External validation of serum biomarkers predicting short-term and mid/long-term relapse in patients with Crohn's disease stopping infliximab.

OBJECTIVE: In patients with Crohn's disease (CD) on combination therapy (infliximab and immunosuppressant) and stopping infliximab (cohort from the study of infliximab diSconTinuation in CrOhn's disease patients in stable Remission on combined therapy with Immunosuppressors (STORI)), the risk of short-term (≤6 months) and mid/long-term relapse (>6 months) was associated with distinct blood protein profiles. Our aim was to test the external validity of this finding in the SPARE cohort (A proSpective Randomized Controlled Trial comParing infliximAb-antimetabolites Combination Therapy to Anti-metabolites monotheRapy and Infliximab monothErapy in Crohn's Disease Patients in Sustained Steroid-free Remission on Combination Therapy). DESIGN: In SPARE, patients with CD in sustained steroid-free clinical remission and on combination therapy were randomly allocated to three arms: continuing combination therapy, stopping infliximab or stopping immunosuppressant. In the baseline serum of the STORI and SPARE (arm stopping infliximab) cohorts, we studied 202 immune-related proteins. The proteins associated with time to relapse (univariable Cox model) were compared between STORI and SPARE. The discriminative ability of biomarkers (individually and combined in pairs) was evaluated by the c-statistic (concordance analysis) which was compared with C-reactive protein (CRP), faecal calprotectin and a previously validated model (CEASE). RESULTS: In STORI and SPARE, distinct blood protein profiles were associated with the risk of short-term (eg, high level: CRP, haptoglobin, interleukin-6, C-type lectin domain family 4 member C) and mid/long-term relapse (eg, low level: Fms-related tyrosine kinase 3 ligand, kallistatin, fibroblast growth factor 2). At external validation, the top 10 biomarker pairs showed a higher c-statistic than the CEASE model, CRP and faecal calprotectin in predicting short-term (0.76-0.80 vs 0.74 vs 0.71 vs 0.69, respectively) and mid/long-term relapse (0.66-0.68 vs 0.61 vs 0.52 vs 0.59, respectively). CONCLUSION: In patients with CD stopping infliximab, we confirm that the risk of short-term and mid/long-term relapse is associated with distinct blood protein profiles showing the potential to guide infliximab withdrawal. TRIAL REGISTRATION NUMBER: NCT00571337 and NCT02177071.

Sliding Windows in Ion Mobility (SWIM): A New Approach to Increase the Resolving Power in Trapped Ion Mobility-Mass Spectrometry Hyphenated with Chromatography.

Over the past decade, the separation efficiency achieved by linear IMS instruments has increased substantially, with state-of-the-art IM technologies, such as the trapped ion mobility (TIMS), the cyclic traveling wave ion mobility (cTWIMS), and the structure for lossless ion manipulation (SLIM) platforms commonly demonstrating resolving powers in excess of 200. However, for complex sample analysis that require front end separation, the achievement of such high resolving power in TIMS is significantly hampered, since the ion mobility range must be broad enough to analyze all the classes of compounds of interest, whereas the IM analysis time must be short enough to cope with the time scale of the preseparation technique employed. In this paper, we introduce the concept of sliding windows in ion mobility (SWIM) for chromatography hyphenated TIMS applications that bypasses the need to use a wide and fixed IM range by using instead narrow and mobile ion mobility windows that adapt to the analytes' ion mobility during chromatographic separation. GC-TIMS-MS analysis of a mixture of 174 standards from several halogenated persistent organic pollutant (POP) classes, including chlorinated and brominated dioxins, biphenyls, and PBDEs, demonstrated that the average IM resolving power could be increased up to 40% when the SWIM mode was used, thereby greatly increasing the method selectivity for the analysis of complex samples.

Surface-assisted laser desorption/ionization mass spectrometry imaging: A review.

