Assisted Laser Desorption Ionization-mass Spectrometry Research Articles

Effect of dynamic exclusion and the use of FAIMS, DIA and MALDI-mass spectrometry imaging with ion mobility on amyloid protein identification

Amyloidosis is a disease characterized by local and systemic extracellular deposition of amyloid protein fibrils where its excessive accumulation in tissues and resistance to degradation can lead to organ failure. Diagnosis is challenging because of approximately 36 different amyloid protein subtypes. Imaging methods like immunohistochemistry and the use of Congo red staining of amyloid proteins for laser capture microdissection combined with liquid chromatography tandem mass spectrometry (LMD/LC–MS/MS) are two diagnostic methods currently used depending on the expertise of the pathology laboratory. Here, we demonstrate a streamlined in situ amyloid peptide spatial mapping by Matrix Assisted Laser Desorption Ionization–Mass Spectrometry Imaging (MALDI-MSI) combined with Trapped Ion Mobility Spectrometry for potential transthyretin (ATTR) amyloidosis subtyping. While we utilized the standard LMD/LC–MS/MS workflow for amyloid subtyping of 31 specimens from different organs, we also evaluated the potential introduction in the MS workflow variations in data acquisition parameters like dynamic exclusion, or testing Data Dependent Acquisition combined with High-Field Asymmetric Waveform Ion Mobility Spectrometry (DDA FAIMS) versus Data Independent Acquisition (DIA) for enhanced amyloid protein identification at shorter acquisition times. We also demonstrate the use of Mascot’s Error Tolerant Search and PEAKS de novo sequencing for the sequence variant analysis of amyloidosis specimens.

Spatial detection and consequences of nonrenal calcitriol production as assessed by targeted mass spectrometry imaging.

The immune benefits of vitamin D3 supplementation beyond calcium and phosphate maintenance are highly clinically debated. Kidney expression of CYP27B1 is the source of endocrine, circulating 1,25(OH)2D3 (active form of vitamin D) that maintains serum calcium and phosphate. 1,25(OH)2D3 may also be made by the CYP27B1 enzyme in nonrenal cells, like immune cells, in a process driven by cellular availability of 25(OH)D3 and inflammation. Due to the endocrine nature of 1,25(OH)2D3 in circulation, it is difficult to discern between these 2 sources. We recently created a regulatory deletion model of Cyp27b1 (M1/M21-DIKO) where mice have normal inflammatory-regulated Cyp27b1 expression in nonrenal tissues (unlike global Cyp27b1-KO) but no expression within the kidney. Here, utilizing on-tissue chemical derivatization and matrix assisted laser desorption ionization-mass spectrometry imaging (MALDI-MSI), we investigated the distribution of 1,25(OH)2D3 and 25(OH)D3 in the kidney, liver, spleen, and thymus. MALDI-MSI demonstrated increased 1,25(OH)2D3 in nonrenal tissues such as the spleen after vitamin D3 supplementation in M1/M21-DIKO mice. Additionally, from this, we found increased Il4 and decreased Tnfa in the spleen after vitamin D3 supplementation. Taken together, these data demonstrate nonrenal production of 1,25(OH)2D3 in vivo and provide a consequence of vitamin D3 supplementation and nonrenal 1,25(OH)2D3 production in cytokine changes.

Library Screening, In Vivo Confirmation, and Structural and Bioinformatic Analysis of Pentapeptide Sequences as Substrates for Protein Farnesyltransferase.

Protein farnesylation is a post-translational modification where a 15-carbon farnesyl isoprenoid is appended to the C-terminal end of a protein by farnesyltransferase (FTase). This process often causes proteins to associate with the membrane and participate in signal transduction pathways. The most common substrates of FTase are proteins that have C-terminal tetrapeptide CaaX box sequences where the cysteine is the site of modification. However, recent work has shown that five amino acid sequences can also be recognized, including the pentapeptides CMIIM and CSLMQ. In this work, peptide libraries were initially used to systematically vary the residues in those two parental sequences using an assay based on Matrix Assisted Laser Desorption Ionization-Mass Spectrometry (MALDI-MS). In addition, 192 pentapeptide sequences from the human proteome were screened using that assay to discover additional extended CaaaX-box motifs. Selected hits from that screening effort were rescreened using an in vivo yeast reporter protein assay. The X-ray crystal structure of CMIIM bound to FTase was also solved, showing that the C-terminal tripeptide of that sequence interacted with the enzyme in a similar manner as the C-terminal tripeptide of CVVM, suggesting that the tripeptide comprises a common structural element for substrate recognition in both tetrapeptide and pentapeptide sequences. Molecular dynamics simulation of CMIIM bound to FTase further shed light on the molecular interactions involved, showing that a putative catalytically competent Zn(II)-thiolate species was able to form. Bioinformatic predictions of tetrapeptide (CaaX-box) reactivity correlated well with the reactivity of pentapeptides obtained from in vivo analysis, reinforcing the importance of the C-terminal tripeptide motif. This analysis provides a structural framework for understanding the reactivity of extended CaaaX-box motifs and a method that may be useful for predicting the reactivity of additional FTase substrates bearing CaaaX-box sequences.

