MALDI Mass Spectrometry Research Articles

Bacterial discrimination by Fourier transform infrared spectroscopy, MALDI-mass spectrometry and whole-genome sequencing.

Aim: Proof-of-concept study, highlighting the clinical diagnostic ability of FT-IR compared with MALDI-TOF MS, combined with WGS. Materials & methods: 104 pathogenic isolates of Neisseriameningitidis, Streptococcus pneumoniae, Streptococcus pyogenes and Staphylococcus aureus were analyzed. Results: Overall prediction accuracy was 99.6% in FT-IR and 95.8% in MALDI-TOF-MS. Analysis of N. meningitidis serogroups was superior in FT-IR compared with MALDI-TOF-MS. Phylogenetic relationship of S. pyogenes was similar by FT-IR and WGS, but not S. aureus or S. pneumoniae. Clinical severity was associated with the zinc ABC transporter and DNA repair genes in S. pneumoniae and cell wall proteins (biofilm formation, antibiotic and complement permeability) in S. aureus via WGS. Conclusion: FT-IR warrants further clinical evaluation as a promising diagnostic tool.

Application of MALDI-MS for characterization of fucoidan hydrolysates and screening of endo-fucoidanase activity

Brown macroalgae synthesize large amounts of fucoidans, sulfated fucose-containing polysaccharides, in the ocean. Fucoidans are of importance for their recently discovered contribution to marine carbon dioxide sequestration and due to their potential applications in biotechnology and biomedicine. However, fucoidans have high intra- and intermolecular diversity that challenges assignment of structure to biological function and the development of applications. Fucoidan-active enzymes may be used to simplify this diversity by producing defined oligosaccharides more applicable for structural refinement, characterization, and structure to function assignment for example via bioassays. In this study, we combined MALDI mass spectrometry with biocatalysis to show that the endo-fucoidanases P5AFcnA and Wv323 can produce defined oligosaccharide structures directly from unrefined macroalgal biomass. P5AFcnA released oligosaccharides from seven commercial fucoidan extracts in addition to unrefined biomass of three macroalgae species indicating a broadly applicable approach reproducible across 10 species. Both MALDI-TOF/TOF and AP-MALDI-Orbitrap systems were used, demonstrating that the approach is not instrument-specific and exploiting their combined high-throughput and high-resolution capabilities. Overall, the combination of MALDI-MS and endo-fucoidanase assays offers high-throughput evaluation of fucoidan samples and also enables extraction of defined oligosaccharides of known structure from unrefined seaweed biomass.

Spatially resolved lipidomics shows conditional transfer of lipids produced by Bacteroides thetaiotaomicron into the mouse gut

The extent to which bacterial lipids produced by the gut microbiota penetrate host tissues is unclear. Here, we combined mass spectrometry approaches to identify lipids produced by the human gut symbiont Bacteroides thetaiotaomicron (B.theta) and spatially track these bacterial lipids in the mouse colon. We characterize 130 B.theta lipids by liquid chromatography-tandem mass spectrometry (LC-MS/MS), using wild-type and mutant B.theta strains to confidently identify lipid structures and their interconnected pathways invitro. Of these, 103 B.theta lipids can be detected and spatially mapped in a single MALDI mass spectrometry imaging run. We map unlabeled bacterial lipids across colon sections of germ-free and specific-pathogen-free (SPF) mice and mice mono-colonized with wild-type or sphingolipid-deficient (BTMUT) B.theta. We observe co-localization of bacterially derived phosphatidic acid with host tissues in BTMUT mice, consistent with lipid penetration into host tissues. These results indicate limited and selective transfer of bacterial lipids to the host.

