Solvent effects on differentiation of mouse brain tissue using laser microdissection 'cut and drop' sampling with direct mass spectral analysis.

RATIONALE: Laser microdissection-liquid vortex capture/electrospray ionization mass spectrometry (LMD-LVC/ESI-MS) has potential for on-line classification of tissue but an investigation into what analytical conditions provide best spectral differentiation has not been conducted. The effects of solvent, ionization polarity, and spectral acquisition parameters on differentiation of mouse brain tissue regions are described. METHODS: Individual 40 × 40 µm microdissections from cortex, white, grey, granular, and nucleus regions of mouse brain tissue were analyzed using different capture/ESI solvents, in positive and negative ion mode ESI, using time-of-flight (TOF)-MS and sequential window acquisitions of all theoretical spectra (SWATH)-MS (a permutation of tandem-MS), and combinations thereof. Principal component analysis-linear discriminant analysis (PCA-LDA), applied to each mass spectral dataset, was used to determine the accuracy of differentiation of mouse brain tissue regions. RESULTS: Mass spectral differences associated with capture/ESI solvent composition manifested as altered relative distributions of ions rather than the presence or absence of unique ions. In negative ion mode ESI, 80/20 (v/v) methanol/water yielded spectra with low signal/noise ratios relative to other solvents. PCA-LDA models acquired using 90/10 (v/v) methanol/chloroform differentiated tissue regions with 100% accuracy while data collected using methanol misclassified some samples. The combination of SWATH-MS and TOF-MS data improved differentiation accuracy. CONCLUSIONS: Combined TOF-MS and SWATH-MS data differentiated white, grey, granular, and nucleus mouse tissue regions with greater accuracy than when solely using TOF-MS data. Using 90/10 (v/v) methanol/chloroform, tissue regions were perfectly differentiated. These results will guide future studies looking to utilize the potential of LMD-LVC/ESI-MS for tissue and disease differentiation.

Integration of Ion Mobility MSE after Fully Automated, Online, High-Resolution Liquid Extraction Surface Analysis Micro-Liquid Chromatography.

Direct analysis by mass spectrometry (imaging) has become increasingly deployed in preclinical and clinical research due to its rapid and accurate readouts. However, when it comes to biomarker discovery or histopathological diagnostics, more sensitive and in-depth profiling from localized areas is required. We developed a comprehensive, fully automated online platform for high-resolution liquid extraction surface analysis (HR-LESA) followed by micro-liquid chromatography (LC) separation and a data-independent acquisition strategy for untargeted and low abundant analyte identification directly from tissue sections. Applied to tissue sections of rat pituitary, the platform demonstrated improved spatial resolution, allowing sample areas as small as 400 µm to be studied, a major advantage over conventional LESA. The platform integrates an online buffer exchange and washing step for removal of salts and other endogenous contamination that originates from local tissue extraction. Our carry over-free platform showed high reproducibility, with an interextraction variability below 30%. Another strength of the platform is the additional selectivity provided by a postsampling gas-phase ion mobility separation. This allowed distinguishing coeluted isobaric compounds without requiring additional separation time. Furthermore, we identified untargeted and low-abundance analytes, including neuropeptides deriving from the pro-opiomelanocortin precursor protein and localized a specific area of the pituitary gland (i.e., adenohypophysis) known to secrete neuropeptides and other small metabolites related to development, growth, and metabolism. This platform can thus be applied for the in-depth study of small samples of complex tissues with histologic features of ∼400 µm or more, including potential neuropeptide markers involved in many diseases such as neurodegenerative diseases, obesity, bulimia, and anorexia nervosa.

Mass spectrometry imaging for clinical research - latest developments, applications, and current limitations.

Mass spectrometry is being used in many clinical research areas ranging from toxicology to personalized medicine. Of all the mass spectrometry techniques, mass spectrometry imaging (MSI), in particular, has continuously grown towards clinical acceptance. Significant technological and methodological improvements have contributed to enhance the performance of MSI recently, pushing the limits of throughput, spatial resolution, and sensitivity. This has stimulated the spread of MSI usage across various biomedical research areas such as oncology, neurological disorders, cardiology, and rheumatology, just to name a few. After highlighting the latest major developments and applications touching all aspects of translational research (i.e. from early pre-clinical to clinical research), we will discuss the present challenges in translational research performed with MSI: data management and analysis, molecular coverage and identification capabilities, and finally, reproducibility across multiple research centers, which is the largest remaining obstacle in moving MSI towards clinical routine.