In the last decades, surface-assisted laser desorption/ionization mass spectrometry (SALDI-MS) has attracted increasing interest due to its unique capabilities, achievable through the nanostructured substrates used to promote the analyte desorption/ionization. While the most widely recognized asset of SALDI-MS is the untargeted analysis of small molecules, this technique also offers the possibility of targeted approaches. In particular, the implementation of SALDI-MS imaging (SALDI-MSI), which is the focus of this review, opens up new opportunities. After a brief discussion of the nomenclature and the fundamental mechanisms associated with this technique, which are still highly controversial, the analytical strategies to perform SALDI-MSI are extensively discussed. Emphasis is placed on the sample preparation but also on the selection of the nanosubstrate (in terms of chemical composition and morphology) as well as its functionalization possibilities for the selective analysis of specific compounds in targeted approaches. Subsequently, some selected applications of SALDI-MSI in various fields (i.e., biomedical, biological, environmental, and forensic) are presented. The strengths and the remaining limitations of SALDI-MSI are finally summarized in the conclusion and some perspectives of this technique, which has a bright future, are proposed in this section.

FT-ICR Mass Spectrometry Imaging at Extreme Mass Resolving Power Using a Dynamically Harmonized ICR Cell with 1ω or 2ω Detection.

MALDI mass spectrometry imaging (MALDI MSI) is a powerful analytical method for achieving 2D localization of compounds from thin sections of typically but not exclusively biological samples. The dynamically harmonized ICR cell (ParaCell) was recently introduced to achieve extreme spectral resolution capable of providing the isotopic fine structure of ions detected in complex samples. The latest improvement in the ICR technology also includes 2ω detection, which significantly reduces the transient time while preserving the nominal mass resolving power of the ICR cell. High-resolution MS images acquired on FT-ICR instruments equipped with 7T and 9.4T superconducting magnets and the dynamically harmonized ICR cell operating at suboptimal parameters suffered severely from the pixel-to-pixel shifting of m/z peaks due to space-charge effects. The resulting profile average mass spectra have depreciated mass measurement accuracy and mass resolving power under the instrument specifications that affect the confidence level of the identified ions. Here, we propose an analytical workflow based on the monitoring of the total ion current to restrain the pixel-to-pixel m/z shift. Adjustment of the laser parameters is proposed to maintain high spectral resolution and mass accuracy measurement within the instrument specifications during MSI analyses. The optimized method has been successfully employed in replicates to perform high-quality MALDI MS images at resolving power (FWHM) above 1,000,000 in the lipid mass range across the whole image for superconducting magnets of 7T and 9.4T using 1 and 2ω detection. Our data also compare favorably with MALDI MSI experiments performed on higher-magnetic-field superconducting magnets, including the 21T MALDI FT-ICR prototype instrument of the NHMFL group at Tallahassee, Florida.

Intertwined metal homeostasis, oxidative and biotic stress responses in the Arabidopsis frd3 mutant.

FRD3 (FERRIC REDUCTASE DEFECTIVE 3) plays a major role in iron (Fe) and zinc (Zn) homeostasis in Arabidopsis. It transports citrate, which enables metal distribution in the plant. An frd3 mutant is dwarf and chlorotic and displays a constitutive Fe-deficiency response and strongly altered metal distribution in tissues. Here, we have examined the interaction between Fe and Zn homeostasis in an frd3 mutant exposed to varying Zn supply. Detailed phenotyping using transcriptomic, ionomic, histochemical and spectroscopic approaches revealed the full complexity of the frd3 mutant phenotype, which resulted from altered transition metal homeostasis, manganese toxicity, and oxidative and biotic stress responses. The cell wall played a key role in these processes, as a site for Fe and hydrogen peroxide accumulation, and displayed modified structure in the mutant. Finally, we showed that Zn excess interfered with these mechanisms and partially restored root growth of the mutant, without reverting the Fe-deficiency response. In conclusion, the frd3 mutant molecular phenotype is more complex than previously described and illustrates how the response to metal imbalance depends on multiple signaling pathways.

Multilabel Per-Pixel Quantitation in Mass Spectrometry Imaging.