Multimodal Spectroscopic Studies to Evaluate the Effect of Nod-Factor-Based Fertilizer on the Maize (Zea mays) Stem.

Maize (Zea mays) is one of the most cultivated plants in the world. Due to the large area, the scale of its production, and the demand to increase the yield, there is a need for new environmentally friendly fertilizers. One group of such candidates is bacteria-produced nodulation (or nod) factors. Limited research has explored the impact of nodulation, factors on maize within field conditions, with most studies restricted to greenhouse settings and early developmental stages. Additionally, there is a scarcity of investigations that elucidate the metabolic alterations in the maize stem due to nod-factor exposure. It was therefore the aim of this study. Maize stem's metabolites and fibers were analyzed with various imaging analytical techniques: matrix assisted laser desorption ionization-mass spectrometry imaging (MALDI-MSI), Raman spectroscopy, attenuated total reflection Fourier transform infrared spectroscopy (ATR FT-IR), and diffuse reflectance infrared Fourier transform spectroscopy. Moreover, the biochemical analyses were used to evaluate the proteins and soluble carbohydrates concentration and total phenolic content. These techniques were used to evaluate the influence of nod factor-based biofertilizer on the growth of a non-symbiotic plant, maize. The biofertilizer increased the grain yield and the stem mass. Moreover, the spectroscopic and biochemical investigation proved the appreciable biochemical changes in the stems of the maize in biofertilizer-treated plants. Noticeable changes were found in the spatial distribution and the increase in the concentration of flavonoids such as maysin, quercetin, and rutin. Moreover, the concentration of cell wall components (fibers) increased. Furthermore, it was shown that the use of untargeted analyses (such as Raman and ATR FT-IR, spectroscopic imaging, and MALDI-MSI) is useful for the investigation of the biochemical changes in plants.

Molecular Techniques and Target Selection for the Identification of Candida spp. in Oral Samples

Candida species are the causative agent of oral candidiasis, with medical devices being platforms for yeast anchoring and tissue colonization. Identifying the infectious agent involved in candidiasis avoids an empirical prescription of antifungal drugs. The application of high-throughput technologies to the diagnosis of yeast pathogens has clear advantages in sensitivity, accuracy, and speed. Yet, conventional techniques for the identification of Candida isolates are still routine in clinical and research settings. Molecular approaches are the focus of intensive research, but conversion into clinic settings requires overcoming important challenges. Several molecular approaches can accurately identify Candida spp.: Polymerase Chain Reaction, Microarray, High-Resolution Melting Analysis, Multi-Locus Sequence Typing, Restriction Fragment Length Polymorphism, Loop-mediated Isothermal Amplification, Matrix Assisted Laser Desorption Ionization-mass spectrometry, and Next Generation Sequencing. This review examines the advantages and disadvantages of the current molecular methods used for Candida spp. Identification, with a special focus on oral candidiasis. Discussion regarding their application for the diagnosis of oral infections aims to identify the most rapid, affordable, accurate, and easy-to-perform molecular techniques to be used as a point-of-care testing method. Special emphasis is given to the difficulties that health care professionals need to overcome to provide an accurate diagnosis.

Characterization of serum N-glycome alterations in seasonal allergic rhinitis using MALDI-TOF-MS: A pilot study

Seasonal allergic rhinitis (SAR) is an inflammatory process. In this pilot study, matrix assisted laser desorption ionization-mass spectrometry (MALDI-MS)-based analyses were conducted to investigate the effects of SAR on serum N-glycome in a small dataset (n = 10 for both SAR patients and controls). It was detected that two N-glycan compositions (H6N5E2L1 and H6N5E1L2) were down-regulated in SAR patients. Additionally, five tri-antennary N-glycan traits, including both galactosylated and sialylated (A3GS and A3GL) and non-fucosylated (A3F0GE, A3F0GL, A3F0GS) ones, were decreased. Furthermore, three high-antennary and fucosylated N-glycan traits (A3F, A3EF and A4F) were increased in SAR patients compared to controls.

