Realm of proteomics in breast cancer management and drug repurposing to alleviate intricacies of treatment.

Breast cancer, a multi-networking heterogeneous disease, has emerged as a serious impediment to progress in clinical oncology. Although technological advancements and emerging cancer research studies have mitigated breast cancer lethality, a precision cancer-oriented solution has not been achieved. Thus, this review will persuade the acquiescence of proteomics-based diagnostic and therapeutic options in breast cancer management. Recently, the evidence of breast cancer health surveillance through imaging proteomics, single-cell proteomics, interactomics, and post-translational modification (PTM) tracking, to construct proteome maps and proteotyping for stage-specific and sample-specific cancer subtyping have outperformed conventional ways of dealing with breast cancer by increasing diagnostic efficiency, prognostic value, and predictive response. Additionally, the paradigm shift in applied proteomics for designing a chemotherapy regimen to identify novel drug targets with minor adverse effects has been elaborated. Finally, the potential of proteomics in alleviating the occurrence of chemoresistance and enhancing reprofiled drugs' effectiveness to combat therapeutic obstacles has been discussed. Owing to the enormous potential of proteomics techniques, the clinical recognition of proteomics in breast cancer management can be achievable and therapeutic intricacies can be surmountable.

Direct mass spectrometric imaging of document handwriting with laser desorption ionization and post ultraviolet photodissociation.

Handwriting represents personal education and physical or psychological states. This work describes a chemical imaging technique for document evaluation that combines laser desorption ionization with post ultraviolet photo-induced dissociation (LDI-UVPD) in mass spectrometry. Taken the advantages of chromophores in ink dyes, handwriting papers were subjected to direct laser desorption ionization without additional matrix materials. It is a surface-sensitive analytical method that uses a low intensity pulsed laser at 355 nm to remove chemical components from very outermost surfaces of overlapped handwritings. Meanwhile, the transfer of photoelectrons to those compounds leads to the ionization and the formation of radical anions. The gentle evaporation and ionization property enable the dissection of chronological orders. Paper documents maintain intact without extensive damages after laser irradiation. The evolving plume resulting from the irradiation of the 355 nm laser is fired by the second ultraviolet laser at 266 nm that is in parallel to the sample surface. In contrast to collision activated dissociation in tandem MS/MS, such post ultraviolet photodissociation generates much more different fragment ions through electron-directed specific cleavages of chemical bonds. LDI-UVPD can not only provide graphic representation of chemical components but also reveal hidden dynamic features such as alterations, pressures and aging.

Gadolinium contrast agents: dermal deposits and potential effects on epidermal small nerve fibers.

Small fiber neuropathy (SFN) affects unmyelinated and thinly myelinated nerve fibers causing neuropathic pain with distal distribution and autonomic symptoms. In idiopathic SFN (iSFN), 30% of the cases, the underlying aetiology remains unknown. Gadolinium (Gd)-based contrast agents (GBCA) are widely used in magnetic resonance imaging (MRI). However, side-effects including musculoskeletal disorders and burning skin sensations were reported. We investigated if dermal Gd deposits are more prevalent in iSFN patients exposed to GBCAs, and if dermal nerve fiber density and clinical parameters are likewise affected. 28 patients (19 females) with confirmed or no GBCA exposure were recruited in three German neuromuscular centers. ISFN was confirmed by clinical, neurophysiological, laboratory and genetic investigations. Six volunteers (two females) served as controls. Distal leg skin biopsies were obtained according to European recommendations. In these samples Gd was quantified by elemental bioimaging and intraepidermal nerve fibers (IENF) density via immunofluorescence analysis. Pain phenotyping was performed in all patients, quantitative sensory testing (QST) only in a subset (15 patients; 54%). All patients reported neuropathic pain, described as burning (n = 17), jabbing (n = 16) and hot (n = 11) and five QST scores were significantly altered. Compared to an equal distribution significantly more patients reported GBCA exposures (82%), while 18% confirmed no exposures. Compared to unexposed patients/controls significantly increased Gd deposits and lower z-scores of the IENF density were confirmed in exposed patients. QST scores and pain characteristics were not affected. This study suggests that GBCA exposure might alter IENF density in iSFN patients. Our results pave the road for further studies investigating the possible role of GBCA in small fiber damage, but more investigations and larger samples are needed to draw firm conclusions.