Distinguishing Turkish pine honey from multi-floral honey through MALDI-MS-based N-glycomics and machine learning

Honey, a multifaceted blend of sugars, amino acids, vitamins, proteins, and minerals, exhibits compositional variability dependent upon the floral source. While previous studies have attempted to categorize honey, the use of glycomic profiles for honey classification remains an unexplored avenue. This investigation seeks to establish a methodology for distinguishing honey types, specifically multi-floral and pine honey, employing mass spectrometry-based glycomic analysis in tandem with machine learning. In this search, seven samples of pine honey and eight samples of multi-floral honey were obtained from diverse regions of Turkey. Subsequently, the proteins within these honey samples were extracted, and glycans were enzymatically released. The released glycans were labeled with 2-aminobenzoic acid (2-AA) and subjected to analysis via matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The glycan profiles of pine and multi-floral honey were determined through these analytical procedures, revealing a total of 76 distinct N-glycan structures. Among these, 13 N-glycan profiles consistently established at high levels across experimental replicates and were incorporated in subsequent analyses. Following the quantification of individual glycan abundances, statistically significant differences in glycan profiles were determined. Notably, N-glycans Hex5HexNAc2, Hex4HexNAc3, and Hex5HexNAc3 displayed considerable differences. Using the 13 N-glycan profiles, an accuracy rate of 93.5% was obtained from machine learning analysis, which increased to 100% when incorporating the identified significantly changed glycans. The most productive models were identified as “subspace and fine k-nearest neighbors (KNN).” The findings underscore the potential of mass spectrometry-based glycomics in conjunction with machine learning as a robust tool for precise honey type classification and its prospective utility in quality control and honey product authentication.

Visualizing the spatial distribution of ustalic acid in the fruiting body of Tricholoma kakishimeji.

Imaging mass spectrometry (IMS) was conducted for the first time using ustalic acid (UA) and the fruiting body of Tricholoma kakishimeji to localize mushroom toxins. The mushroom materials were systematically collected in Japan, and analysis of the cross sections of the materials at a resolution of 120μm using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-IMS) revealed the localization of UA and its biogenically related metabolites. MALDI-IMS confirmed that UA was predominantly located on the entire surface of the fruiting body and accumulated in higher amounts in younger fruiting bodies than in mature ones. UA is the first toxic secondary metabolite in the genus Tricholoma locally identified using IMS in mushrooms.

Antibody-Drug Conjugate Made of Zoledronic Acid and the Anti-CD30 Brentuximab-Vedotin Exert Anti-Lymphoma and Immunostimulating Effects.

Relevant advances have been made in the management of relapsed/refractory (r/r) Hodgkin Lymphomas (HL) with the use of the anti-CD30 antibody-drug conjugate (ADC) brentuximab-vedotin (Bre-Ved). Unfortunately, most patients eventually progress despite the excellent response rates and tolerability. In this report, we describe an ADC composed of the aminobisphosphonate zoledronic acid (ZA) conjugated to Bre-Ved by binding the free amino groups of this antibody with the phosphoric group of ZA. Liquid chromatography-mass spectrometry, inductively coupled plasma-mass spectrometry, and matrix-assisted laser desorption ionization-mass spectrometry analyses confirmed the covalent linkage between the antibody and ZA. The novel ADC has been tested for its reactivity with the HL/CD30+ lymphoblastoid cell lines (KMH2, L428, L540, HS445, and RPMI6666), showing a better titration than native Bre-Ved. Once the HL-cells are entered, the ADC co-localizes with the lysosomal LAMP1 in the intracellular vesicles. Also, this ADC exerted a stronger anti-proliferative and pro-apoptotic (about one log fold) effect on HL-cell proliferation compared to the native antibody Bre-Ved. Eventually, Bre-Ved-ZA ADC, in contrast with the native antibody, can trigger the proliferation and activation of cytolytic activity of effector-memory Vδ2 T-lymphocytes against HL-cell lines. These findings may support the potential use of this ADC in the management of r/r HL.

Identification of Mycobacterium abscessus using the peaks of ribosomal protein L29, L30 and hemophore-related protein by MALDI-MS proteotyping

Mycobacteroides (Mycobacterium) abscessus, which causes a variety of infectious diseases in humans, is becoming detected more frequently in clinical specimens as cases are spreading worldwide. Taxonomically, M. abscessus is composed of three subspecies of M. abscessus subsp. abscessus, M. abscessus subsp. bolletii, and M. abscessus subsp. massiliense, with different susceptibilities to macrolides. In order to identify rapidly these three subspecies, we determined useful biomarker proteins, including ribosomal protein L29, L30, and hemophore-related protein, for distinguishing the subspecies of M. abscessus using the matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) profiles. Thirty-three clinical strains of M. abscessus were correctly identified at the subspecies-level by the three biomarker protein peaks. This study ultimately demonstrates the potential of routine MALDI-MS-based laboratory methods for early identification and treatment for M. abscessus infections.

