-
1.
Metabolite elucidation of 2-fluoro-deschloroketamine (2F-DCK) using molecular networking across three complementary in vitro and in vivo models.
Gicquel, T, Pelletier, R, Richeval, C, Gish, A, Hakim, F, Ferron, PJ, Mesli, V, Allorge, D, Morel, I, Gaulier, JM
Drug testing and analysis. 2022;(1):144-153
-
-
Free full text
-
Abstract
This work first aims to investigate metabolites of 2-fluoro-deschloroketamine (2F-DCK), a new arylcyclohexylamine derivatives (a group of dissociative ketamine-based substances) using two in vitro experimental approaches, and to compare obtained results by means of molecular networking. Metabolites of 2F-DCK were investigated using both human liver microsomes (HLMs) and hepatic (HepaRG) cell line incubates using molecular networking approach: 2F-DCK pure substance was incubated with HLMs for up to 1 h at two concentrations (100 and 500 μM) and with HepaRG cells for two time periods (8 and 24 h) at one concentration (20 μM). In vitro obtained results were subsequently applied to a 2F-DCK-related fatality case. In vitro-produced metabolites were investigated using high-resolution accurate mass spectrometry using Orbitrap mass analyzer technology. Thirteen metabolites were in vitro produced and several metabolic pathways can be postulated. Seven additional metabolites were found in post-mortem samples (bile and urine) of the case, comprising three Phase II metabolites, which appear to be minor in vivo metabolites. HLMs and HepaRG cell models appear to be complementary and obtained data allowed the identification of several specific 2F-DCK metabolites in biological samples. In practical terms, observed metabolic ratios suggested that nor-2F-DCK (208.1137 m/z) and a hydrogenated metabolite (224.1443 m/z) could be proposed as reliable metabolites to be recorded in HRMS libraries in order to improve detection of 2F-DCK use.
-
2.
A Practical Guide to Small Protein Discovery and Characterization Using Mass Spectrometry.
Ahrens, CH, Wade, JT, Champion, MM, Langer, JD
Journal of bacteriology. 2022;(1):e0035321
Abstract
Small proteins of up to ∼50 amino acids are an abundant class of biomolecules across all domains of life. Yet due to the challenges inherent in their size, they are often missed in genome annotations, and are difficult to identify and characterize using standard experimental approaches. Consequently, we still know few small proteins even in well-studied prokaryotic model organisms. Mass spectrometry (MS) has great potential for the discovery, validation, and functional characterization of small proteins. However, standard MS approaches are poorly suited to the identification of both known and novel small proteins due to limitations at each step of a typical proteomics workflow, i.e., sample preparation, protease digestion, liquid chromatography, MS data acquisition, and data analysis. Here, we outline the major MS-based workflows and bioinformatic pipelines used for small protein discovery and validation. Special emphasis is placed on highlighting the adjustments required to improve detection and data quality for small proteins. We discuss both the unbiased detection of small proteins and the targeted analysis of small proteins of interest. Finally, we provide guidelines to prioritize novel small proteins, and an outlook on methods with particular potential to further improve comprehensive discovery and characterization of small proteins.
-
3.
Critical role of mass spectrometry proteomics in tear biomarker discovery for multifactorial ocular diseases (Review).
Ma, JYW, Sze, YH, Bian, JF, Lam, TC
International journal of molecular medicine. 2021;(5)
-
-
Free full text
-
Abstract
The tear film is a layer of body fluid that maintains the homeostasis of the ocular surface. The superior accessibility of tears and the presence of a high concentration of functional proteins make tears a potential medium for the discovery of non‑invasive biomarkers in ocular diseases. Recent advances in mass spectrometry (MS) have enabled determination of an in‑depth proteome profile, improved sensitivity, faster acquisition speed, proven variety of acquisition methods, and identification of disease biomarkers previously lacking in the field of ophthalmology. The use of MS allows efficient discovery of tear proteins, generation of reproducible results, and, more importantly, determines changes of protein quantity and post‑translation modifications in microliter samples. The present review compared techniques for tear collection, sample preparation, and acquisition applied for the discovery of tear protein markers in normal subjects and multifactorial conditions, including dry eye syndrome, diabetic retinopathy, thyroid eye disease and primary open‑angle glaucoma, which require an early diagnosis for treatment. It also summarized the contribution of MS to early discovery by means of disease‑related protein markers in tear fluid and the potential for transformation of the tear MS‑based proteome to antibody‑based assay for future clinical application.