Matrix-assisted laser-desorption/ionization-mass spectrometric imaging of psilocybin and its analogues in psychedelic mushrooms using a cesium chloride-coated target plate.

Fungi with hallucinogenic properties and neurotoxicity have been listed as prohibited drugs in recent years, but there is a lack of in situ quantification of psilocybin and analogues in these samples to avoid the decomposition of these psychoactive tryptamines in time-consuming sample preparation. In this study, matrix-assisted laser-desorption/ionization (MALDI)-Fourier transform ion cyclotron resonance (FT ICR) mass spectrometric imaging (MSI) was used to analyze the distribution of psilocybin and its analogues in hallucinogenic Psilocybe mushrooms. A cesium chloride (CsCl)-coated target plate was prepared to improve the detection sensitivity and reduce the interference of other compounds or decomposition products with very similar m/z values in MALDI-FT ICR MS analysis. Psilocybin and other tryptamines with structurally similar compounds, including psilocin, baeocystin, tryptophan, tryptamine, and aeruginascin, were identified and imaged in the psilocybe tissue section; the semiquantitative analysis of the distribution of psilocybin was also investigated using a homemade 75-well CsCl-coated plate; and the target plate can be placed on the mass spectrometry target carrier along with the indium-tin oxide (ITO) conductive slide, which can simultaneously carry out matrix vapor deposition, thus ensuring the parallelism between the standards and samples in the pretreatment experiment and MSI. The contents of psilocybin and its analogues in the psilocybe tissue section can be evaluated from the color changes corresponding to different concentration standard curves. Furthermore, a comprehensive comparison between MALDI-FT ICR MS and ultra-performance liquid chromatography-quadrupole time of flight mass spectrometry (UPLC-Q/TOF MS) analysis was performed for quantification and validation. This study reduces the decomposition in time-consuming sample pretreatment and provides a powerful tool for drug abuse control and forensic analysis.

Matrix-assisted laser desorption/ionization mass spectrometric imaging the spatial distribution of biodegradable vascular stents using a self-made semi-quantitative target plate.

In recent years, the development and optimization of biodegradable coronary stents have become the research focus of many medical device manufacturers and scientific research institutions since they can be completely degraded and absorbed, and they restore vascular function. However, there is a lack of in situ quantification of these stents spatially in tissue in vivo. In this study, matrix-assisted laser desorption/ionization (MALDI) Fourier transform ion cyclotron resonance (FT ICR) and time-of-flight (TOF) mass spectrometric imaging (MSI) were used to analyze the time-dependent distributions of a biodegradable vascular scaffold, which consisted of copolymers of lactic acid and glycolic acid (PLGA) and its degradation products in cross-sections and longitudinal sections of blood vessels. The MALDI-MSI methods for analyzing the distribution of PLGA and its derivatives in vivo were established by optimizing the conditions of sample pretreatment and mass spectrometry (MS). In order to semi-quantify the contents of PLGA degradation products in blood vessels, self-made stainless-steel and indium tin oxide (ITO) target plates were developed to compare and establish the standard curves for semi-quantitative analysis. The target plate can be placed on the target carrier of MS simultaneously with the conductive slide, which can simultaneously carry out vapor deposition or spray on the substrate, to ensure the parallelism of the pretreatment experiments between the standards and the actual vascular samples. The proposed method provided a powerful tool for evaluating the distributions and degradation process of biological stent materials in the coronary artery, as well as provided technical support for the research and development of degradable biological stents and product optimization.

A new update of MALDI-TOF mass spectrometry in lipid research.

Matrix-assisted laser desorption and ionization (MALDI) mass spectrometry (MS) is an indispensable tool in modern lipid research since it is fast, sensitive, tolerates sample impurities and provides spectra without major analyte fragmentation. We will discuss some methodological aspects, the related ion-forming processes and the MALDI MS characteristics of the different lipid classes (with the focus on glycerophospholipids) and the progress, which was achieved during the last ten years. Particular attention will be given to quantitative aspects of MALDI MS since this is widely considered as the most serious drawback of the method. Although the detailed role of the matrix is not yet completely understood, it will be explicitly shown that the careful choice of the matrix is crucial (besides the careful evaluation of the positive and negative ion mass spectra) in order to be able to detect all lipid classes of interest. Two developments will be highlighted: spatially resolved Imaging MS is nowadays well established and the distribution of lipids in tissues merits increasing interest because lipids are readily detectable and represent ubiquitous compounds. It will also be shown that a combination of MALDI MS with thin-layer chromatography (TLC) enables a fast spatially resolved screening of an entire TLC plate which makes the method competitive with LC/MS.

