Per E. Andrén

Fine Mapping the Spatial Distribution and Concentration of Unlabeled Drugs within Tissue Micro-Compartments Using Imaging Mass Spectrometry

Readouts that define the physiological distributions of drugs in tissues are an unmet challenge and at best imprecise, but are needed in order to understand both the pharmacokinetic and pharmacodynamic properties associated with efficacy. Here we demonstrate that it is feasible to follow the in vivo transport of unlabeled drugs within specific organ and tissue compartments on a platform that applies MALDI imaging mass spectrometry to tissue sections characterized with high definition histology. We have tracked and quantified the distribution of an inhaled reference compound, tiotropium, within the lungs of dosed rats, using systematic point by point MS and MS/MS sampling at 200 µm intervals. By comparing drug ion distribution patterns in adjacent tissue sections, we observed that within 15 min following exposure, tiotropium parent MS ions (mass-to-charge; m/z 392.1) and fragmented daughter MS/MS ions (m/z 170.1 and 152.1) were dispersed in a concentration gradient (80 fmol-5 pmol) away from the central airways into the lung parenchyma and pleura. These drug levels agreed well with amounts detected in lung compartments by chemical extraction. Moreover, the simultaneous global definition of molecular ion signatures localized within 2-D tissue space provides accurate assignment of ion identities within histological landmarks, providing context to dynamic biological processes occurring at sites of drug presence. Our results highlight an important emerging technology allowing specific high resolution identification of unlabeled drugs at sites of in vivo uptake and retention.

Internet-based cognitive behaviour therapy for obsessive–compulsive disorder: a randomized controlled trial

Background Cognitive behaviour therapy (CBT) is an effective treatment for obsessive–compulsive disorder (OCD) but access to CBT is limited. Internet-based CBT (ICBT) with therapist support is potentially a more accessible treatment. There are no randomized controlled trials testing ICBT for OCD. The aim of this study was to investigate the efficacy of ICBT for OCD in a randomized controlled trial. Method Participants (n=101) diagnosed with OCD were randomized to either 10 weeks of ICBT or to an attention control condition, consisting of online supportive therapy. The primary outcome measure was the Yale–Brown Obsessive Compulsive Scale (YBOCS) administered by blinded assessors. Results Both treatments lead to significant improvements in OCD symptoms, but ICBT resulted in larger improvements than the control condition on the YBOCS, with a significant between-group effect size (Cohens d) of 1.12 (95% CI 0.69–1.53) at post-treatment. The proportion of participants showing clinically significant improvement was 60% (95% CI 46–72) in the ICBT group compared to 6% (95% CI 1–17) in the control condition. The results were sustained at follow-up. Conclusions ICBT is an efficacious treatment for OCD that could substantially increase access to CBT for OCD patients. Replication studies are warranted.

Heat Stabilization of the Tissue Proteome: A New Technology for Improved Proteomics

After tissue or body fluid sampling, proteases and other protein-modifying enzymes can rapidly change composition of the proteome. As a direct consequence, analytical results will reflect a mix of in vivo proteome and ex vivo degradation products. Vital information about the presampling state may be destroyed or distorted, leading to variation between samples and incorrect conclusions. Sample stabilization and standardization of sample handling can reduce or eliminate this problem. Here, a novel tissue stabilization system which utilizes a combination of heat and pressure under vacuum was used to stop degradation in mouse brain tissue immediately after sampling. It was found by biochemical assays that enzymatic activity was reduced to background levels in stabilized samples. Western blot analysis confirmed that post-translational phosphorylations of analyzed proteins were stable and conserved for up to 2 h at room temperature and that peptide extracts were devoid of abundant protein degradation fragments. The combination of reduced complexity and proteolytic inactivation enabled mass spectrometric identification of several neuropeptides and endogenous peptides including modified species at higher levels compared to nonstabilized samples. The tissue stabilizing system ensures reproducible and rapid inactivation of enzymes. Therefore, the system provides a powerful improvement to proteomics by greatly reducing the complexity and dynamic range of the proteome in tissue samples and enables enhanced possibilities for discovery and analysis of clinically relevant protein/peptide biomarkers.