-
4.
Discrimination of French wine brandy origin by PTR-MS headspace analysis using ethanol ionization and sensory assessment.
Malfondet, N, Brunerie, P, Le Quéré, JL
Analytical and bioanalytical chemistry. 2021;(12):3349-3368
Abstract
The headspace volatile organic compound (VOC) fingerprints (volatilome) of French wine brandies were investigated by proton transfer reaction time-of-flight mass spectrometry (PTR-ToF-MS). Protonated ethanol chemical ionization was used with dedicated experimental conditions that were previously validated for model wines. These included a reference vial containing a hydro-alcoholic solution with the same ethanol content (20% v/v) as the diluted sample spirits, which was used to establish steady-state ionization conditions. A low electric field strength to number density ratio E/N (85 Td) was used in the drift tube in order to limit the fragmentation of the protonated analytes. The obtained headspace fingerprints were used to investigate the origin of French brandies produced within a limited geographic production area. Brandies of two different vintages (one freshly distilled and one aged for 14 years in French oak barrels) were successfully classified according to their growth areas using unsupervised (principal component analysis, PCA) and supervised (partial least squares regression discriminant analysis, PLS-DA) multivariate analyses. The models obtained by PLS-DA allowed the identification of discriminant volatile compounds that were mainly characterised as key aroma compounds of wine brandies. The discrimination was supported by sensory evaluation conducted with free sorting tasks. The results showed that this ethanol ionization method was suitable for direct headspace analysis of brandies. They also demonstrated its ability to distinguish French brandies according to their growth areas, and this effect on brandy VOC composition was confirmed at a perceptive level.
-
5.
Leveraging orthogonal mass spectrometry based strategies for comprehensive sequencing and characterization of ribosomal antimicrobial peptide natural products.
Moyer, TB, Parsley, NC, Sadecki, PW, Schug, WJ, Hicks, LM
Natural product reports. 2021;(3):489-509
-
-
Free full text
-
Abstract
Covering: Up to July 2020Ribosomal antimicrobial peptide (AMP) natural products, also known as ribosomally synthesized and post-translationally modified peptides (RiPPs) or host defense peptides, demonstrate potent bioactivities and impressive complexity that complicate molecular and biological characterization. Tandem mass spectrometry (MS) has rapidly accelerated bioactive peptide sequencing efforts, yet standard workflows insufficiently address intrinsic AMP diversity. Herein, orthogonal approaches to accelerate comprehensive and accurate molecular characterization without the need for prior isolation are reviewed. Chemical derivatization, proteolysis (enzymatic and chemical cleavage), multistage MS fragmentation, and separation (liquid chromatography and ion mobility) strategies can provide complementary amino acid composition and post-translational modification data to constrain sequence solutions. Examination of two complex case studies, gomesin and styelin D, highlights the practical implementation of the proposed approaches. Finally, we emphasize the importance of heterogeneous AMP peptidoforms that confer varying biological function, an area that warrants significant further development.
-
6.
Integrated mass spectrometry-based multi-omics for elucidating mechanisms of bacterial virulence.
