Differential compartmental processing and phosphorylation of pathogenic human tau and native mouse tau in the line 66 model of frontotemporal dementia.

Synapse loss is associated with motor and cognitive decline in multiple neurodegenerative disorders, and the cellular redistribution of tau is related to synaptic impairment in tauopathies, such as Alzheimer's disease and frontotemporal dementia. Here, we examined the cellular distribution of tau protein species in human tau overexpressing line 66 mice, a transgenic mouse model akin to genetic variants of frontotemporal dementia. Line 66 mice express intracellular tau aggregates in multiple brain regions and exhibit sensorimotor and motor learning deficiencies. Using a series of anti-tau antibodies, we observed, histologically, that nonphosphorylated transgenic human tau is enriched in synapses, whereas phosphorylated tau accumulates predominantly in cell bodies and axons. Subcellular fractionation confirmed that human tau is highly enriched in insoluble cytosolic and synaptosomal fractions, whereas endogenous mouse tau is virtually absent from synapses. Cytosolic tau was resistant to solubilization with urea and Triton X-100, indicating the formation of larger tau aggregates. By contrast, synaptic tau was partially soluble after Triton X-100 treatment and most likely represents aggregates of smaller size. MS corroborated that synaptosomal tau is nonphosphorylated. Tau enriched in the synapse of line 66 mice, therefore, appears to be in an oligomeric and nonphosphorylated state, and one that could have a direct impact on cognitive function.

Multiplex LA-ICP-MS bio-imaging of brain tissue of a parkinsonian mouse model stained with metal-coded affinity-tagged antibodies and coated with indium-spiked commercial inks as internal standards.

BACKGROUND: Immunohistochemistry techniques represent a powerful tool to detect and quantify disease related proteins. Improvements were accomplished by tagged antibodies using laser ablation and inductively coupled plasma mass spectrometry (LA-ICP-MS). However, these approaches are effected by day-to-variations due to instrumental drift. NEW METHOD: Brain tissue from line 62, a Parkinson's disease model, and control mice were incubated with four antibodies relevant to the disease and standardized to three house-keeping proteins. In addition, a new standardization approach was developed and the results compared. This new approach consisted of coating specimens with gelatin and printing an indium-doped ink with a commercial ink jet printer. Furthermore, the method was evaluated for different ablation spot sizes with respect to resolution and signal-to-noise ratio. RESULTS: Normalization using house-keeping proteins led to high background signals even at high resolution. Normalization using indium-doped ink improved the signal-to-noise ratio even when small laser spot sizes were used and further improved by overlaying tissue specimen with gelatin. COMPARISON WITH EXISTING METHODS: Line 62 mice had more α-Synuclein and gliosis but decreased numbers of neurons, as found by conventional immunohistochemistry. These data are in line with the results obtained by LA-ICP-MS with indium standardization. However, differences between L62 and controls for tyrosine hydroxylase were only detected by LA-ICP-MS. CONCLUSIONS: Internal standardisation using indium-doped inks is an effective method to overcome day-to-day variations and instrumental drifts. The new approach results in an increased signal-to-noise ratio and only under these conditions small but significant changes were detected, as seen for tyrosine hydroxylase.

Promoting medical competencies through a didactic tutor qualification programme - a qualitative study based on the CanMEDS Physician Competency Framework.

BACKGROUND: In peer-led tutorial courses, qualified medical students ("tutors") provide their peers with opportunities to deepen their theoretical knowledge effectively and to practice clinical skills already in preclinical semesters. At the Medical Faculty of Heidelberg University, a structured medical didactic qualification programme prepares and trains future tutors for their responsibilities. This programme consists of four modules: 1. medical didactics and group leadership, 2. subject-specific training, 3. performance of tutorial courses as well as 4. collegial advice and reflection on the tutors' activities. The aim of this study is to systematically analyse and present the development of role competencies for medical tutors based on the CanMEDS Physician Competency Framework through the didactic qualification programme. METHODS: We applied a qualitative research approach to detect CanMEDS role competencies acquisition within the tutor qualification programme. The CanMEDS framework describes key competencies, grouped thematically under seven professional roles. Two tutors and three training coordinators independently assigned the individual modules of the tutor qualification programme to the key competencies of the CanMEDS framework. Tutors and training coordinators compared and discussed the allocations within the groups in a consensus finding process. All authors analysed the findings in order to find out the so-called "hidden curriculum". The views of both groups are presented separately. RESULTS: The training programme promotes the acquisition of competencies in all seven CanMEDS roles. The roles of the scholar and the leader are promoted in all modules. In addition, the first and fourth module focus predominately on the role of the collaborator, the second on the role of the medical expert and communicator, and the fourth on the role of the professional. CONCLUSIONS: The systematic analysis through assignment of the CanMEDS roles to the individual modules of the tutor qualification programme documents the comprehensive acquisition of competencies, not only with regard to the tutor activity, but generally with regard to the later role of the physician. The reflection on one's own competency acquisition can support the promotion of corresponding competencies in the qualification programme and their transfer into the professional practice later.