Cutaneous squamous cell carcinoma characterized by MALDI mass spectrometry imaging in combination with machine learning

Cutaneous squamous cell carcinoma (SCC) is an increasingly prevalent global health concern. Current diagnostic and surgical methods are reliable, but they require considerable resources and do not provide metabolomic insight. Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) enables detailed, spatially resolved metabolomic analysis of tissue samples. Integrated with machine learning, MALDI-MSI could yield detailed information pertaining to the metabolic alterations characteristic for SCC. These insights have the potential to enhance SCC diagnosis and therapy, improving patient outcomes while tackling the growing disease burden. This study employs MALDI-MSI data, labelled according to histology, to train a supervised machine learning model (logistic regression) for the recognition and delineation of SCC. The model, based on data acquired from discrete tumor sections (n = 25) from a mouse model of SCC, achieved a predictive accuracy of 92.3% during cross-validation on the labelled data. A pathologist unacquainted with the dataset and tasked with evaluating the predictive power of the model in the unlabelled regions, agreed with the model prediction for over 99% of the tissue areas. These findings highlight the potential value of integrating MALDI-MSI with machine learning to characterize and delineate SCC, suggesting a promising direction for the advancement of mass spectrometry techniques in the clinical diagnosis of SCC and related keratinocyte carcinomas.

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.

Direct saliva analysis by MALDI mass spectrometry: A workflow suitable for future clinical applications

Direct saliva analysis by MALDI mass spectrometry: A workflow suitable for future clinical applications

Abstract 1063: G Protein 12 Alpha Subunit Covalent Binding To Alpha-snap And Its Potential Role In Von Willebrand Factor Secretion

Rationale: α-SNAP is part of a large 20S complex composed of 3 SNARE, 4 α-SNAP, and 6 N-ethylmaleimide sensitive factor proteins that together regulate endothelial cell Weibel Palade body fusion and exocytosis of von Willebrand factor (vWF). We previously showed that Gα12 is essential for basal and thrombin-induced vWF secretion, that GST-α-SNAP pulldown of Gα12 is dependent on N-terminal domain aa 10-15 in Gα12, and that a myristoylated 6 aa α-SNAP Binding Domain Gα12 peptide (Myr-SBD6) reduces vWF secretion, thrombo-inflammation, and lethality in septic mice. Here, using direct binding assays (Surface Plasmon Resonance (SPR), AlphaLISA, and mass spectrometry (MS)), we assessed the molecular underpinnings of SBD6 and modified higher affinity peptide (SBD15) binding directly to α-SNAP. Methods: GST-α-SNAP was incubated alone or with SBD6, SBD15-PEG-biotin, or SBD6 with C to S mutation and then evaluated by Maldi MS. SPR was performed for both SBD6 and SBD15 to assess binding affinity ( K D ) and kinetics ( k a and k d ). After demonstrating the covalent linkage of α-SNAP with SBD15-PEG-biotin, cysteine mapping was performed to identify the SBD binding site on α-SNAP. Free cysteines in SBD15-biotin conjugated α-SNAP were capped with N-ethylmaleimide, disulfide bonds were reduced with DTT, reduced Cys residues were capped with iodoacetamide (IAA), and tryptic peptide digests were analyzed by LC/MS/MS. Results: Maldi MS studies showed that SBD15-PEG-biotin exhibited covalent binding to GST-α-SNAP. Further, proteomic analysis of Cys mapped fragments identified a peptide containing C103, KADPQEAINC 103 LMR, that was modified by NEM in control samples and shifted in the presence of SBD15, indicating modification by IAA in α-SNAP - SBD15 conjugates. Further, the C103S α-SNAP mutant was unable to bind to SBD6, and C11S-SBD15-PEG-biotin was unable to bind to α-SNAP. Conclusion: We propose that amino-terminal Gα12 Cys11 covalently binds to α-SNAP Cys103 and that this plays a critical role in the mechanism regulating vWF secretion. The discovery of this disulfide binding mechanism between Gα12 and α-SNAP opens up new avenues for therapeutic intervention in thrombotic disorders associated with elevated plasma vWF.