Man, L, Klare, WP, Dale, AL, Cain, JA, Cordwell, SJ
Biochemical Society transactions. 2021;(5):1905-1926
Abstract
Despite being considered the simplest form of life, bacteria remain enigmatic, particularly in light of pathogenesis and evolving antimicrobial resistance. After three decades of genomics, we remain some way from understanding these organisms, and a substantial proportion of genes remain functionally unknown. Methodological advances, principally mass spectrometry (MS), are paving the way for parallel analysis of the proteome, metabolome and lipidome. Each provides a global, complementary assay, in addition to genomics, and the ability to better comprehend how pathogens respond to changes in their internal (e.g. mutation) and external environments consistent with infection-like conditions. Such responses include accessing necessary nutrients for survival in a hostile environment where co-colonizing bacteria and normal flora are acclimated to the prevailing conditions. Multi-omics can be harnessed across temporal and spatial (sub-cellular) dimensions to understand adaptation at the molecular level. Gene deletion libraries, in conjunction with large-scale approaches and evolving bioinformatics integration, will greatly facilitate next-generation vaccines and antimicrobial interventions by highlighting novel targets and pathogen-specific pathways. MS is also central in phenotypic characterization of surface biomolecules such as lipid A, as well as aiding in the determination of protein interactions and complexes. There is increasing evidence that bacteria are capable of widespread post-translational modification, including phosphorylation, glycosylation and acetylation; with each contributing to virulence. This review focuses on the bacterial genotype to phenotype transition and surveys the recent literature showing how the genome can be validated at the proteome, metabolome and lipidome levels to provide an integrated view of organism response to host conditions.
-
7.
Sample Preparation Free Mass Spectrometry Using Laser-Assisted Rapid Evaporative Ionization Mass Spectrometry: Applications to Microbiology, Metabolic Biofluid Phenotyping, and Food Authenticity.
Cameron, SJS, Perdones-Montero, A, Van Meulebroek, L, Burke, A, Alexander-Hardiman, K, Simon, D, Schaffer, R, Balog, J, Karancsi, T, Rickards, T, et al
Journal of the American Society for Mass Spectrometry. 2021;(6):1393-1401
Abstract
Mass spectrometry has established itself as a powerful tool in the chemical, biological, medical, environmental, and agricultural fields. However, experimental approaches and potential application areas have been limited by a traditional reliance on sample preparation, extraction, and chromatographic separation. Ambient ionization mass spectrometry methods have addressed this challenge but are still somewhat restricted in requirements for sample manipulation to make it suitable for analysis. These limitations are particularly restrictive in view of the move toward high-throughput and automated analytical workflows. To address this, we present what we consider to be the first automated sample-preparation-free mass spectrometry platform utilizing a carbon dioxide (CO2) laser for sample thermal desorption linked to the rapid evaporative ionization mass spectrometry (LA-REIMS) methodology. We show that the pulsatile operation of the CO2 laser is the primary factor in achieving high signal-to-noise ratios. We further show that the LA-REIMS automated platform is suited to the analysis of three diverse biological materials within different application areas. First, clinical microbiology isolates were classified to species level with an accuracy of 97.2%, the highest accuracy reported in current literature. Second, fecal samples from a type 2 diabetes mellitus cohort were analyzed with LA-REIMS, which allowed tentative identification of biomarkers which are potentially associated with disease pathogenesis and a disease classification accuracy of 94%. Finally, we showed the ability of the LA-REIMS system to detect instances of adulteration of cooking oil and determine the geographical area of production of three protected olive oil products with 100% classification accuracy.
-
8.
Quantitative density gradient analysis by mass spectrometry (qDGMS) and complexome profiling analysis (ComPrAn) R package for the study of macromolecular complexes.
Páleníková, P, Harbour, ME, Ding, S, Fearnley, IM, Van Haute, L, Rorbach, J, Scavetta, R, Minczuk, M, Rebelo-Guiomar, P
Biochimica et biophysica acta. Bioenergetics. 2021;(6):148399
Abstract
Many cellular processes involve the participation of large macromolecular assemblies. Understanding their function requires methods allowing to study their dynamic and mechanistic properties. Here we present a method for quantitative analysis of native protein or ribonucleoprotein complexes by mass spectrometry following their separation by density - qDGMS. Mass spectrometric quantitation is enabled through stable isotope labelling with amino acids in cell culture (SILAC). We provide a complete guide, from experimental design to preparation of publication-ready figures, using a purposely-developed R package - ComPrAn. As specific examples, we present the use of sucrose density gradients to inspect the assembly and dynamics of the human mitochondrial ribosome (mitoribosome), its interacting proteins, the small subunit of the cytoplasmic ribosome, cytoplasmic aminoacyl-tRNA synthetase complex and the mitochondrial PDH complex. ComPrAn provides tools for analysis of peptide-level data as well as normalization and clustering tools for protein-level data, dedicated visualization functions and graphical user interface. Although, it has been developed for the analysis of qDGMS samples, it can also be used for other proteomics experiments that involve 2-state labelled samples separated into fractions. We show that qDGMS and ComPrAn can be used to study macromolecular complexes in their native state, accounting for the dynamics inherent to biological systems and benefiting from its proteome-wide quantitative and qualitative capability.