Derivatization Strategies for the Detection of Triamcinolone Acetonide in Cartilage by Using Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging.

Osteoarthritis (OA), characterized by degeneration of the cartilaginous tissue in articular joints, severely impairs mobility in many people worldwide. The degeneration is thought to be mediated by inflammatory processes occurring in the tissue of the joint, including the cartilage. Intra-articular administered triamcinolone acetonide (TAA) is one of the drug treatments employed to ameliorate the inflammation and pain that characterizes OA. However, the penetration and distribution of TAA into the avascular cartilage is not well understood. We employed matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI), which has been previously used to directly monitor the distribution of drugs in biological tissues, to evaluate the distribution of TAA in human cartilage after in vitro incubation. Unfortunately, TAA is not easily ionized by regular electrospray ionization (ESI) or MALDI. To overcome this problem, we developed an on-tissue derivatization method with Girard's reagent T (GirT) in human incubated cartilage being able to study its distribution and quantify the drug abundance (up to 3.3 ng/µL). Our results demonstrate the depth of penetration of a corticosteroid drug in human OA cartilage using MALDI-MSI.

The last step in cocaine biosynthesis is catalyzed by a BAHD acyltransferase.

The esterification of methylecgonine (2-carbomethoxy-3ß-tropine) with benzoic acid is the final step in the biosynthetic pathway leading to the production of cocaine in Erythoxylum coca. Here we report the identification of a member of the BAHD family of plant acyltransferases as cocaine synthase. The enzyme is capable of producing both cocaine and cinnamoylcocaine via the activated benzoyl- or cinnamoyl-Coenzyme A thioesters, respectively. Cocaine synthase activity is highest in young developing leaves, especially in the palisade parenchyma and spongy mesophyll. These data correlate well with the tissue distribution pattern of cocaine as visualized with antibodies. Matrix-assisted laser-desorption ionization mass spectral imaging revealed that cocaine and cinnamoylcocaine are differently distributed on the upper versus lower leaf surfaces. Our findings provide further evidence that tropane alkaloid biosynthesis in the Erythroxylaceae occurs in the above-ground portions of the plant in contrast with the Solanaceae, in which tropane alkaloid biosynthesis occurs in the roots.

Use of advantageous, volatile matrices enabled by next-generation high-speed matrix-assisted laser desorption/ionization time-of-flight imaging employing a scanning laser beam.

RATIONALE: In mass spectrometry imaging (MSI) it is often desirable to analyse the same sample in both polarities to extract the most information. However, many matrices that produce high-quality spectra in matrix-assisted laser desorption/ionization (MALDI) are volatile, greatly limiting their use in long imaging experiments. We demonstrate that using a new high speed MALDI-MSI instrument, volatile matrices, including those that produce intense lipid signals in both positive and negative ion mode, can now be effectively used in MSI. METHODS: A prototype Bruker rapifleX MALDI Tissuetyper™ time-of-flight (TOF) instrument was used for high-speed imaging. This allows acquisition rates up to 50 pixels/s made possible by use of a 10 kHz laser and two rotating mirrors that allow the laser beam to be moved over, and synchronised with, the rapidly moving sample. MSI experiments were performed on mouse brain sections using non-vacuum stable dithranol and 2,6-dihydroxyacetophenone (DHA) matrices with pixel sizes ranging from 10 × 10 µm(2) to 50 × 50 µm(2). RESULTS: Both DHA and dithranol produced rich, complementary lipid spectra in both positive and negative ion modes. Due to the rapid acquisition speed of the instrument, both matrices could be effectively used for MSI despite their volatility. For example, an entire mouse brain could be imaged consecutively in both positive and negative ion mode with 50 × 50 µm(2) pixels in ~35 min. We demonstrate that these speeds make possible both faster and higher resolution imaging of biological tissues on practical timescales. CONCLUSIONS: These high acquisition speeds now make possible whole new classes of matrices that are unstable under high vacuum for MALDI-MSI studies. This provides researchers with far greater range and flexibility in choosing the best matrix for the given sample and analytes that they wish to detect. In addition, such instruments allow MSI to be performed at higher resolution across larger areas on practical time scales.