Ambient Mass Spectrometry Imaging of Small Molecules from Cells and Tissues.

New methods to analyze cells and tissues in ambient condition without any harsh chemical fixation or physical freezing and drying are summarized in this report. The first approach, an atmospheric pressure mass spectrometry imaging method, is based on laser ablation in atmospheric pressure assisted by atmospheric plasma and nanomaterials such as nanoparticles and graphene to enhance laser ablation. The second one is based on secondary ion mass spectrometry (SIMS) imaging of live cells in solution capped with single-layer graphene to preserve intact and hydrated biological samples even under ultrahigh vacuum for SIMS bio-imaging in solution.

Mass Spectrometric Biosensing: A Powerful Approach for Multiplexed Analysis of Clinical Biomolecules.

Rapid and sensitive detection of clinical biomolecules in a multiplexed fashion is of great importance for accurate diagnosis of diseases. Mass spectrometric (MS) approaches are exceptionally suitable for clinical analysis due to its high throughput, high sensitivity, and reliable qualitative and quantitative capabilities. To break through the bottleneck of MS technique for detecting high-molecular-weight substances with low ionization efficiency, the concept of mass spectrometric biosensing has been put forward by adopting mass spectrometric chips to recognize the targets and mass spectrometry to detect the signals switched by the recognition. In this review, the principle of mass spectrometric sensing, the construction of different mass tags used for biosensing, and the typical combination mode of mass spectrometric imaging (MSI) technique are summarized. Future perspectives including the design of portable matching platforms, exploitation of novel mass tags, development of effective signal amplification strategies, and standardization of MSI methodologies are proposed to promote the advancements and practical applications of mass spectrometric biosensing.

Proteomics-based identification of TMED9 is linked to vascular invasion and poor prognoses in patients with hepatocellular carcinoma.

BACKGROUND: Due to the difficulties in early diagnosing and treating hepatocellular carcinoma (HCC), prognoses for patients remained poor in the past decade. In this study, we established a screening model to discover novel prognostic biomarkers in HCC patients. METHODS: Candidate biomarkers were screened by liquid chromatography with tandem mass spectrometry (LC-MS/MS) analyses of five HCC normal (N)/tumor (T) paired tissues and preliminarily verified them through several in silico database analyses. Expression levels and functional roles of candidate biomarkers were respectively evaluated by immunohistochemical staining in N/T paired tissue (n = 120) and MTS, colony formation, and transwell migration/invasion assays in HCC cell lines. Associations of clinicopathological features and prognoses with candidate biomarkers in HCC patients were analyzed from GEO and TCGA datasets and our recruited cohort. RESULTS: We found that the transmembrane P24 trafficking protein 9 (TMED9) protein was elevated in HCC tissues according to a global proteomic analysis. Higher messenger (m)RNA and protein levels of TMED9 were observed in HCC tissues compared to normal liver tissues or pre-neoplastic lesions. The TMED9 mRNA expression level was significantly associated with an advanced stage and a poor prognosis of overall survival (OS, p = 0.00084) in HCC patients. Moreover, the TMED9 protein expression level was positively correlated with vascular invasion (p = 0.026), OS (p = 0.044), and disease-free survival (p = 0.015) in our recruited Taiwanese cohort. In vitro, manipulation of TMED9 expression in HCC cells significantly affected cell migratory, invasive, proliferative, and colony-forming abilities. CONCLUSIONS: Ours is the first work to identify an oncogenic role of TMED9 in HCC cells and may provide insights into the application of TMED9 as a novel predictor of clinical outcomes and a potential therapeutic target in patients with HCC.

Transcriptomic and Metabolomic Analyses Provide Insights into the Upregulation of Fatty Acid and Phospholipid Metabolism in Tomato Fruit under Drought Stress.