In quantitative mass spectrometry imaging (MSI), the gold standard adds a single structural homologue of the target compound at a known concentration to the sample. This internal standard enables to map the detected intensity of the target molecule against an external calibration curve. This approach, however, ignores local noise levels and disproportional ion suppression effects, which might depend on the concentration of the target compound. To overcome these issues, we propose a novel approach that applies several isotopically labeled versions, each at a different concentration, to the sample. This allows creating individual internal calibration curves for every MSI pixel. As proof of principle, we have quantified an endogenous peptide of histone H4 by matrix-assisted laser desorption/ionization-Q-MSI (MALDI-Q-MSI), using a mixture of three isotopically labeled versions. The usage of a fourth label allowed us to compare the gold standard to our multilabel approach. We observed substantial heterogeneity in ion suppression across the tissue, which disclosed itself as varying slopes in the per-pixel regression analyses. These slopes were histology-dependent and differed from each other by up to a factor of 4. The results were validated by liquid chromatography-mass spectrometry (LC-MS), exhibiting a high agreement between LC-MS and MALDI-Q-MSI (Pearson correlation r = 0.87). A comparison between the multilabel and single-label approaches revealed a higher accuracy for the multilabel method when the local target compound concentration differed too much from the concentration of the single label. In conclusion, we show that the multilabel approach provides superior quantitation compared to a single-label approach, in case the target compound is inhomogeneously distributed at a wide concentration range in the tissue.

Adaptive Pixel Mass Recalibration for Mass Spectrometry Imaging Based on Locally Endogenous Biological Signals.

Mass spectrometry imaging (MSI) is a powerful and convenient method for revealing the spatial chemical composition of different biological samples. Molecular annotation of the detected signals is only possible if a high mass accuracy is maintained over the entire image and the m/z range. However, the change in the number of ions from pixel-to-pixel of the biological samples could lead to small fluctuations in the detected m/z-values, called mass shift. The use of internal calibration is known to offer the best solution to avoid, or at least to reduce, mass shifts. Their "a priori" selection for a global MSI acquisition is prone to false positive detection and therefore to poor recalibration. To fill this gap, this work describes an algorithm that recalibrates each spectrum individually by estimating its mass shift with the help of a list of pixel-specific internal calibrating ions, automatically generated in a data-adaptive manner (https://github.com/LaRoccaRaphael/MSI_recalibration). Through a practical example, we applied the methodology to a zebrafish whole-body section acquired at a high mass resolution to demonstrate the impact of mass shift on data analysis and the capability of our algorithm to recalibrate MSI data. In addition, we illustrate the broad applicability of the method by recalibrating 31 different public MSI data sets from METASPACE from various samples and types of MSI and show that our recalibration significantly increases the numbers of METASPACE annotations (gaining from 20 up to 400 additional annotations), particularly the high-confidence annotations with a low false discovery rate.

Dual-polarity SALDI FT-ICR MS imaging and Kendrick mass defect data filtering for lipid analysis.

Lipids are biomolecules of crucial importance involved in critical biological functions. Yet, lipid content determination using mass spectrometry is still challenging due to their rich structural diversity. Preferential ionisation of the different lipid species in the positive or negative polarity is common, especially when using soft ionisation mass spectrometry techniques. Here, we demonstrate the potency of a dual-polarity approach using surface-assisted laser desorption/ionisation coupled to Fourier transform-ion cyclotron resonance (SALDI FT-ICR) mass spectrometry imaging (MSI) combined with Kendrick mass defect data filtering to (i) identify the lipids detected in both polarities from the same tissue section and (ii) show the complementarity of the dual-polarity data, both regarding the lipid coverage and the spatial distributions of the various lipids. For this purpose, we imaged the same mouse brain section in the positive and negative ionisation modes, on alternate pixels, in a SALDI FT-ICR MS imaging approach using gold nanoparticles (AuNPs) as dual-polarity nanosubstrates. Our study demonstrates, for the first time, the feasibility of (i) a dual-polarity SALDI-MSI approach on the same tissue section, (ii) using AuNPs as nanosubstrates combined with a FT-ICR mass analyser and (iii) the Kendrick mass defect data filtering applied to SALDI-MSI data. In particular, we show the complementarity in the lipids detected both in a given ionisation mode and in the two different ionisation modes. Graphical abstract.

Mass shift in mass spectrometry imaging: comprehensive analysis and practical corrective workflow.