Phosphorylation of the outer membrane mitochondrial protein OM64 influences protein import into mitochondria.

Mitochondrial localized proteins are mostly synthesized in the cytosol and translocated across the outer mitochondrial membrane via the translocase of the outer membrane (TOM) complex. Although the channel protein is conserved among eukaryotes, the receptor proteins are more divergent and show features specific to the plant lineage. OM64, which is a paralogue of the chloroplast docking protein Toc64, is unique to plants. However, due to the presence of a cytosolic exposed TPR domain it might functionally replace yeast/mammalian Tom70, which is not found in plant mitochondria, by interacting with the C-terminal (M)EEVD motif of the heat shock proteins Hsp90 and Hsp70. In this study, we show that OM64 is phosphorylated within its TPR domain. Using isothermal titration calorimetry it could be demonstrated that phosphorylation reduces the binding affinity of OM64 to Hsp90. Moreover, in vivo expression of genes encoding different OM64 variants in planta revealed that phosphorylation of OM64 impairs the import efficiency of the mitochondrial preprotein pFAD, a subunits of the mitochondrial ATP synthase. In summary, our data provide significant insight into the fine-tuning mechanisms of mitochondrial protein import mediated by phosphorylation of the cytosolic exposed receptor protein OM64.

Sex differences in murine myocardium are not exclusively regulated by gonadal hormones.

We investigated sex differences in cardiac protein patterns of intact and castrated mice using proteomics and 1D and 2D immunoblotting. To exclude differences concerning developmental aspects gonadectomy was conducted in mature mice at the age of three months. The main sex-related regulation in the protein pattern of the myocardium occurred for proteins involved in metabolic processes whereas only few proteins involved in other pathways underwent a regulation. Many regulated proteins (2/3) displayed a characteristic V form, which means that these proteins are up- or down-regulated in sexually mature compared to young mice and are back-regulated after castration, emphasizing a direct regulation by gonadal hormones. Several other spots (1/3) showed the same male/female regulation or a drastic increase in male/female spot intensity ratio after castration, suggesting either a regulation independent of sex hormones or a removal of an inhibiting feedback mechanism by gonadectomy. Technically, we found that it cannot be expected that a single spot contains only one protein species and that one protein is present in only one spot. We thus propose for proteomic investigations to identify/quantify all spots of a 2-DE pattern to obtain information about protein speciation and its potential importance for function and pathology. BIOLOGICAL SIGNIFICANCE: Sex related differences in cardiovascular disease, including risk factors, disease manifestation and outcomes, are far from being well understood, and improved biological understanding of these differences in the healthy myocardium is of great importance. We investigated sex related changes of myocardial protein pattern in intact and castrated mice at different ages and found metabolic proteins to be highly regulated, some of which independently from gonadal hormones.

Dual Internal Standards with Metals and Molecules for MALDI Imaging of Kidney Lipids.

The quest for internal standards useful in MALDI imaging studies goes on to get not only lateral distribution but also reliable relative quantitative information. We developed a method based on application of matrix and dual internal standards to allow intra- and intersample normalization of lipids intensities in kidney sections of control and cisplatin-treated Wistar rats. An inkjet printer was used to deposit a custom-prepared ink with DHB as MALDI matrix, a primary lipids-based internal standard, and a spiked lanthanide as a secondary internal standard. We applied different laser energy and varied the amounts of matrix-internal standards mixture to evaluate the normalization potential of the internal standards. Successful correction of intensity artifacts caused by instrumental drifts was possible, but not those resulting from uneven matrix application. ICP-MS absolute quantification of the lanthanide in the printed layer ensured the reproducibility of the matrix and internal standards application with RSD of 10-15%. Internal standard-normalized data allowed intrasample modification of the MALDI image to make it compatible with the optical image. Normalization to internal standards corrected a 2-fold difference in lipids intensity, which allowed a meaningful comparison of tissue lipids in control and cisplatin-treated kidneys. More importantly, normalization of lipid relative abundances based on the same adduct type (H+, Na+, and K+) for analyte and internal standard corrected for different ionization efficiencies showing a realistic signal level and enabling reliable comparison of different samples on relative quantitative basis.