Transcriptome and metabolome analysis in tomato (Solanum lycopersicum) fruits cultivated under drought conditions showed that drought stress promoted fatty acid synthesis and increased the content of fatty acids in fruits. The accumulation of some phospholipids composed of palmitic acid and oleic acid also was significantly increased, especially in seeds. Moreover, inositol, which is a component of cell membranes and cell walls, was increased through the activity of the myoinositol monophosphatase 1-mediated pathway. In mature fruits, the levels of metabolic regulators such as ß-alanine and 4-aminobutyric acid were elevated. These results showed that these compounds are drought-responsive and enhance drought tolerance and subsequently they could enhance the nutritional value and health benefits of tomato fruit.

Highly Multiplexed Immunohistochemical MALDI-MS Imaging of Biomarkers in Tissues.

Immunohistochemistry (IHC) combined with fluorescence microscopy provides an important and widely used tool for researchers and pathologists to image multiple biomarkers in tissue specimens. However, multiplex IHC using standard fluorescence microscopy is generally limited to 3-5 different biomarkers, with hyperspectral or multispectral methods limited to 8. We report the development of a new technology based on novel photocleavable mass-tags (PC-MTs) for facile antibody labeling, which enables highly multiplexed IHC based on MALDI mass spectrometric imaging (MALDI-IHC). This approach significantly exceeds the multiplexity of both fluorescence- and previous cleavable mass-tag-based methods. Up to 12-plex MALDI-IHC was demonstrated on mouse brain, human tonsil, and breast cancer tissues specimens, reflecting the known molecular composition, anatomy, and pathology of the targeted biomarkers. Novel dual-labeled fluorescent PC-MT antibodies and label-free small-molecule mass spectrometric imaging greatly extend the capability of this new approach. MALDI-IHC shows promise for use in the fields of tissue pathology, tissue diagnostics, therapeutics, and precision medicine.

Biosynthetic Intermediate Probes for Visualizing and Identifying the Biosynthetic Enzymes of Plant Metabolites.

Plant metabolites play important roles in both plant physiology and drug discovery. Taking advantage of new emerging technologies such as next generation sequencing (NGS), whole genome assembly, bioinformatics, omics-based strategies have been demonstrated as popular and powerful ways to elucidate complex metabolic pathways in plants. In this viewpoint, biosynthetic intermediates probes have been proposed as the potentinal tools to study the plant natural product biosynthesis via chemical proteomics appoaches or transcriptome analysis.

Monitoring of adsorption and transfer of organochlorines in soybean seeds and sprouts with mass spectrometric imaging.

Development of analytical techniques that can monitor the adsorption, transfer and in-situ distribution of environmental pollutants in agricultural products is essential to ensure the implementation of stringent food safety standards for consumer protection. A mass spectrometric imaging approach is described herein to investigate the dynamic changes and spatial distributions of 4, 4'-DDT (dichlorodiphenyltrichloroethane) in soybean seeds and sprouts during the growth. Soy beans seeds incubated in DDT containing water were sliced in every 20 µm and directly blotted on the surface of a compressed thin film of (Bi2O3)0.07(CoO)0.03(ZnO)0.9 nanoparticles. Endogenous molecules and exogenous DDT compounds in soy bean seeds were ionized and dissociated by photoelectrons that are generated on surfaces of semiconductor nanoparticles upon the irradiation of the 3rd harmonic (355 nm) of Nd3+:YAG laser. Structural identification is achieved by the interpretation of fragment ions resulting from electron-initiated specific bond cleavages or hole oxidization. Mass spectrometric images reveal increased quantities of DDT residues in soy bean seeds and sprouts during the growth. It provides an in situ way without extensive sample preparation to monitor the transfer and distribution of exogenous pollutants as well as the possible impacts on plant growth.

The rhizosphere signature on the cell motility, biofilm formation and secondary metabolite production of a plant-associated Lysobacter strain.

Lysobacter spp. are common bacterial inhabitants of the rhizosphere of diverse plant species. However, the impact of the rhizosphere conditions on their physiology is still relatively understudied. To provide clues on the behaviour of Lysobacter spp. in this ecological niche, we investigated the physiology of L. capsici AZ78 (AZ78), a biocontrol strain isolated from tobacco rhizosphere, on a common synthetic growth medium (LBA) and on a growth medium containing components of the plant rhizosphere (RMA). The presence of a halo surrounding the AZ78 colony on RMA was a first visible effect related to differences in growth medium composition and it corresponded to the formation of a large outer ring. The lower quantity of nutrients available in RMA as compared with LBA was associated to a higher expression of a gene encoding cAMP-receptor-like protein (Clp), responsible for cell motility and biofilm formation regulation. AZ78 cells on RMA were motile, equipped with cell surface appendages and organised in small groups embedded in a dense layer of fibrils. Metabolic profiling by mass spectrometry imaging revealed increased diversity of analytes produced by AZ78 on RMA as compared with LBA. In particular, putative cyclic lipodepsipeptides, polycyclic tetramate macrolactams, cyclic macrolactams and other putative secondary metabolites with antibiotic activity were identified. Overall, the results obtained in this study shed a light on AZ78 potential to thrive in the rhizosphere by its ability to move, form biofilm and release secondary metabolites.