-
9.
Unravelling the Allosteric Targeting of PHGDH at the ACT-Binding Domain with a Photoactivatable Diazirine Probe and Mass Spectrometry Experiments.
Spillier, Q, Ravez, S, Dochain, S, Vertommen, D, Thabault, L, Feron, O, Frédérick, R
Molecules (Basel, Switzerland). 2021;(2)
Abstract
The serine biosynthetic pathway is a key element contributing to tumor proliferation. In recent years, targeting of phosphoglycerate dehydrogenase (PHGDH), the first enzyme of this pathway, intensified and revealed to be a promising strategy to develop new anticancer drugs. Among attractive PHGDH inhibitors are the α-ketothioamides. In previous work, we have demonstrated their efficacy in the inhibition of PHGDH in vitro and in cellulo. However, the precise site of action of this series, which would help the rational design of new inhibitors, remained undefined. In the present study, the detailed mechanism-of-action of a representative α-ketothioamide inhibitor is reported using several complementary experimental techniques. Strikingly, our work led to the identification of an allosteric site on PHGDH that can be targeted for drug development. Using mass spectrometry experiments and an original α-ketothioamide diazirine-based photoaffinity probe, we identified the 523Q-533F sequence on the ACT regulatory domain of PHGDH as the binding site of α-ketothioamides. Mutagenesis experiments further documented the specificity of our compound at this allosteric site. Our results thus pave the way for the development of new anticancer drugs using a completely novel mechanism-of-action.
-
10.
Metabolomic Profiling of Human Urine Samples Using LC-TIMS-QTOF Mass Spectrometry.
Di Poto, C, Tian, X, Peng, X, Heyman, HM, Szesny, M, Hess, S, Cazares, LH
Journal of the American Society for Mass Spectrometry. 2021;(8):2072-2080
Abstract
The identification of metabolites in biological samples is challenging due to their chemical and structural diversity. Ion mobility spectrometry (IMS) separates ionized molecules based on their mobility in a carrier buffer gas giving information about the ionic shape by measuring the rotationally averaged collision cross-section (CCS) value. This orthogonal descriptor, in combination with the m/z, isotopic pattern distribution, and MS/MS spectrum, has the potential to improve the identification of molecular molecules in complex mixtures. Urine metabolomics can reveal metabolic differences, which arise as a result of a specific disease or in response to therapeutic intervention. It is, however, complicated by the presence of metabolic breakdown products derived from a wide range of lifestyle and diet-related byproducts, many of which are poorly characterized. In this study, we explore the use of trapped ion mobility spectrometry (TIMS) via LC parallel accumulation with serial fragmentation (PASEF) for urine metabolomics. A total of 362 urine metabolites were characterized from 80 urine samples collected from healthy volunteers using untargeted metabolomics employing HILIC and RP chromatography. Additionally, three analytes (Trp, Phe, and Tyr) were selected for targeted quantification. Both the untargeted and targeted data was highly reproducible and reported CCS measurements for identified metabolites were robust in the presence of the urine matrix. A comparison of CCS values among different laboratories was also conducted, showing less than 1.3% ΔCCS values across different platforms. This is the first report of a human urine metabolite database compiled with CCS values experimentally acquired using an LC-PASEF TIMS-qTOF platform.