Quantification in MALDI-MS imaging: what can we learn from MALDI-selected reaction monitoring and what can we expect for imaging?

Quantification by mass spectrometry imaging (Q-MSI) is one of the hottest topics of the current discussions among the experts of the MS imaging community. If MSI is established as a powerful qualitative tool in drug and biomarker discovery, its reliability for absolute and accurate quantification (QUAN) is still controversial. Indeed, Q-MSI has to deal with several fundamental aspects that are difficult to control, and to account for absolute quantification. The first objective of this manuscript is to review the state-of-the-art of Q-MSI and the current strategies developed for absolute quantification by direct surface sampling from tissue sections. This includes comments on the quest for the perfect matrix-matched standards and signal normalization approaches. Furthermore, this work investigates quantification at a pixel level to determine how many pixels must be considered for accurate quantification by ultraviolet matrix-assisted laser desorption/ionization (MALDI), the most widely used technique for MSI. Particularly, this study focuses on the MALDI-selected reaction monitoring (SRM) in rastering mode, previously demonstrated as a quantitative and robust approach for small analyte and peptide-targeted analyses. The importance of designing experiments of good quality and the use of a labeled compound for signal normalization is emphasized to minimize the signal variability. This is exemplified by measuring the signal for cocaine and a tryptic peptide (i.e., obtained after digestion of a monoclonal antibody) upon different experimental conditions, such as sample stage velocity, laser power and frequency, or distance between two raster lines. Our findings show that accurate quantification cannot be performed on a single pixel but requires averaging of at least 4-5 pixels. The present work demonstrates that MALDI-SRM/MSI is quantitative with precision better than 10-15 %, which meets the requirements of most guidelines (i.e., in bioanalysis or toxicology) for quantification of drugs or peptides from tissue homogenates.

Gas-phase separation of drugs and metabolites using modifier-assisted differential ion mobility spectrometry hyphenated to liquid extraction surface analysis and mass spectrometry.

The present work describes an alternative generic approach to LC-MS for the analysis of drugs of abuse as well as their metabolites in post-mortem tissue samples. The platform integrates liquid extraction surface analysis (LESA) for analytes tissue extraction followed by differential ion mobility spectrometry (DMS) mass spectrometry for analytes gas phase separation. Detection is performed on a triple quadrupole linear ion trap using the selected reaction monitoring mode for quantification as well as product ion scan mode for structural confirmatory analyses. The major advantages of the platform are that neither chromatographic separation nor extensive sample preparation are required. In DMS the combination of a high separation voltage (i.e., up to 4 kV) together with organic modifiers (e.g., alcohols, acetonitrile, acetone) added in the drift gas is required to achieve the separation of isomeric metabolites, such as the ones of cocaine and tramadol. DMS also separates morphine from its glucuronide metabolites, which allows for preventing the overestimation of morphine in case of fragmentation of the glucuronides in the atmospheric-to-vacuum interface of the mass spectrometer. Cocaine, opiates, opioids, amphetamines, benzodiazepines and several of their metabolites could be identified in post-mortem human kidney and muscle tissue based on simultaneous screening and confirmatory analysis in data-dependent acquisition mode using an analyte-dependent compensation voltage to selectively transmit ions through the DMS cell to the mass analyzer. Quantitative performance of the LESA-DMS-MS platform was evaluated for cocaine and two of its metabolites spotted onto a tissue section using deuterated internal standard. Analyte's responses were linear from 2 to 1000 pg on tissue corresponding to a limit of detection in the order of nanograms of analyte per gram of tissue. Accuracy and precision based on QC sample was found to be less than 10%. Replicate analyses of cocaine and its metabolites in forensic samples showed an intra- and inter-sections variability of less than 25%.