Brain Lipid Dynamics in Amyloid Precursor Protein/Presenilin 1 Mouse Model of Early Alzheimer's Disease by Desorption Electrospray Ionization and Matrix Assisted Laser Desorption Ionization-Mass Spectrometry Imaging Techniques.

Alzheimer's disease (AD) is closely associated with lipid metabolism dysfunction. However, space distribution and metabolism of aberrant lipids in the brain of early-stage AD mouse remain unclear. In our current work, a novel lipidomics method based on mass spectrometry imaging was developed to visually disclose molecular perturbation and characterize space distribution in the brain of double transgenic amyloid precursor protein/presenilin 1 mouse (2 and 3 months old). Significant changes were detected, including phosphatidylethanolamines, phosphatidylcholines, fatty acids, lysophospholipids, and glycerides in AD mouse brain. The results in this study suggest that these significantly altered lipid metabolic pathways (glycerophospholipid metabolism) may be implicated in early-stage AD. Our work deepens the understanding of the physio-pathologic mechanism of early-stage AD.

Differential N-glycosylation profiling of formalin-fixed paraffin-embedded (FFPE) invasive ductal carcinoma tissues using MALDI-TOF-MS.

Invasive ductal carcinoma (IDC) is the most common type of breast cancer. As dynamic changes of the glycome are closely associated with complex diseases, they have become a focal point of cancer research involving predictive and prognostic markers. Formalin-fixed paraffin-embedded (FFPE) clinical specimens are representative of the tumor environment and are thus utilized in studies on cancer related research and biomarker discovery. Further studies on differential N-glycosylation profiling of IDC cancer tissues are necessary in order to understand the biological role of glycans in cancer and to evaluate their predictive ability. In this study, matrix assisted laser desorption ionization-mass spectrometry (MALDI-MS)-based analyses were conducted for determining differential N-glycosylation patterns of IDC. Two different derivatization methods, namely, 2-aminobenzoic acid (2-AA) labeling and linkage-specific sialic acid esterification, were used for the analysis of N-glycans. Forty-seven 2-AA labeled and fifty ethyl esterified N-glycans were identified by MALDI-MS. In statistical analyses conducted for 2-AA-labeled N-glycans, the relative amounts of 32 N-glycans and prevalence of 15 N-glycan traits showed significant (p < 0.05) differences between cancer and normal tissues; and in such analyses for the ethyl-esterified N-glycans, the relative amounts of 27 N-glycans and prevalence of 17 N-glycan traits showed significant (p < 0.05) differences between them. It was found that mainly high mannose N-glycans, including H5N2, H6N2, and H7N2, and two fucosylated compositions (H3N3F1 and H5N5F1) showed strong discrimination between IDC and controls. In addition, compared with the controls, high mannose N-glycans were observed to be up-regulated in IDC whereas bisecting N-glycans were down-regulated.

Chip-based SALDI-MS for rapid determination of intracellular ratios of glutathione to glutathione disulfide

Alterations in the ratio of glutathione (GSH) to glutathione disulfide (GSSG) reveal the cell living state and are associated with a variety of diseases. In this study, an Au NPs grafted nanoporous silicon chip was used for surface assisted laser desorption ionization-mass spectrometry (SALDI-MS) detection of GSH. Due to the bond interaction between thiol of GSH and Au NPs modified on the chip surfaces, GSH could be captured from the complex cellular lysate. Meanwhile, the composite nanostructures of Au NPs grafted porous silicon surface presented good desorption/ionization efficiency for GSH detection. The GSH levels in different tumor cells were successfully detected. Chip-based SALDI-MS was optimized for quantification of intracellular GSH/GSSG ratio changing under drug stimulation in liver tumor cells, GSSG was reduced to GSH by reductant of tris(2-carboxyethyl)phosphine (TCEP) and isotope-labeling GSH was as an internal standard. It was found that the increasing concentration of drug irinotecan and hypoxia culture condition caused the rapid consumption of GSH and a decrease of GSH/GSSG ratio in liver tumor cells. The developed SALDI-MS method provided a convenient way to accurately measure and rapidly monitor cellular GSH value and the ratios of GSH/GSSG.

