In order to quantify small molecules at the early stage of drug discovery, we developed a quantitation approach based on mass spectrometry imaging (MSI) using matrix-assisted laser ...desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) without the use of a labeled compound. We describe a method intended to respond to the main challenges encountered in quantification through MALDI imaging dedicated to whole-body or single heterogeneous organ samples (brain, eye, liver). These include the high dependence of the detected signal on the matrix deposition, the MALDI ionization yield of specific target molecules, and lastly, the ion suppression effect on the tissue. To address these challenges, we based our approach on the use of a normalization factor called the TEC (Tissue Extinction Coefficient). This factor takes into account the ion suppression effect that is both tissue- and drug-specific. Through this protocol, the amount of drug per gram of tissue was determined, which in turn, was compared with other analytical techniques such as Liquid Chromatography-Mass spectrometry (LC-MS/MS).
This article is part of a Special Issue entitled: Imaging Mass Spectrometry: A User’s Guide to a New Technique for Biological and Biomedical Research.
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► Attempt to address the challenge of tissue suppression effect. ► Introduction of a normalization factor called the TEC. ► Definition of a methodology for quantitative analysis using MSI. ► Drugs quantitation in different tissue sample.
Generation of skin distribution profiles and reliable determination of drug molecule concentration in the target region are crucial during the development process of topical products for treatment of ...skin diseases like psoriasis and atopic dermatitis. Imaging techniques like mass spectrometric imaging (MSI) offer sufficient spatial resolution to generate meaningful distribution profiles of a drug molecule across a skin section. In this study, we use matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) to generate quantitative skin distribution profiles based on tissue extinction coefficient (TEC) determinations of four different molecules in cross sections of human skin explants after topical administration. The four drug molecules: roflumilast, tofacitinib, ruxolitinib, and LEO 29102 have different physicochemical properties. In addition, tofacitinib was administrated in two different formulations. The study reveals that with MALDI-MSI, we were able to observe differences in penetration profiles for both the four drug molecules and the two formulations and thereby demonstrate its applicability as a screening tool when developing a topical drug product. Furthermore, the study reveals that the sensitivity of the MALDI-MSI techniques appears to be inversely correlated to the drug molecules’ ability to bind to the surrounding tissues, which can be estimated by their Log D values.
Graphical abstract
Hyaluronic acid (HA) is a major component of the skin, contributing to tissue hydration and biomechanical properties. As HA content in the skin decreases with age, formulas containing HA are widely ...used in cosmetics and HA injections in aesthetic procedures to reduce the signs of aging. To prove the beneficial effects of these treatments, efficient quantification of HA levels in the skin is necessary, but remains difficult. A new analytical method has been developed based on matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) to quantify HA content in cross sections of human skin explants. A standardized and reproducible chemical entity (3 dimeric motifs or 6-mer) quantifiable by MALDI-MSI was produced by enzymatic hydrolysis using a specific hyaluronidase (H1136) in HA solution. This enzymatic digestion was carried out on skin sections before laser desorption, enabling the detection of HA. Histological coloration allowed us to localize the epidermis and the dermis on skin sections and, by comparison with the MALDI molecular image, to calculate the relative HA concentrations in these tissue areas. Skin explants were treated topically using a formula containing HA or its placebo, and the HA distribution profiles were compared with those obtained from untreated explants. A significant increase in HA was shown in each skin layer following topical application of the formula containing HA versus placebo and untreated samples (average of 126±40% and 92±40%, respectively). The MALDI-MSI technique enabled the quantification and localization of all HA macromolecules (endogenous and exogenous) on skin sections and could be useful for determining the efficacy of new cosmetic products designed to fight the signs of aging.
A decade after its inception, MALDI imaging mass spectrometry has become a unique technique in the proteomics arsenal for biomarker hunting in a variety of diseases. At this stage of development, it ...is important to ask whether we can consider this technique to be sufficiently developed for routine use in a clinical setting or an indispensable technology used in translational research. In this report, we consider the contributions of MALDI imaging mass spectrometry and profiling technologies to clinical studies. In addition, we outline new directions that are required to align these technologies with the objectives of clinical proteomics, including: 1) diagnosis based on profile signatures that complement histopathology, 2) early detection of disease, 3) selection of therapeutic combinations based on the individual patient's entire disease-specific protein network, 4) real time assessment of therapeutic efficacy and toxicity, 5) rational redirection of therapy based on changes in the diseased protein network that are associated with drug resistance, and 6) combinatorial therapy in which the signaling pathway itself is viewed as the target rather than any single “node” in the pathway.