MALDI mass spectrometry imaging (MSI) allows the mapping and the tentative identification of compounds based on their m/z value. In typical MSI, a spectrum is taken at incremental 2D coordinates (pixels) across a sample surface. Single pixel mass spectra show the resolving power of the mass analyzer. Mass shift, i.e., variations of the m/z of the same ion(s), may occur from one pixel to another. The superposition of shifted masses from individual pixels peaks apparently degrades the resolution and the mass accuracy in the average spectrum. This leads to low confidence annotations and biased localization in the image. Besides the intrinsic performances of the analyzer, the sample properties (local composition, thickness, matrix deposition) and the calibration method are sources of mass shift. Here, we report a critical analysis and recommendations to mitigate these sources of mass shift. Mass shift 2D distributions were mapped to illustrate its effect and explore systematically its origin. Adapting the sample preparation, carefully selecting the data acquisition settings, and wisely applying post-processing methods (i.e., m/z realignment or individual m/z recalibration pixel by pixel) are key factors to lower the mass shift and to improve image quality and annotations. A recommended workflow, resulting from a comprehensive analysis, was successfully applied to several complex samples acquired on both MALDI ToF and MALDI FT-ICR instruments.

Rapid visualization of lipopeptides and potential bioactive groups of compounds by combining ion mobility and MALDI imaging mass spectrometry.

Mass spectrometry imaging (MSI) has become a powerful method for mapping metabolite distribution in a tissue. Applied to bacterial colonies, MSI has a bright future, both for the discovery of new bioactive compounds and for a better understanding of bacterial antibiotic resistance mechanisms. Coupled with separation techniques such as ion mobility mass spectrometry (IM-MS), the identification of metabolites directly on the image is now possible and does not require additional analysis such as HPLC-MS/MS. In this article, we propose to apply a semi-targeted workflow for rapid IM-MSI data analysis focused on the search for bioactive compounds. First, chemically-related compounds showing a repetitive mass unit (i.e. lipids and lipopeptides) were targeted based on the Kendrick mass defect analysis. The detected groups of potentially bioactive compounds were then confirmed by fitting their measured ion moibilites to their measured m/z values. Using both their m/z and ion mobility values, the selected groups of compounds were identified using the available databases and finally their distribution was observed on the image. Using this workflow on a co-culture of bacteria, we were able to detect and localize bioactive compounds involved in the microbial interaction.

Radical-Pairing Interactions in a Molecular Switch Evidenced by Ion Mobility Spectrometry and Infrared Ion Spectroscopy.

The digital revolution sets a milestone in the progressive miniaturization of working devices and in the underlying advent of molecular machines. Foldamers involving mechanically entangled components with modular secondary structures are among the most promising designs for molecular switch-based applications. Characterizing the nature and dynamics of their intramolecular network following the application of a stimulus is the key to their performance. Here, we use non-dissociative electron transfer as a reductive stimulus in the gas phase and probe the consecutive co-conformational transitions of a donor-acceptor oligorotaxane foldamer using electrospray mass spectrometry interfaced with ion mobility and infrared ion spectroscopy. A comparison of collision cross section distributions for analogous closed-shell and radical molecular ions sheds light on their respective formation energetics, while variations in their respective infrared absorption bands evidence changes in intramolecular organization as the foldamer becomes more compact. These differences are compatible with the advent of radical-pairing interactions.

Discovery of biomarker candidates associated with the risk of short-term and mid/long-term relapse after infliximab withdrawal in Crohn's patients: a proteomics-based study.

OBJECTIVE: A subset of Crohn's disease (CD) patients experiences mid/long-term remission after infliximab withdrawal. Biomarkers are needed to identify those patients. DESIGN: New biomarkers of relapse were searched in the baseline serum of CD patients stopping infliximab when they were under combined therapy (antimetabolite and infliximab) and stable clinical remission (diSconTinuation in CrOhn's disease patients in stable Remission on combined therapy with Immunosuppressors cohort, n=102). From shotgun proteomics experiment (discovery step), biomarker candidates were identified and further targeted by selected reaction monitoring (verification step). The dataset was stratified to search for markers of short-term (<6 months) or mid/long-term relapse (>6 months). The risk of relapse and the predicting capacity associated with biomarker candidates were evaluated using univariate Cox model and log-rank statistic, respectively. To test their complementary predicting capacity, biomarker candidates were systematically combined in pairs. RESULTS: Distinct biomarker candidates were associated with the risk (HR) of short-term (15 proteins, 2.9

MS Imaging-Guided Microproteomics for Spatial Omics on a Single Instrument.