Quantitative MALDI-MS and Imaging of Fungicide Pyrimethanil in Strawberries with 2-Nitrophloroglucinol as an Effective Matrix.

This work explores the use of 2-nitrophloroglucinol (2-NPG) as a matrix for quantitative analysis of the fungicide Pyrimethanil (PYM) in strawberries using matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) and imaging. 2-NPG was selected for PYM analysis for optimum sensitivity and precision compared to common matrices α-cyano-4-hydroxylcinnamic acid (CHCA) and 2,5-dihydroxybenzoic acid (DHB). PYM-sprayed strawberries were frozen 0, 1, 3, and 4 days after treatment and sectioned for MALDI imaging. The remaining part of each strawberry was processed using quick easy cheap effective rugged and safe (QuEChERS) extraction and analyzed by MALDI-MS and ultraperformance liquid chromatography multireaction-monitoring (UPLC-MRM). MALDI-MS showed comparable performance to UPLC-MRM in calibration, LOD/LOQ, matrix effect, and recovery, with the benefit of fast analysis. The MALDI imaging results demonstrated that PYM progressively penetrated the interior of the strawberry over time and the PYM concentration on tissue measured by MALDI imaging correlated linearly with MALDI-MS and UPLC-MRM measurements and accounts for 79% MALDI-MS and 85% UPLC-MRM values on average. Additionally, quartz crystal microbalance (QCM) was introduced as a new approach to determine strawberry tissue mass per area for MALDI imaging absolute quantitation with sensitive, direct, and localized measurements. This work demonstrates the first example of absolute quantitative MALDI imaging of pesticides in a heterogeneous plant tissue. The novel use of the 2-NPG matrix in quantitative MALDI-MS and imaging could be applied to other analytes, and the new QCM tissue mass per area method is potentially useful for quantitative MALDI imaging of heterogeneous tissues in general.

Elucidating Gender-Specific Distribution of Imipramine, Chloroquine, and Their Metabolites in Mice Kidney Tissues through AP-MALDI-MSI.

Knowledge of gender-specific drug distributions in different organs are of great importance for personalized medicine and reducing toxicity. However, such drug distributions have not been well studied. In this study, we investigated potential differences in the distribution of imipramine and chloroquine, as well as their metabolites, between male and female kidneys. Kidneys were collected from mice treated with imipramine or chloroquine and then subjected to atmospheric pressure matrix-assisted laser desorption ionization-mass spectrometry imaging (AP-MALDI-MSI). We observed differential distributions of the drugs and their metabolites between male and female kidneys. Imipramine showed prominent distributions in the cortex and medulla in male and female kidneys, respectively. Desipramine, one of the metabolites of imipramine, showed significantly higher (*** p < 0.001) distributions in the medulla of the male kidney compared to that of the female kidney. Chloroquine and its metabolites were accumulated in the pelvis of both male and female kidneys. Interestingly, they showed a characteristic distribution in the medulla of the female kidney, while almost no distributions were observed in the same areas of the male kidney. For the first time, our study revealed that the distributions of imipramine, chloroquine, and their metabolites were different in male and female kidneys.

Matrix- and surface-assisted laser desorption/ionization-mass spectrometry analysis of fingermark components for forensic studies: current trends and future prospects.

The chemical analysis of fingermarks (FMs) has attracted considerable attention in the realm of forensic investigations. Techniques based on direct ionization of a sample by laser irradiation, specifically matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS), have provided excellent figures of merit for analyzing high molecular-weight compounds. However, it can be challenging to analyze low molecular-weight compounds using MALDI-MS owing to potential interference produced by the organic matrices in the low molecular-weight region, which can impede the detection of small molecules (m/z < 700Da). Alternately, surface-assisted laser desorption/ionization-mass spectrometry (SALDI-MS) has shown great promise for small molecules analysis owing to the unique properties of the nanostructures used, particularly, minimal chemical background in low m/z region improved theproduction of ions involved in this method. The advancement of MALDI-MS and SALDI-MS has propelled their application in the analysis of FM components, focused on gaining deep insights into individual traits. This review aims to outline the current role of MALDI-MS and SALDI-MS in the chemical analysis of FMs. It also describes the latest achievements in forensic intelligence derived from fingermark analysis using these powerful methods. The accomplishments include the understanding of certain characteristics and lifestyles of donors. The review offers a comprehensive overview of the challenges and demands in this field. It suggests potential enhancements in this rapidly expanding domain to bridge the gap between research and practical police casework.