Printing metal-spiked inks for LA-ICP-MS bioimaging internal standardization: comparison of the different nephrotoxic behavior of cisplatin, carboplatin, and oxaliplatin.

The study of the distribution of the cytostatic drugs cisplatin, carboplatin, and oxaliplatin along the kidney may help to understand their different nephrotoxic behavior. Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) allows the acquisition of trace element images in biological tissues. However, results obtained are affected by several variations concerning the sample matrix and instrumental drifts. In this work, an internal standardization method based on printing an Ir-spiked ink onto the surface of the sample has been developed to evaluate the different distributions and accumulation levels of the aforementioned drugs along the kidney of a rat model. A conventional ink-jet printer was used to print fresh sagittal kidney tissue slices of 4 µm. A reproducible and homogenous deposition of the ink along the tissue was observed. The ink was partially absorbed on top of the tissue. Thus, this approach provides a pseudo-internal standardization, due to the fact that the ablation sample and internal standard take place subsequently and not simultaneously. A satisfactory normalization of LA-ICP-MS bioimages and therefore a reliable comparison of the kidney treated with different Pt-based drugs were achieved even for tissues analyzed on different days. Due to the complete ablation of the sample, the transport of the ablated internal standard and tissue to the inductively coupled plasma-mass spectrometry (ICP-MS) is practically taking place at the same time. Pt accumulation in the kidney was observed in accordance to the dosages administered for each drug. Although the accumulation rate of cisplatin and oxaliplatin is high in both cases, their Pt distributions differ. The strong nephrotoxicity observed for cisplatin and the absence of such side effect in the case of oxaliplatin could explain these distribution differences. The homogeneous distribution of oxaliplatin in the cortical and medullar areas could be related with its higher affinity for cellular transporters such as MATE2-k.

Application of MeCAT-Click labeling for protein abundance characterization of E. coli after heat shock experiments.

In a proof of concept study, metal-coded affinity tags based on click chemistry (MeCAT-Click) were used to analyze the proteome of Escherichia coli (E. coli) in response to heat stress. This allows high labeling efficiency, high detection sensitivity, and multiplex capabilities, which are pivotal for its application to protein quantification. Two approaches are presented for relative quantification of differentially lanthanide-labeled proteins. The first approach uses isotope-labeling, where ESI-MS was utilized to quantify the differentially labeled proteins from different states of E. coli. With this approach, 14 proteins were found with changed abundance, among them five proteins upregulated. In the second approach, differentially labeled samples were separated by two dimensional gel electrophoresis (2-DE) and scanned by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). Comparison of the signal intensities of the different lanthanides was used to quantify different sample states. Based on this information, ESI-MS was used to identify the proteins with different abundance. The sensitivity of LA-ICP-MS allowed us to find one upregulated protein that was nearly invisible by silver staining ("Probable replication endonuclease from retron EC67"). The advantage of this approach is to locate low abundant proteins with differential expression using LA-ICP-MS, which may be overlooked otherwise. BIOLOGICAL SIGNIFICANCE: This paper demonstrates the successful application of a novel metal labeling strategy to quantify the proteins from complex biological samples. In comparison with former metal labeling strategies, it reduces the steric hindrance and improves the labeling efficiency during the labeling process, which ensure its successful application. This methodology is compatible with both molecular and elemental mass spectrometry. ESI-MS/MS in combination with software-based search allows the identification and relative quantification of labeled proteins. In addition, LA-ICP-MS helps to locate the labeled proteins in 2-DE gels with superior detection capability, thus, target proteins with low abundance can be precisely followed. Its excellent sensitivity allows one to track the proteins of interest that are barely visible by silver staining.

Introducing a curricular program culminating in a certificate for training peer tutors in medical education.