A neuroproteomic approach to targeting neuropeptides in the brain

A novel universal neuropeptide display approach in the mass range of 300–5000 Da was developed to complement two‐dimensional gel electrophoresis in the analysis of peptides and small proteins from brain tissue samples. For the analysis of neuropeptides we utilized on‐line nanoscale capillary reversed phase liquid chromatography and electrospray ionization quadrupole‐time of flight mass spectrometry. The method was employed for the analysis of a large number of peptides from three specific rat brain regions. Approximately 1500 peptides from each brain region were detected in the same analysis. Several of these peptides were sequenced using collision‐induced dissociation and identified by database search tools. In addition, a method for comparing peptide elution profiles between samples was developed, to provide two‐ and three‐dimensional computer graphics of the profiles and to pinpoint differences for statistical measurements. Among the characterized peptides were fragments from proteins such as hemoglobin, α‐synuclein, stathmin, cyclophilin, actin, NADH dehydrogenase, cytochrome c oxidase and prosomatostatin, as well as the bioactive neuropeptides VV‐hemorphin‐4, and LVV‐hemorphin‐7. The present study showed that the combination of nanoscale reversed phase liquid chromatography and high‐resolution tandem mass spectrometry provides a novel and powerful approach to investigate a large number of peptides and protein fragments in the brain.

Evidence for a role of the 5-HT1B receptor and its adaptor protein, p11, in l-DOPA treatment of an animal model of Parkinsonism

Parkinsons disease (PD) is characterized by a progressive degeneration of substantia nigra dopaminergic neurons projecting to the striatum. Restoration of dopamine transmission by l-DOPA relieves symptoms of PD but causes prominent side effects. There is a strong serotonin innervation of the striatum by serotonergic neurons that remains relatively preserved in PD. The study of this innervation has been largely neglected. Here, we demonstrate that chronic l-DOPA administration to 6-OHDA-lesioned rodents increases, via D1 receptors, the levels of the 5-HT1B receptor and its adaptor protein, p11, in dopamine-denervated striatonigral neurons. Using unilaterally 6-OHDA-lesioned p11 WT and KO mice, it was found that administration of a selective 5-HT1B receptor agonist, CP94253, inhibited l-DOPA-induced rotational behavior and abnormal involuntary movements in a p11-dependent manner. These data reveal an l-DOPA-induced negative-feedback mechanism, whereby the serotonin system may influence the symptomatology of Parkinsonism.

SwePep, a Database Designed for Endogenous Peptides and Mass Spectrometry

A new database, SwePep, specifically designed for endogenous peptides, has been constructed to significantly speed up the identification process from complex tissue samples utilizing mass spectrometry. In the identification process the experimental peptide masses are compared with the peptide masses stored in the database both with and without possible post-translational modifications. This intermediate identification step is fast and singles out peptides that are potential endogenous peptides and can later be confirmed with tandem mass spectrometry data. Successful applications of this methodology are presented. The SwePep database is a relational database developed using MySql and Java. The database contains 4180 annotated endogenous peptides from different tissues originating from 394 different species as well as 50 novel peptides from brain tissue identified in our laboratory. Information about the peptides, including mass, isoelectric point, sequence, and precursor protein, is also stored in the database. This new approach holds great potential for removing the bottleneck that occurs during the identification process in the field of peptidomics. The SwePep database is available to the public.

Novel mass spectrometry imaging software assisting labeled normalization and quantitation of drugs and neuropeptides directly in tissue sections

MALDI MS imaging has been extensively used to produce qualitative distribution maps of proteins, peptides, lipids, small molecule pharmaceuticals and their metabolites directly in biological tissue sections. There is growing demand to quantify the amount of target compounds in the tissue sections of different organs. We present a novel MS imaging software including protocol for the quantitation of drugs, and for the first time, an endogenous neuropeptide directly in tissue sections. After selecting regions of interest on the tissue section, data is read and processed by the software using several available methods for baseline corrections, subtractions, denoising, smoothing, recalibration and normalization. The concentrations of in vivo administered drugs or endogenous compounds are then determined semi-automatically using either external standard curves, or by using labeled compounds, i.e., isotope labeled analogs as standards. As model systems, we have quantified the distribution of imipramine and tiotropium in the brain and lung of dosed rats. Substance P was quantified in different mouse brain structures, which correlated well with previously reported peptide levels. Our approach facilitates quantitative data processing and labeled standards provide better reproducibility and may be considered as an efficient tool to quantify drugs and endogenous compounds in tissue regions of interest.