Distinguishing methicillin-resistant Staphylococcus aureus from methicillin-sensitive strains by combining Fe3O4 magnetic nanoparticle-based affinity mass spectrometry with a machine learning strategy

Pathogenic bacteria, including drug-resistant variants such as methicillin-resistant Staphylococcus aureus (MRSA), can cause severe infections in the human body. Early detection of MRSA is essential for clinical diagnosis and proper treatment, considering the distinct therapeutic strategies for methicillin-sensitive S. aureus (MSSA) and MRSA infections. However, the similarities between MRSA and MSSA properties present a challenge in promptly and accurately distinguishing between them. This work introduces an approach to differentiate MRSA from MSSA utilizing matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) in conjunction with a neural network-based classification model. Four distinct strains of S. aureus were utilized, comprising three MSSA strains and one MRSA strain. The classification accuracy of our model ranges from ~ 92 to ~ 97% for each strain. We used deep SHapley Additive exPlanations to reveal the unique feature peaks for each bacterial strain. Furthermore, Fe3O4 MNPs were used as affinity probes for sample enrichment to eliminate the overnight culture and reduce the time in sample preparation. The limit of detection of the MNP-based affinity approach toward S. aureus combined with our machine learning strategy was as low as ~ 8 × 103 CFU mL−1. The feasibility of using the current approach for the identification of S. aureus in juice samples was also demonstrated.Graphical

MALDI-mass spectrometry imaging as a new technique for detecting non-heme iron in peripheral tissues via caudal vein injection of deferoxamine.

As one of the most common iron-chelating agents, deferoxamine (DFO) rapidly chelates iron in the body. Moreover, it does not compete for the iron characteristic of hemoglobin in the blood cells, which is common in the clinical treatment of iron poisoning. Iron is a trace element necessary to maintain organism normal life activities. Iron deficiency can lead to anemia, whereas iron overload can cause elevated levels of cellular oxidative stress and cell damage. As a consequence, detection of the iron content in tissues and blood is of great significance. The traditional techniques for detecting the iron content include inductively coupled plasma-mass spectrometry and atomic absorption spectrometry, which cannot be used for imaging purposes. Laser ablation-ICP-MS and synchrotron radiation micro-X-ray fluorescence can map the concentration and distribution of iron in tissues. However, these methods can only be used to measure the total iron levels in blood or tissues. In recent years, due to the deepening understanding of iron metabolism, diseases related to iron overload have attracted increasing attention. Therefore, we took advantage of the properties of DFO in terms of chelating iron and investigated different sampling times following DFO injection in the tail vein of mice. We used mass spectrometry imaging (MSI) technology to detect the DFO and ferrioxamine content in the blood and different tissues to indirectly characterize the non-heme iron content.

Frontal Cortex Lipid Alterations During the Onset of Alzheimer's Disease.

Although sporadic Alzheimer's disease (AD) is a neurodegenerative disorder of unknown etiology, familial AD is associated with specific gene mutations. A commonality between these forms of AD is that both display multiple pathogenic events including cholinergic and lipid dysregulation. We aimed to identify the relevant lipids and the activity of their related receptors in the frontal cortex and correlating them with cognition during the progression of AD. MALDI-mass spectrometry imaging (MSI) and functional autoradiography was used to evaluate the distribution of phospholipids/sphingolipids and the activity of cannabinoid 1 (CB1), sphingosine 1-phosphate 1 (S1P1), and muscarinic M2/M4 receptors in the frontal cortex (FC) of people that come to autopsy with premortem clinical diagnosis of AD, mild cognitive impairment (MCI), and no cognitive impairment (NCI). MALDI-MSI revealed an increase in myelin-related lipids, such as diacylglycerol (DG) 36:1, DG 38:5, and phosphatidic acid (PA) 40:6 in the white matter (WM) in MCI compared to NCI, and a downregulation of WM phosphatidylinositol (PI) 38:4 and PI 38:5 levels in AD compared to NCI. Elevated levels of phosphatidylcholine (PC) 32:1, PC 34:0, and sphingomyelin 38:1 were observed in discrete lipid accumulations in the FC supragranular layers during disease progression. Muscarinic M2/M4 receptor activation in layers V-VI decreased in AD compared to MCI. CB1 receptor activity was upregulated in layers V-VI, while S1P1 was downregulated within WM in AD relative to NCI. FC WM lipidomic alterations are associated with myelin dyshomeostasis in prodromal AD, suggesting WM lipid maintenance as a potential therapeutic target for dementia.