Evaluation of the Role of Novel Aprotic Dimethyl Sulfoxide in Cutaneous Antisepsis Protocols Prior to Blood Donor Phlebotomy with Isopropyl Alcohol, Povidone Iodine or Chlorhexidine.

The study was planned to measure the reduction of the load of bacterial flora on the blood donor's arm quantitatively using a three step protocol of donor arm cleansing incorporating either 70% isopropyl alcohol (IPA) or 5% w/v povidone iodine (PVI, 0.5% w/v available iodine) or 4% chlorhexidine gluconate (CHG) with or without the addition of 5% dimethyl sufloxide (DMSO). Single blind randomized study after obtaining ethical clearance, using the Miles and Misra technique for quantification and matrix assisted laser desorption ionization-mass spectrometry for identification of colony morphotypes on blood donor's skin. The mean pre-cleansingcolony forming units (CFUs) was 89,318 and mean post-cleansingCFUs was 132, with a mean reduction of 99.85% with a mean log reduction of 3.24 (95% CI 2.01-4.47) at a P value of <0.0001. The post-cleansing CFUs was reduced to zero in all 34 samples in the protocol using CHG with DMSO, in 23 of 31 samples in the protocol using PVI with DMSO and 19 of 29 samples in the protocol using IPA with DMSO. The difference in means of the reduction of CFUs in protocols using CHG with DMSO compared with protocols using PVI or IPA with DMSO and PVI or IPA without DMSO was statistically significant with P value of 0.006, 0.0009, 0.015 and 0.05 respectively. The enhanced cutaneous antisepsis effect of CHG when complimented with DMSO in presence of IPA using the three step protocol of donor arm cleansing could stimulate more research and utilization of this as an additional safety towards the prevention of the problem of bacterial contamination of blood and blood components.

Biospectroscopy, biospectrometry and imaging of Ilex paraguariensis. Basis for non-destructive quality evaluation using artificial vision.

Yerba mate (YM) is massively produced and consumed as an infusion in South America and is gaining popularity all over the world. This product is obtained from the dried leaves of Ilex paraguariensis Saint Hilaire, mixed with fragments of its dried branches. For its commercialization YM must have a minimum percentage of leaves according to a standard classification. Until now, composition quantification has been mechanically performed, thus development of new methods is still pending. In this work a quantification method using solid-phase molecular fluorescence, alternately diffuse reflectance spectroscopy and an imaging technique using a scanner have been proposed. Strong differences between the spectroscopic properties of leaves and sticks were observed and in all the cases linear correlations between the processed signals and composition were observed. Interesting differences in chemical composition of YM leaves and sticks were additionally obtained in this work by means of total phenol content quantification, ultraviolet matrix assisted laser-desorption ionization mass spectrometry and ultraviolet laser-desorption ionization mass spectrometry.

Isolation and screening of Weissella strains for their potential use as starter during attiéké production

Description of the subject. Variability observed in sensory characteristics of “attiéké” results from an uncontrolled fermentation process due to the use of artisanal starters. Objectives. This study aims to screen microbial strains for their use during fermentation of cassava dough into attieké. Method. Technological properties of three lactic acid bacteria (LAB) isolated from artisanal starters were highlighted in vitro, and these LAB were identified as either Weissella cibaria or Weissella confusa by Matrix Assisted Laser Desorption Ionisation-Mass Spectrometry (MALDI-TOF MS). Results. The three Weissella isolates Wc 69, Wc 21 and Wc 114 induced in less than 42 h a decrease below 4.2 (main food safety factor) of the initial pH of MRS (de Man, Rogosa and Sharpe) broth. These strains are osmotolerant, present alpha-amylase activity and ferment the indigestible sugar raffinose. Moreover isolates Wc 114 and Wc 69 are thermotolerant, while Wc 114 presents a pectinase activity necessary for cassava dough softening. Conclusions. Considering their technological properties, the three isolated Weissella strains could be suitable in optimizing and standardizing the quality of attieké.

Development of operational protocols for the analysis of primary and secondary fingermark lifts by MALDI-MS imaging

Eight years of intensive research have demonstrated that Matrix Assisted Laser Desorption Ionisation-Mass Spectrometry Profiling and Imaging (MALDI-MSP and MSI) are powerful tools to gather intelligence around a suspect lifestyle, directly from the identifying ridges of a latent fingermark.