Autobiography of an Analytical Chemist.

Most of my research directions were opportunistic. Having worked with lasers in the early stages of laser applications in analytical chemistry, attending conferences, workshops, and administrative meetings that were not exactly aligned with our own research, locating to a building or in a department that housed scientists with different backgrounds, having certain specialized equipment at the right time, and having funding agencies that were broad-minded clearly contributed to my ventures into diverse fields. Most of all, it had to be the many eager minds that I have had the fortune to work with. I have always tried to suggest research topics that might be interesting to the individual coworker rather than something straight out of my own research proposals. Only then did each person actually own the project rather than consider it a chore. After all, we work in the field of analytical chemistry, in which almost anything we do can fit in.

Investigating time dependent brain distribution of nevirapine via mass spectrometric imaging.

Central nervous system (CNS) HIV infection causes brain tissue inflammation and tissue deficit that contributes to neuroAIDS. This complication is escalated by the blood-brain barrier (BBB), which prevents easy access to antiretroviral drugs entering the CNS. In this study the aims were to investigate the BBB membrane penetration and brain localization patterns of Nevirapine (NVP) using Imaging Mass Spectrometry (MSI). Sprague-Dawley rats received an intraperitoneal dose of NVP (50 mg kg-1). Plasma and brain samples were harvested, and mass spectrometric methods were then applied to determine the pharmacokinetic properties and localization patterns of NVP in the brain. The pharmacokinetic parameters demonstrated a rapid bio-distribution of NVP in plasma and brain. The plasma Cmax was 6320 ng mL-1 and the brain Cmax was 1923 ng mL-1, both at a Tmax of 0.25 h. MSI of coronal brain sections showed that NVP penetrated and localized in the brain regions implicated with the development of HIV associated neurodegeneration. NVP has great potential to combat neuroAIDS.

Electron Acceptive Mass Tag for Mass Spectrometric Imaging-Guided Synergistic Targeting to Mice Brain Glutamate Receptors.

Dysfunctional glutamate receptors (GluRs) have been implicated in neurological disorders and injuries. Hetero-tetrameric assemblies of different GluR subunits or splicing variants have distinct spatiotemporal expression patterns and pharmacological properties. Mass spectrometric imaging of GluRs-targeted small molecules is important for determining the regional preferences of these compounds. We report herein the development of a mass tag covalently bonded with glutamate or N-methyl-d-aspartate that functions as both an electron acceptor to generate mass spectrometric signals on irradiated (Bi2O3)0.07(CoO)0.03(ZnO)0.9 nanoparticles with the third harmonic (355 nm) of Nd3+:YAG laser and as the core component to target bilobed clamshell-like structures of GluRs. In this approach, different molecules produce the same tag ion. It provides a new avenue for quantitative assessment of spatial densities of different compounds, which cannot be achieved with well-established stable isotope labeling technique due to different ionization efficiency of different compounds. Various coexisting endogenous molecules are also simultaneously detected for investigation of overall physiological changes induced by these compounds. Because semiconductors do not generate background peaks, this method eliminates interferences from organic matrix materials that are used in regular MALDI (matrix assisted laser desorption ionization). The localized ionization provides high spatial resolution that can be down to sub-micrometers.

Molecules and elements for quantitative bioanalysis: The allure of using electrospray, MALDI, and ICP mass spectrometry side-by-side.