Synthesis of new representatives of A3B-type carboranylporphyrins based on meso-tetra(pentafluorophenyl)porphyrin transformations.

A carboranylporphyrin of A3B-type bearing a single pentafluorophenyl ring was prepared through the regioselective nucleophilic aromatic substitution reaction of the p-fluorine atoms in 5,10,15,20-tetrakis(pentafluorophenyl)porphyrin with 9-mercapto-m-carborane. The reaction of this porphyrin with sodium azide led to the selective substitution of the p-fluorine atom in the pentafluorophenyl substituent with an azide functionality which upon reduction with SnCl2 resulted in the formation of the corresponding porphyrin with an amino group. Pentafluorophenyl-substituted A3B-porphyrins were studied and transformed to thiol and amino-substituted compounds allowing for the preparation of porphyrins with different reactive groups such as hydroxy and amino derivatives capable for further functionalization and conjugation of these porphyrins to other substrates. In addition, conjugates containing maleimide or biotin entities in the structure of carborane A3B-porphyrin were also synthesized based on the amino-substituted A3B-porphyrin. The structures of the prepared carboranylporphyrins were determined by UV-vis, IR, 1H, 19F, 11B NMR spectroscopic data and MALDI mass spectrometry.

Unveiling the regulatory mechanisms of nodules development and quality formation in Panax notoginseng using multi-omics and MALDI-MSI

IntroductionRenowned for its role in traditional Chinese medicine, Panax notoginseng exhibits healing properties including bidirectional regulatory effects on hematological system diseases. However, the presence of nodular structures near the top of the main root, known as nail heads, may impact the quality of the plant's valuable roots. ObjectivesIn this paper, we aim to systematically analyze nail heads to identify their potential correlation with P. notoginseng quality. Additionally, we will investigate the molecular mechanisms behind nail head development. MethodsMorphological characteristics and anatomical features were analyzed to determine the biological properties of nail heads. Active component analysis and MALDI mass spectrometry imaging (MALDI-MSI) were performed to determine the correlation between nail heads and P. notoginseng quality. Phytohormone quantitation, MALDI-MSI, RNA-seq, and Arabidopsis transformation were conducted to elucidate the mechanisms of nail head formation. Finally, protein-nucleic acid and protein–protein interactions were investigated to construct a transcriptional regulatory network of nodule development and quality formation. ResultsOur analyses have revealed that nail heads originate from an undeveloped lateral root. The content of ginsenosides was found to be positively associated with the amount of nail heads. Ginsenoside Rb1 specifically accumulated in the cortex of nail heads, while IAA, tZR and JAs also showed highest accumulation in the nodule. RNA-seq analysis identified PnIAA14 and PnCYP735A1 as inhibitors of lateral root development. PnMYB31 and PnMYB78 were found to form binary complexes with PnbHLH31 to synergistically regulate the expression of PnIAA14, PnCYP735A1, PnSS, and PnFPS. ConclusionOur study details the major biological properties of nodular structures in P. notoginseng and outlines their impact on the quality of the herb. It was also determined that PnMYB31- and PnMYB78-PnbHLH31 regulate phytohormones and ginsenosides accumulation, further affecting plant development and quality. This research provides insights for quality evaluation and clinical applications of P. notoginseng.

Spatial mapping of gemcitabine and its pharmacologically active metabolites in tissues using MALDI mass spectrometry imaging

Spatial mapping of gemcitabine and its pharmacologically active metabolites in tissues using MALDI mass spectrometry imaging