Rapid and widespread distribution of doxycycline in rat brain: a mass spectrometric imaging study

1. The penetration of tetracyclines into the brain has been widely documented. The aim of this work was to develop a matrix assisted laser desorption ionization-mass spectrometry imaging (MALDI MSI) method for the molecular histology of doxycycline (DOX) in the healthy rat brain.2. The time-dependent distribution was investigated after an i.p. dose of 25 mg/kg at 0, 5, 30, 120, 240, 360 and 480 min postdose. LCMS/MS was used to quantify the drug in plasma and brain homogenates and MALDI MSI was used to determine the distribution of the analyte.3. Within the first-hour postdose, the drug showed slow accumulation into the plasma and brain tissues. DOX brain concentration gradually increased and reached a peak (Cmax) of 1034.9 ng/mL at 240 min postdose, resulting in a brain plasma ratio of 31%. The images acquired by MSI matched the quantification results and clearly showed drug distribution over the entire rat brain coronal section from 5 min and its slow elimination after 360-min postdose.4. Our findings confirm that MALDI MSI provides an advanced, label-free and faster alternative technique for xenobiotic distribution such as DOX in tissues, making it an essential drug discovery tool for other possible neuroprotective agents.

Development of a three component complex to increase isoniazid efficacy against isoniazid resistant and nonresistant Mycobacterium tuberculosis.

The bacterium responsible for causing tuberculosis has evolved resistance to antibiotics used to treat the disease, resulting in new multidrug resistant Mycobacterium tuberculosis (MDR-TB) and extensively drug resistant M. tuberculosis (XDR-TB) strains. Analytical techniques (1)H and (13)C Nuclear Magnetic Resonance (NMR), Fourier Transform-Ion Cyclotron Resonance with Electrospray Ionization (FT-ICR/ESI), and Matrix Assisted Laser Desorption Ionization-Mass Spectrometry (MALDI-TOF-MS) were used to study different aspects of the Cu(II)-polyethylene glycol (PEG-3350)-sucrose-isoniazid and Cu(II)-polyethylene glycol (PEG3350)-glucose-isoniazid complexes. The Cu(II) cation, sucrose or glucose, and the aggregate formed by PEG primarily serve as a composite drug delivery agent for the frontline antibiotic, however the improvement in MIC values produced with the CU-PEG-SUC-INH complex suggest an additional effect. Several Cu-PEG-SUC-INH complex variations were tested against INH resistant and nonresistant strains of M. tuberculosis.

Biosynthesis of Gold Nanoparticles and Identification of Capping Agent Using Gas Chromatography-Mass Spectrometry and Matrix Assisted Laser Desorption Ionization-Mass Spectrometry.

In the present study, gold nanoparticles (AuNPs) were synthesized using leaf extract of Syzygium jambolanum and capping agent has been explored. The synthesized AuNPs have been characterized using UV-visible spectroscopy (UV-vis), Fourier transform infrared spectroscopy (FTIR), high resolution transmission electron microscopy (HRTEM) and atomic force microscopic (AFM) analysis. The AuNPs show intense surface plasmon resonance (SPR) band at 528 nm and were found to be spherical and hexagonal in shape with particle size ranging from 20-30 nm. Transmission electron microscopy and atomic force microscopy were used to analyze the surface morphology of synthesized AuNPs. The capping ligand has been evaluated using matrix assisted laser desorption ionization-mass spectrometry (MALDI-MS) and gas chromatography-mass spectrometry (GC-MS) analysis.

Comparison of methods for the analysis of therapeutic immunoglobulin G Fc-glycosylation profiles—Part 2: Mass spectrometric methods

To monitor the Fc glycosylation of therapeutic immunoglobulin G in bioprocess development, product characterization and release analytics, reliable techniques for glycosylation analysis are needed. Several analytical methods are suitable for this application. We recently presented results comparing detection methods for glycan analysis that are separation-based, but did not include mass spectrometry (MS). In the study reported here, we comprehensively compared MS-based methods for Fc glycosylation profiling of an IgG biopharmaceutical. A therapeutic antibody reference material was analyzed 6-fold on 2 different days, and the methods investigated were compared with respect to precision, accuracy, throughput and analysis time. Emphasis was put on the detection and quantitation of sialic acid-containing glycans. Eleven MS methods were compared to hydrophilic interaction liquid chromatography of 2-aminobenzamide labeled glycans with fluorescence detection, which served as a reference method and was also used in the first part of the study. The methods compared include electrospray MS of the heavy chain and Fc part after limited digestion, liquid chromatography MS of a tryptic digest, porous graphitized carbon chromatography MS of released glycans, electrospray MS of glycopeptides, as well as matrix assisted laser desorption ionization MS of glycans and glycopeptides. Most methods showed excellent precision and accuracy. Some differences were observed with regard to the detection and quantitation of low abundant glycan species like the sialylated glycans and the amount of artefacts due to in-source decay.