To understand biological processes, not only reliable identification, but quantification of constituents in biological processes play a pivotal role. This is especially true for the proteome: protein quantification must follow protein identification, since sometimes minute changes in abundance tell the real tale. To obtain quantitative data, many sophisticated strategies using electrospray and MALDI mass spectrometry (MS) have been developed in recent years. All of them have advantages and limitations. Several years ago, we started to work on strategies, which are principally capable to overcome some of these limits. The fundamental idea is to use elemental signals as a measure for quantities. We began by replacing the radioactive 32 P with the "cold" natural 31 P to quantify modified nucleotides and phosphorylated peptides and proteins and later used tagging strategies for quantification of proteins more generally. To do this, we introduced Inductively Coupled Plasma Mass Spectrometry (ICP-MS) into the bioanalytical workflows, allowing not only reliable and sensitive detection but also quantification based on isotope dilution absolute measurements using poly-isotopic elements. The detection capability of ICP-MS becomes particularly attractive with heavy metals. The covalently bound proteins tags developed in our group are based on the well-known DOTA chelate complex (1,4,7,10-tetraazacyclododecane-N,N',N″,N‴-tetraacetic acid) carrying ions of lanthanoides as metal core. In this review, I will outline the development of this mutual assistance between molecular and elemental mass spectrometry and discuss the scope and limitations particularly of peptide and protein quantification. The lanthanoide tags provide low detection limits, but offer multiplexing capabilities due to the number of very similar lanthanoides and their isotopes. With isotope dilution comes previously unknown accuracy. Separation techniques such as electrophoresis and HPLC were used and just slightly adapted workflows, already in use for quantification in bioanalysis. Imaging mass spectrometry (MSI) with MALDI and laser ablation ICP-MS complemented the range of application in recent years.

Spatiotemporal Dynamics of Molecular Messaging in Bacterial Co-Cultures Studied by Multimodal Chemical Imaging.

Microbial community behavior is coupled to a set of genetically-regulated chemical signals that correlate with cell density - the quorum sensing (QS) system - and there is growing appreciation that the QS-regulated behavior of bacteria is chemically, spatially, and temporally complex. In addition, while it has been known for some time that different species use different QS networks, we are beginning to appreciate that different strains of the same bacterial species also differ in their QS networks. Here we combine mass spectrometric imaging (MSI) and confocal Raman microscopy (CRM) approaches to investigate co-cultures involving different strains (FRD1 and PAO1C) of the same species (Pseudomonas aeruginosa) as well as those involving different species (P. aeruginosa and E. coli). Combining MSI and CRM makes it possible to supersede the limits imposed by individual imaging approaches and enables the spatial mapping of individual bacterial species and their microbial products within a mixed bacterial community growing in situ on surfaces. MSI is used to delineate the secretion of a specific rhamnolipid surfactant as well as alkyl quinolone (AQ) messengers between FRD1 and PAO1C strains of P. aeruginosa, showing that the spatial distribution and production rate of AQ messengers in PAO1C far outstrips that of FRD1. In the case of multiple species, CRM is used to show that the prolific secretion of AQs by the PAO1C strain of P. aeruginosa is used to mediate its interaction with co-cultured E. coli.

Ion fragmentations via photoelectron activated radical relays and competed hole oxidization on semiconductor nanoparticles for mass spectrometry.

Structural identification is challenging in mass spectrometric imaging because of inadequate sample quantities and limited sampling time in each pixel for tandem mass spectrometry (MS/MS) experiments, which are usually used for the generation of fragment ions. We report herein the observation of a cascade of highly specific chemical bond cleavages via a low-energy photoelectron activated radical relays and a competed hole oxidization on surfaces of (Bi2O3)0.07(CoO)0.03(ZnO)0.9 semiconductor nanoparticles irradiated with the 3rd harmonic (355 nm) of the Nd3+: YAG laser. Distinguished from high energy electron impact (EI), this approach generates gaseous radical anions through the exothermic capture of low-energy tunneling electrons that are not able to cause extensive vibrational excitations. It was found not only original radical center but also secondary or even tertiary radical centers cause specific bond cleavages exclusively on α positions. The original radical center directly activates the cleavages of α-positioned chemical bonds that cause the formation of secondary radical centers. Ion fragmentations proceed along the newly formed radical centers that further activate the cleavages of their α-positioned chemical bonds. Using 8 compounds, we have demonstrated various radical reactions involved in desulfonation, cyclization, and ring contraction reactions as well as competed hole oxidization-generated hydroxyl radical substitution reactions. The interpretable fragment ions provide unambiguous experimental evidences for structural elucidation of drug residues and metabolites in mass spectrometric imaging of tissue slices without tandem mass spectrometry (MS/MS).