Mass spectrometry imaging (MSI) allows investigating the spatial distribution of chemical compounds directly in biological tissues. As the analytical depth of MSI is limited, MSI needs to be coupled to more sensitive local extraction-based omics approaches to achieve a comprehensive molecular characterization. For this, it is important to retain the spatial information provided by MSI for follow-up omics studies. It has been shown that regiospecific MSI data can be used to guide a laser microdissection system for ultra-sensitive liquid chromatography-mass spectrometry (LC-MS) analyses. So far, this combination has required separate and specialized mass spectrometry (MS) instrumentation. Recent advances in dual-source instrumentation, harboring both matrix assisted laser/desorption ionization (MALDI) and electrospray ionization (ESI) sources, promise state-of-the-art MSI and liquid-based proteomic capabilities on the same MS instrument. This study demonstrates that such an instrument can offer both fast lipid-based MSI at high mass and high lateral resolution and sensitive LC-MS on local protein extracts from the exact same tissue section.

Combination of Capillary Zone Electrophoresis-Mass Spectrometry, Ion Mobility-Mass Spectrometry, and Theoretical Calculations for Cysteine Connectivity Identification in Peptides Bearing Two Intramolecular Disulfide Bonds.

Disulfide bonds between cysteine residues are commonly involved in the stability of numerous peptides and proteins and are crucial for providing biological activities. In such peptides, the appropriate cysteine connectivity ensures the proper conformation allowing an efficient binding to their molecular targets. Disulfide bond connectivity characterization is still challenging and is a critical issue in the analysis of structured peptides/proteins targeting pharmaceutical or pharmacological utilizations. This study describes the development of new and fast gas-phase and in-solution electrophoretic methods coupled to mass spectrometry to characterize the cysteine connectivity of disulfide bonds. For this purpose, disulfide isomers of three peptides bearing two intramolecular disulfide bonds but different cysteine connectivity have been investigated. Capillary zone electrophoresis and ion mobility both coupled to mass spectrometry were used to perform the separation in both aqueous and gas phases, respectively. The separation efficiency of each technique has been critically evaluated and compared. Finally, theoretical calculations were performed to support and explain the experimental data based on the predicted physicochemical properties of the different peptides.

Recommendations for reporting ion mobility Mass Spectrometry measurements.

Here we present a guide to ion mobility mass spectrometry experiments, which covers both linear and nonlinear methods: what is measured, how the measurements are done, and how to report the results, including the uncertainties of mobility and collision cross section values. The guide aims to clarify some possibly confusing concepts, and the reporting recommendations should help researchers, authors and reviewers to contribute comprehensive reports, so that the ion mobility data can be reused more confidently. Starting from the concept of the definition of the measurand, we emphasize that (i) mobility values (K0 ) depend intrinsically on ion structure, the nature of the bath gas, temperature, and E/N; (ii) ion mobility does not measure molecular surfaces directly, but collision cross section (CCS) values are derived from mobility values using a physical model; (iii) methods relying on calibration are empirical (and thus may provide method-dependent results) only if the gas nature, temperature or E/N cannot match those of the primary method. Our analysis highlights the urgency of a community effort toward establishing primary standards and reference materials for ion mobility, and provides recommendations to do so. © 2019 The Authors. Mass Spectrometry Reviews Published by Wiley Periodicals, Inc.

Characterization of New Fengycin Cyclic Lipopeptide Variants Produced by Bacillus amyloliquefaciens (ET) Originating from a Salt Lake of Eastern Algeria.