Introduction
Amorolfine 5% lacquer is an established topical treatment for fungal infection of the nails. The success of topical therapy for onychomycosis depends on whether the permeated drug ...concentration in the deep nail bed is retained above the effective antifungal minimum inhibitory concentration (MIC). We compared the penetration profile of amorolfine and a new topical formula of terbinafine in human mycotic toenails using matrix-assisted laser desorption ionization mass spectrometry imaging–Fourier transform ion cyclotron resonance (MALDI-FTICR) imaging.
Methods
Amorolfine 5% lacquer and terbinafine 7.8% lacquer were applied to mycotic nails (
n
= 17); nail sections were prepared, and MALDI-FTICR analysis was performed. Based on the MICs of amorolfine and terbinafine needed to kill 90% (MIC
90
) of
Trichophyton rubrum
, the fold differences between the MIC
90
and the antifungal concentrations in the nails (the multiplicity of the MIC
90
) were calculated overall and for the keratin-unbound fractions.
Results
Both amorolfine and terbinafine penetrated the entire thickness of the nail. The mean concentration across the entire nail section 3 h following terbinafine treatment was 1414 μg/g of tissue (equivalent to 4.9 mM) compared with 780 μg/g (2.5 mM) following amorolfine treatment (not significantly different;
p
= 0.878). The median multiplicity of the MIC
90
was significantly higher in amorolfine- than terbinafine-treated nails overall (191 vs. 48;
p
= 0.010) and for the keratin-unbound fractions only (7.4 vs. 0.8;
p
= 0.002).
Conclusion
In this ex vivo study, MALDI-FTICR demonstrated that, although amorolfine 5% and terbinafine 7.8% had similar distribution profiles, both penetrating from the surface to the nail bed, the concentration of amorolfine in the nail was significantly higher than that of terbinafine relative to their respective MIC
90
values. Clinical studies are required to determine whether these effects translate to a clinical difference in treatment success.
Introduction
Onychomycosis is a fungal infection of the nails that can be challenging to treat. Here, matrix-assisted laser desorption ionization–Fourier transform ion cyclotron resonance ...(MALDI-FTICR) imaging was applied to the quantitative analysis of the penetration profile of the antifungal compound, amorolfine, in human mycotic toenails. The amorolfine profile was compared with those of three other antifungals, ciclopirox, naftifine, and tioconazole.
Methods
Antifungal compounds (amorolfine 5% lacquer, ciclopirox 8% lacquer, naftifine 1% solution, and tioconazole 28% solution) were applied to mycotic nails (
n
= 42). Nail sections were prepared, and MALDI-FTICR analysis was performed on the sections at a spatial resolution of 70 μm to compare the distribution profiles. Based on the minimum inhibitory concentrations of the four test compounds needed to kill 90% (MIC
90
) of the fungal organism,
Trichophyton rubrum
, the fold differences between the MIC
90
and the antifungal concentrations in the nails (termed the multiplicity of the MIC
90
) were calculated for each.
Results
The penetration profiles indicated higher concentrations of amorolfine and ciclopirox in the deeper layers of the nails 3 h after treatment, compared with naftifine and tioconazole. The mean concentrations across the entire nail sections at 3 h were significantly different among the four antifungals: amorolfine, 2.46 mM; ciclopirox, 0.95 mM; naftifine, 0.63 mM; and tioconazole, 1.36 mM (
p
= 0.016;
n
= 8 per compound). The median multiplicity of the MIC
90
at 3 h was 191-fold for amorolfine, tenfold for ciclopirox, 52-fold for naftifine, and 208-fold for tioconazole.
Conclusion
In this study, MALDI-FTICR was successfully applied to the quantitative analysis of antifungal distribution in human mycotic nails. The findings suggest that amorolfine penetrates deeper layers of the nail and accumulates at concentrations far exceeding the MIC needed to exert antimycotic activity.