Bacillus subtilis subsp. subtilis CBMDC3f with antimicrobial activity against Gram-positive foodborne pathogenic bacteria: UV-MALDI-TOF MS analysis of its bioactive compounds.

In this work a new Bacillus sp. strain, isolated from honey, was characterized phylogenetically. Its antibacterial activity against three relevant foodborne pathogenic bacteria was studied; the main bioactive metabolites were analyzed using ultraviolet matrix assisted laser desorption-ionization mass spectrometry (UV-MALDI MS). Bacillus CBMDC3f was phylogenetically characterized as Bacillus subtilis subsp. subtilis after rRNA analysis of the 16S subunit and the gyrA gene (access codes Genbank JX120508 and JX120516, respectively). Its antibacterial potential was evaluated against Listeria monocytogenes (9 strains), B. cereus (3 strains) and Staphylococcus aureus ATCC29213. Its cell suspension and cell-free supernatant (CFS) exerted significant anti-Listeria and anti-S. aureus activities, while the lipopeptides fraction (LF) also showed anti-B. cereus effect. The UV-MALDI-MS analysis revealed surfactin, iturin and fengycin in the CFS, whereas surfactin predominated in the LF. The CFS from CBMDC3f contained surfactin, iturin and fengycin with four, two and four homologues per family, respectively, whereas four surfactin, one iturin and one fengycin homologues were identified in the LF. For some surfactin homologues, their UV-MALDI-TOF/TOF (MS/MS; Laser Induced Decomposition method, LID) spectra were also obtained. Mass spectrometry analysis contributed with relevant information about the type of lipopeptides that Bacillus strains can synthesize. From our results, surfactin would be the main metabolite responsible for the antibacterial effect.

On plate graphite supported sample processing for simultaneous lipid and protein identification by matrix assisted laser desorption ionization mass spectrometry

The simultaneous identification of lipids and proteins by matrix assisted laser desorption ionization-mass spectrometry (MALDI-MS) after direct on-plate processing of micro-samples supported on colloidal graphite is demonstrated. Taking advantages of large surface area and thermal conductivity, graphite provided an ideal substrate for on-plate proteolysis and lipid extraction. Indeed proteins could be efficiently digested on-plate within 15min, providing sequence coverages comparable to those obtained by conventional in-solution overnight digestion. Interestingly, detection of hydrophilic phosphorylated peptides could be easily achieved without any further enrichment step. Furthermore, lipids could be simultaneously extracted/identified without any additional treatment/processing step as demonstrated for model complex samples such as milk and egg. The present approach is simple, efficient, of large applicability and offers great promise for protein and lipid identification in very small samples.

Rapid incorporation of glucosinolates as a strategy used by a herbivore to prevent activation by myrosinases

Various plants have a binary defence system that consists of a substrate and a glucosidase, which is activated upon tissue disruption thereby forming reactive hydrolysis products. Insects feeding on such plants have to overcome this binary defence system or prevent the activation. In this study, we investigated the strategy used by a herbivore to deal with such binary defence. We studied, how the larvae of the sawfly Athalia rosae (Hymenoptera: Tenthredinidae) circumvent the activation of glucosinolates by myrosinase enzymes, which are found in their Brassicaceae host plants. Myrosinase activities were low in the front part of the larval gut but activities increased over the gut passage. In contrast, the glucosinolates were only highly concentrated in the first gut part and were rapidly incorporated into the haemolymph before the food reached the second half of the gut. Thus, the uptake and concentration of glucosinolates, i.e., sequestration, must occur in the front part of the gut. Using Matrix Assisted Laser Desorption Ionization-Mass Spectrometry Imaging (MALDI-MSI), we could demonstrate that the incorporated glucosinolate sinalbin circulates in the haemolymph where it accumulates around the Malpighian tubules. This study highlights the pivotal role of the gut of an adapted herbivore as a regulatory functional organ to cope with plant toxins. MALDI-MSI turned out as a highly useful technique to visualise glucosinolates in a herbivore, which has to deal with plants exhibiting a binary defence system, and may be applied to follow the fate of plant metabolites in other insect species in the future.