Fengycin antibiotic displays a strong antifungal activity and inhibits the growth of a wide range of plant pathogens especially filamentous fungi. The main objective of the present study is to characterize fengycin variants produced by B. amyloliquefaciens strain (ET). LC-MS analysis of fengycin extracts has shown several molecular ion peaks corresponding to conventional fengycin homologues (MH + : m/z 1463.9; 1491.9; 1506) and some new ones (MH + : m/z 1433; 1447; 1461; and 1477). Further characterization of these precursor ions was carried out by LC-MS.MS analysis. Reporter fragment ions were observed (named A and B), they correspond to the cleavage of Orn2-Tyr3 (A), Glu1-Orn2 (B), and used for identifying fengycin variants. The reporter fragment couple ions [A/B] at [m/z 966.5/1080.5] and [m/z 994.4 /1108.5] represent fengycin A and B, respectively. The diagnostic ions at ([m/z 980/1094]) may correspond to fengycin C3, D, S or B2. Interestingly, unknown diagnostic product ions at [m/z 951/1065] and [m/z 979/1093] were detected for the first time in this study which prove that they correspond to new fengycin variants, named fengycin X and fengycin Y, respectively. The fengycin X results from a substitution of the glutamine amino acid (Q), at position 8 of the fengycin A peptide part, by an isoleucine (I) or a leucine (L) residue. This mutation should be the same in fengycin Y but compared to fengycin B.

Green mamba peptide targets type-2 vasopressin receptor against polycystic kidney disease.

Polycystic kidney diseases (PKDs) are genetic disorders that can cause renal failure and death in children and adults. Lowering cAMP in cystic tissues through the inhibition of the type-2 vasopressin receptor (V2R) constitutes a validated strategy to reduce disease progression. We identified a peptide from green mamba venom that exhibits nanomolar affinity for the V2R without any activity on 155 other G-protein-coupled receptors or on 15 ionic channels. Mambaquaretin-1 is a full antagonist of the V2R activation pathways studied: cAMP production, beta-arrestin interaction, and MAP kinase activity. This peptide adopts the Kunitz fold known to mostly act on potassium channels and serine proteases. Mambaquaretin-1 interacts selectively with the V2R through its first loop, in the same manner that aprotinin inhibits trypsin. Injected in mice, mambaquaretin-1 increases in a dose-dependent manner urine outflow with concomitant reduction of urine osmolality, indicating a purely aquaretic effect associated with the in vivo blockade of V2R. CD1-pcy/pcy mice, a juvenile model of PKD, daily treated with 13 [Formula: see text]g of mambaquaretin-1 for 99 d, developed less abundant (by 33%) and smaller (by 47%) cysts than control mice. Neither tachyphylaxis nor apparent toxicity has been noted. Mambaquaretin-1 represents a promising therapeutic agent against PKDs.

Rapid Visualization of Chemically Related Compounds Using Kendrick Mass Defect As a Filter in Mass Spectrometry Imaging.

Kendrick mass defect (KMD) analysis is widely used for helping the detection and identification of chemically related compounds based on exact mass measurements. We report here the use of KMD as a criterion for filtering complex mass spectrometry data set. The method allow automated, easy and efficient data processing, enabling the reconstruction of 2D distributions of families of homologous compounds from MSI images. We show that KMD filtering, based on in-house software, is suitable and robust for high resolution (full width at half-maximum, fwhm, at m/z 410 of 20 000) and very high-resolution (fwhm, at m/z 410 of 160 000) MSI data. This method has been successfully applied to two different types of samples, bacteria cocultures, and brain tissue sections.

Bacillus licheniformis peptidoglycan characterization by CZE-MS: Assessment with the benchmark RP-HPLC-MS method.

Peptidoglycan or murein is an essential polymer found in bacterial cell wall. It is a dynamic structure that is continuously remodeled or modified during bacterial cell growth or in presence of cell wall stresses. These modifications are still poorly understood mainly due to the peptidoglycan, which is rather non-soluble, and the difficulties to separate the hydrophilic glycopeptides (muropeptides) by reversed phase liquid chromatography, generated by the enzymatic digestion using mutanolysin, an N-acetyl-muramidase, cleaving the ß1→4 bound between N-acetylglucosamine and N-acetylmuramic acid. Here, we report the use of CZE-MS for an easy and fast screening of muropeptides generated by the action of muramidase on the Bacillus licheniformis cell wall. Electron transfer and CID-MS were also used to unambiguously identify and localize the presence or the absence of amidation and acetylation moieties on muropeptide variants. The reference method to analyse muropeptides by reversed phase chromatography was also tested and the advantages and disadvantages of both methods were evaluated.