Development and Evaluation of Normalization Methods for Label-free Relative Quantification of Endogenous Peptides

The performances of 10 different normalization methods on data of endogenous brain peptides produced with label-free nano-LC-MS were evaluated. Data sets originating from three different species (mouse, rat, and Japanese quail), each consisting of 35–45 individual LC-MS analyses, were used in the study. Each sample set contained both technical and biological replicates, and the LC-MS analyses were performed in a randomized block fashion. Peptides in all three data sets were found to display LC-MS analysis order-dependent bias. Global normalization methods will only to some extent correct this type of bias. Only the novel normalization procedure RegrRun (linear regression followed by analysis order normalization) corrected for this type of bias. The RegrRun procedure performed the best of the normalization methods tested and decreased the median S.D. by 43% on average compared with raw data. This method also produced the smallest fraction of peptides with interblock differences while producing the largest fraction of differentially expressed peaks between treatment groups in all three data sets. Linear regression normalization (Regr) performed second best and decreased median S.D. by 38% on average compared with raw data. All other examined methods reduced median S.D. by 20–30% on average compared with raw data.

Qualitative and Quantitative MALDI Imaging of the Positron Emission Tomography Ligands Raclopride (a D2 Dopamine Antagonist) and SCH 23390 (a D1 Dopamine Antagonist) in Rat Brain Tissue Sections Using a Solvent-Free Dry Matrix Application Method

The distributions of positron emission tomography (PET) ligands in rat brain tissue sections were analyzed by matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI). The detection of the PET ligands was possible following the use of a solvent-free dry MALDI matrix application method employing finely ground dry α-cyano-4-hydroxycinnamic acid (CHCA). The D2 dopamine receptor antagonist 3,5-dichloro-N-{[(2S)-1-ethylpyrrolidin-2-yl]methyl}-2-hydroxy-6-methoxybenzamide (raclopride) and the D1 dopamine receptor antagonist 7-chloro-3-methyl-1-phenyl-1,2,4,5-tetrahydro-3-benzazepin-8-ol (SCH 23390) were both detected at decreasing abundance at increasing period postdosing. Confirmation of the compound identifications and distributions was achieved by a combination of mass-to-charge ratio accurate mass, isotope distribution, and MS/MS fragmentation imaging directly from tissue sections (performed using MALDI TOF/TOF, MALDI q-TOF, and 12T MALDI-FT-ICR mass spectrometers). Quantitative data was obtained by comparing signal abundances from tissues to those obtained from quantitation control spots of the target compound applied to adjacent vehicle control tissue sections (analyzed during the same experiment). Following a single intravenous dose of raclopride (7.5 mg/kg), an average tissue concentration of approximately 60 nM was detected compared to 15 nM when the drug was dosed at 2 mg/kg, indicating a linear response between dose and detected abundance. SCH 23390 was established to have an average tissue concentration of approximately 15 μM following a single intravenous dose at 5 mg/kg. Both target compounds were also detected in kidney tissue sections when employing the same MSI methodology. This study illustrates that a MSI may well be readily applied to PET ligand research development when using a solvent-free dry matrix coating.

Chiral separation of local anaesthetics by a capillary electrophoresis/partial filling technique coupled on-line to micro-electrospray mass spectrometry

A novel procedure for analysing chiral compounds is described in which a capillary electrophoresis/partial filling technique was coupled on-line to microelectrospray mass spectrometry. Enantiomers of bupivacaine and ropivacaine were separated using methyl-β-cyclodetrin, a non-volatile buffer additive, as a chiral selector. To avoid introduction of the selector into the ion source, the capillary was coated with polyacrylamide to minimize the electroosmotic flow. Prior to sample injection, the capillary was partially filled with cyclodextrin dissolved in acetate buffer. When 30 kV was applied, the positively charged enantiomers migrated towards the mass spectrometer through the zone containing the neutral selector, which acted as a pseudo-stationary phase. A racemic mixture of bupivacaine and ropivacaine was successfully separated and an impurity of 0.25% of (R)-ropivacaine in (S)-ropivacaine could be detected.