botulinum neurotoxins (BoNTs) are bacterial proteins that cause botulism, a life-threatening disease. The Endopep-MS assay permits rapid detection and serotypic differential diagnosis of BoNTs. The serotype-specific nature of this assay requires that each serum sample be aliquoted and individually tested, which in addition to the limited volume of clinical samples, especially in infants, points to the need for a multiplex assay. However, previous attempts to develop such an assay have been challenging, mainly due to inhibition of BoNT/A activity by the BoNT/E peptide substrate. BoNT/A and BoNT/E share the same native target protein as their substrate. We hypothesized that the steric interference between the BoNT/A and BoNT/E substrate peptides is responsible for the difficulty in simultaneously assaying these two toxins. To explore the basis for steric interference, we used the reported structure of BoNT/A in complex with SNAP-25 and modelled the structure of BoNT/E with SNAP-25. Following this thorough structural analysis, we designed a new peptide substrate for BoNT/A that maintained the assay sensitivity and allowed, for the first time, simultaneous detection of the three most abundant human botulinum serotypes. Adopting the multiplex assay will minimize the required sample volume and assay time for botulinum detection while maintaining the superior Endopep-MS assay performance.

Optimization of SNAP-25-derived peptide substrate for improved detection of botulinum A in the Endopep-MS assay.

Botulinum neurotoxins (BoNTs) are the most toxic proteins in nature. Endopeptidase-mass-spectrometry (Endopep-MS) is used as a specific and rapid in-vitro assay to detect BoNTs. In this assay, immunocaptured toxin cleaves a serotype-specific-peptide-substrate, and the cleavage products are then detected by MS. Here we describe the design of a new peptide substrate for improved detection of BoNT type A (BoNT/A). Our strategy was based on reported BoNT/A-SNAP-25 interactions integrated with analysis method efficiency considerations. Integration of the newly designed substrate led to a 10-fold increase in the assay sensitivity both in buffer and in clinically relevant samples.

Early, Real-Time Medical Diagnosis of Botulism by Endopeptidase-Mass Spectrometry.

Botulinum toxin was detected in patient serum using Endopeptidase-mass-spectrometry assay, although all conventional tests provided negative results. Antitoxin was administered, resulting in patient improvement. Implementing this highly sensitive and rapid assay will improve preparedness for foodborne botulism and deliberate exposure.

A new peptide substrate for enhanced botulinum neurotoxin type B detection by endopeptidase-liquid chromatography-tandem mass spectrometry/multiple reaction monitoring assay.

Botulinum neurotoxins (BoNTs) are the most toxic proteins in nature. Rapid and sensitive detection of BoNTs is achieved by the endopeptidase-mass spectrometry (Endopep-MS) assay. In this assay, BoNT cleaves a specific peptide substrate and the cleaved products are analyzed by MS. Here we describe the design of a new peptide substrate for improved detection of BoNT type B (BoNT/B) in the Endopep-MS assay. Our strategy was based on reported BoNT/B-substrate interactions integrated with analysis method efficiency considerations. Incorporation of the new peptide led to a 5-fold increased sensitivity of the assay both in buffer and in a clinically relevant human spiked serum.

Improved detection of botulinum type E by rational design of a new peptide substrate for endopeptidase-mass spectrometry assay.

Botulinum neurotoxins (BoNTs) are the most toxic substances known to humans. Endopeptidase-mass spectrometry (Endopep-MS) is used as a specific and rapid in vitro assay to detect BoNTs. In this assay, immunocaptured toxin cleaves a serotype-specific peptide substrate, and the cleavage products are then detected by MS. To further improve the sensitivity of the assay, we report here the rational design of a new substrate peptide for the detection of botulinum neurotoxin type E (BoNT/E). Our strategy was based on previously reported structural interactions integrated with analysis method efficiency considerations. Integration of the newly designed substrate has led to a more than one order of magnitude increased sensitivity of the assay.

Degradation of sulfur mustard on KF/Al2O3 supports: insights into the products and the reactions mechanisms.

The degradation of the warfare agent sulfur mustard (HD) adsorbed onto KF/Al(2)O(3) sorbents is described. These processes were explored by MAS NMR, using (13)C-labeled sulfur mustard (HD*) and LC-MS techniques. Our study on the detoxification of this blister agent showed the formation of nontoxic substitution and less-toxic elimination products (t(1/2) = 3.5-355 h). Interestingly, the reaction rates were found to be affected by MAS conditions, i.e., by a centrifugation effect. The products and the mechanisms of these processes are discussed.

Dual labeling of metabolites for metabolome analysis (DLEMMA): A new approach for the identification and relative quantification of metabolites by means of dual isotope labeling and liquid chromatography-mass spectrometry.

Advanced metabolomics technologies are anticipated to permit the identification and quantification of metabolites at the whole-metabolome scale. Yet, most of the metabolites either remain unknown or cannot be identified unambiguously. Moreover, the present approaches suffer from inaccuracies in relative quantification because of sample preparation and matrix effects. Here we present Dual Labeling of Metabolites for Metabolome Analysis (DLEMMA) as a valuable tool, which with analogy to DNA array assays enables the identification and relative quantification of differential metabolites in a single sample. DLEMMA was demonstrated as an efficient method for reducing the number of possible chemical structures assigned that exhibit the same elemental composition. Its strength was exemplified by the discovery of 10 novel Tryptophan derivatives. Furthermore, employing DLEMMA by feeding two Phenylalanine-labeled precursors, we could detect differential metabolites between transgenic and control plants. The accuracy of relative quantification is also enhanced since DLEMMA provides identical matrixes for both samples, thus avoiding the effects of different complex biological matrixes on electrospray ionization. Hence, DLEMMA will complement and contribute to the advancement of metabolomics technologies and boost metabolic pathway discovery in diverse organisms.

Gene expression and metabolism in tomato fruit surface tissues.

The cuticle, covering the surface of all primary plant organs, plays important roles in plant development and protection against the biotic and abiotic environment. In contrast to vegetative organs, very little molecular information has been obtained regarding the surfaces of reproductive organs such as fleshy fruit. To broaden our knowledge related to fruit surface, comparative transcriptome and metabolome analyses were carried out on peel and flesh tissues during tomato (Solanum lycopersicum) fruit development. Out of 574 peel-associated transcripts, 17% were classified as putatively belonging to metabolic pathways generating cuticular components, such as wax, cutin, and phenylpropanoids. Orthologs of the Arabidopsis (Arabidopsis thaliana) SHINE2 and MIXTA-LIKE regulatory factors, activating cutin and wax biosynthesis and fruit epidermal cell differentiation, respectively, were also predominantly expressed in the peel. Ultra-performance liquid chromatography coupled to a quadrupole time-of-flight mass spectrometer and gas chromatography-mass spectrometry using a flame ionization detector identified 100 metabolites that are enriched in the peel tissue during development. These included flavonoids, glycoalkaloids, and amyrin-type pentacyclic triterpenoids as well as polar metabolites associated with cuticle and cell wall metabolism and protection against photooxidative stress. Combined results at both transcript and metabolite levels revealed that the formation of cuticular lipids precedes phenylpropanoid and flavonoid biosynthesis. Expression patterns of reporter genes driven by the upstream region of the wax-associated SlCER6 gene indicated progressive activity of this wax biosynthetic gene in both fruit exocarp and endocarp. Peel-associated genes identified in our study, together with comparative analysis of genes enriched in surface tissues of various other plant species, establish a springboard for future investigations of plant surface biology.