Several species of sea cucumbers of the family Holothuriidae possess a particular mechanical defense system called the Cuvierian tubules (Ct). It is also a chemical defense system as triterpene ...glycosides (saponins) appear to be particularly concentrated in Ct. In the present study, the precise localization of saponins in the Ct of Holothuria forskali is investigated. Classical histochemical labeling using lectin was firstly performed but did not generate any conclusive results. Thus, MALDI mass spectrometry Imaging (MALDI-MSI) was directly applied and completed by statistical multivariate tests. A comparison between the tubules of relaxed and stressed animals was realized.
These analyses allowed the detection of three groups of ions, corresponding to the isomeric saponins of the tubules. Saponins detected at m/z 1287 and 1303 were the most abundant and were apparently localized in the connective tissue of the tubules of both relaxed and stressed individuals. Saponins at m/z 1125 and 1141 were detected in lower amount and were present in tissues of relaxed animals. Finally, saponin ions at 1433, 1449, 1463 and 1479 were observed in some Ct of stressed holothuroids in the outer part of the connective tissue. The saponin group m/z 14xx seems therefore to be stress-specific and could originate from modifications of the saponins with m/z of 11xx.
All the results taken together indicate a complex chemical defense mechanism with, for a single organ, different sets of saponins originating from different cell populations and presenting different responses to stress. The present study also reflects that MALDI-MSI is a valuable tool for chemical ecology studies in which specific chemical signalling molecules like allelochemicals or pheromones have to be tracked. This report represents one of the very first studies using these tools to provide a functional and ecological understanding of the role of natural products from marine invertebrates.
Available imaging systems for use in preclinical toxicology studies increasingly show utility as important tools in the toxicologic pathologist’s armamentarium, permit longitudinal evaluation of ...functional and morphological changes in tissues, and provide important information such as organ and lesion volume not obtained by conventional toxicology study parameters. Representative examples of practical imaging applications in toxicology research and preclinical studies are presented for ultrasound, positron emission tomography/single-photon emission computed tomography, optical, magnetic resonance imaging, and matrix-assisted laser desorption ionization—imaging mass spectrometry imaging. Some of the challenges for making imaging systems good laboratory practice–compliant for regulatory submission are presented. Use of imaging data on a case-by-case basis as part of safety evaluation in regulatory submissions is encouraged.
Stromal corneal dystrophies are a group of hereditary disorders caused by mutations in the TGFBI gene. The mutant TGFBIp is prone to protein aggregation and the mutant protein gets deposited in the ...cornea, leading to severe visual impairment. The mutations lead to a corneal specific protein aggregation suggesting the involvement of tissue-specific factors. The exact molecular mechanism of the process of tissue-specific protein aggregation remains to be elucidated. Differential proteolysis of mutant TGFBIp is a critical component of the disease pathology. The differential proteolysis gives rise to shorter peptides that are highly aggregation-prone and initiate the aggregation cascade. Analyzing the proteolytic processing of the different TGFBIp mutant may provide insight to aid in understanding the amyloid aggregation mechanism. We developed a MALDI-MSI methodology to identify expression and spatial localization of TGFBIp peptides in the cornea. Corneal tissue samples were collected from both control and dystrophic patients (with 2 different mutations), embedded in OCT and sectioned. The sections were trypsin digested and subjected to mass spectrometry imaging using a targeted approach to detect TGFBIp. MALDI-MSI identified peptides from TGFBIp that co-localized with the amyloid corneal deposits. In addition to the relative abundance data, the specific location of the peptides across the corneal sections as molecular signatures was also identified. Spatial distribution and intensity of the TGFBIp peptides showed differences between diseased and control models but also between the two LCD phenotypes. The TGFBIp peptide with m/z of 787.474 and m/z of 1179.579 showed increased expression in both LCD mutants compared to the controls. The peptide with m/z of 929.5 showed increased expression in the LCD phenotype with H626R mutation while the peptide with m/z of 1315.802 was abundant in the sample with R124C mutation. This initial report of 2D spatial protein signature and localization of TGFBIp may be expanded to other mutations to understand the proteolytic patterns of TGFBIp in different mutations.
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