Single hair cocaine consumption monitoring by mass spectrometric imaging.

Matrix-assisted laser desorption/ionization mass spectrometric imaging (MALDI-MSI) was used to image the distribution of cocaine and its metabolites in intact single hair samples from chronic users down to a concentration of 5 ng/mg. Acquisitions were performed in rastering mode, at a speed of 1 mm/s and in the selected reaction monitoring (SRM) mode on a MALDI triple quadrupole linear ion trap fitted with a high repetition rate laser (1 kHz). Compared to traditional methods based on LC-MS/MS or GC-MS(/MS) which require to segment the hair to obtain spatial resolution, MALDI-MSI, with a straightforward sample preparation beforehand, allowed obtaining a spatial resolution of 1 mm and thus the chronological information about cocaine consumption contained in a single intact hair over several months could be monitored. The analysis time of an intact single hair sample of 6 cm is approximately of 6 min. Cocaine and its metabolites benzoylecgonine, ethylcocaine, and norcocaine were investigated in nine sets of hair samples for forensic purposes. The analyses were accomplished by spraying α-cyano-4-hydroxycinnamic acid (CHCA), 4-chloro-α-cyano-cinnamic acid (Cl-CCA), or (E)-2-cyano-3-(naphthalen-2-yl)acrylic acid (NpCCA) as MALDI matrices. We also propose a rapid strategy for sensitive confirmatory analyses with both MS/MS and MS(3) experiments performed directly on intact hair samples. Since only part of the hair strand is analyzed, additional analyses are possible at any time on the remaining hair from the strand.

Mass Spectrometry Imaging of Drugs of Abuse in Hair.

Hair testing is a powerful tool routinely used for the detection of drugs of abuse. The analysis of hair is highly advantageous as it can provide prolonged drug detectability versus that in biological fluids and chronological information about drug intake based on the average growth of hair. However, current methodology requires large amounts of hair samples and involves complex time-consuming sample preparation followed by gas or liquid chromatography coupled with mass spectrometry. Mass spectrometry imaging is increasingly being used for the analysis of single hair samples, as it provides more accurate and visual chronological information in single hair samples.Here, two methods for the preparation of single hair samples for mass spectrometry imaging are presented.The first uses an in-house built cutting apparatus to prepare longitudinal sections, the second is a method for embedding and cryo-sectioning hair samples in order to prepare cross-sections all along the hair sample.

Alternative CHCA-based matrices for the analysis of low molecular weight compounds by UV-MALDI-tandem mass spectrometry.

Analysis of low molecular weight compounds (LMWC) in complex matrices by vacuum matrix-assisted laser desorption/ionization (MALDI) often suffers from matrix interferences, which can severely degrade limits of quantitation. It is, therefore, useful to have available a range of suitable matrices, which exhibit complementary regions of interference. Two newly synthesized α-cyanocinnamic acid derivatives are reported here; (E)-2-cyano-3-(naphthalen-2-yl)acrylic acid (NpCCA) and (2E)-3-(anthracen-9-yl)-2-cyanoprop-2enoic acid (AnCCA). Along with the commonly used α-cyano-4-hydroxycinnamic acid (CHCA), and the recently developed 4-chloro-α-cyanocinnamic acid (Cl-CCA) matrices, these constitute a chemically similar series of matrices covering a range of molecular weights, and with correspondingly differing ranges of spectral interference. Their performance was compared by measuring the signal-to-noise ratios (S/N) of 47 analytes, mostly pharmaceuticals, with the different matrices using the selected reaction monitoring (SRM) mode on a triple quadrupole instrument equipped with a vacuum MALDI source. AnCCA, NpCCA and Cl-CCA were found to offer better signal-to-noise ratios in SRM mode than CHCA, but Cl-CCA yielded the best results for 60% of the compounds tested. To better understand the relative performance of this matrix series, the proton affinities (PAs) were measured using the kinetic method. Their relative values were: AnCCA > CHCA > NpCCA > Cl-CCA. This ordering is consistent with the performance data. The synthesis of the new matrices is straightforward and they provide (1) tunability of matrix background interfering ions and (2) enhanced analyte response for certain classes of compounds.