AIM: Student tutorials are now firmly anchored in medical education. However, to date there have only been isolated efforts to establish structured teacher training for peer tutors in medicine. To close this gap, a centralized tutor training program for students, culminating in an academic certificate, was implemented at Heidelberg University Medical School. The program also counts within the scope of the post-graduate Baden-Württemberg Certificate in Academic Teaching (Baden-Württemberg Zertifikat für Hochschuldidaktik). METHOD: Based on a needs assessment, a modular program comprised of four modules and a total of 200 curricular units was developed in cooperation with the Department for Key Competencies and Higher Education at Heidelberg University and implemented during the 2010 summer semester. This program covers not only topic-specific training sessions, but also independent teaching and an integrated evaluation of the learning process that is communicated to the graduates in the form of structured feedback. In addition, to evaluate the overall concept, semi-structured interviews (N=18) were conducted with the program graduates. RESULTS: To date, 495 tutors have been trained in the basic module on teaching medicine, which is rated with a mean overall grade of 1.7 (SW: 0.6) and has served as Module I of the program since 2010. A total of 17% (N=83) of these tutors have gone on to enroll in the subsequent training modules of the program; 27 of them (m=12, f=15) have already successfully completed them. Based on qualitative analyses, it is evident that the training program certificate and its applicability toward the advanced teacher training for university instructors pose a major incentive for the graduates. For successful program realization, central coordination, extensive coordination within the medical school, and the evaluation of the attained skills have proven to be of particular importance. CONCLUSION: The training program contributes sustainably to both quality assurance and professionalism, as well as to solving the issue of resources in medical education. The introduction and continued development of similar programs is desirable.

Site-selective azide incorporation into endogenous RNase A via a "chemistry" approach.

Site-selective labeling of endogenous proteins represents a major challenge in chemical biology, mainly due to the absence of unique reactive groups that can be addressed selectively. Recently, we have shown that surface-exposed lysine residues of two endogenous proteins and a peptide exhibit subtle changes in their individual reactivities. This feature allows the modification of a single residue in a highly site-selective fashion if kinetically controlled labeling conditions are applied. In order to broaden the scope of the "kinetically-controlled protein labeling" (KPL) approach and highlight additional applications, the water-soluble bioorthogonal reagent, biotin-TEO-azido-NHS (11), is developed which enables the site-selective introduction of an azido group onto endogenous proteins/peptides. This bioconjugation reagent features a biotin tag for affinity purification, an azido group for bioorthogonal labeling, a TEO (tetraethylene oxide) linker acting as a spacer and to impart water solubility and an N-hydroxysuccinimidyl (NHS) ester group for reacting with the exposed lysine residue. As a proof of concept, the native protein ribonuclease A (RNase A) bearing ten available lysine residues at the surface is furnished with a single azido group at Lys 1 in a highly site-selective fashion yielding azido-(K1)RNase A. The K1 site-selectivity is demonstrated by the combined application and interpretation of high resolution MALDI-ToF mass spectroscopy, tandem mass spectroscopy and extracted ion chromatography (XIC). Finally, the water soluble azide-reactive phosphine probe, rho-TEO-phosphine (21) (rho: rhodamine), has been designed and applied to attach a chromophore to azido-(K1)RNase A via Staudinger ligation at physiological pH indicating that the introduced azido group is accessible and could be addressed by other established azide-reactive bioorthogonal reaction schemes.

Monitoring CSF proteome alterations in amyotrophic lateral sclerosis: obstacles and perspectives in translating a novel marker panel to the clinic.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a fatal disorder of the motor neuron system with poor prognosis and marginal therapeutic options. Current clinical diagnostic criteria are based on electrophysiological examination and exclusion of other ALS-mimicking conditions. Neuroprotective treatments are, however, most promising in early disease stages. Identification of disease-specific CSF biomarkers and associated biochemical pathways is therefore most relevant to monitor disease progression, response to neuroprotective agents and to enable early inclusion of patients into clinical trials. METHODS AND FINDINGS: CSF from 35 patients with ALS diagnosed according to the revised El Escorial criteria and 23 age-matched controls was processed using paramagnetic bead chromatography for protein isolation and subsequently analyzed by MALDI-TOF mass spectrometry. CSF protein profiles were integrated into a Random Forest model constructed from 153 mass peaks. After reducing this peak set to the top 25%, a classifier was built which enabled prediction of ALS with high accuracy, sensitivity and specificity. Further analysis of the identified peptides resulted in a panel of five highly sensitive ALS biomarkers. Upregulation of secreted phosphoprotein 1 in ALS-CSF samples was confirmed by univariate analysis of ELISA and mass spectrometry data. Further quantitative validation of the five biomarkers was achieved in an 80-plex Multiple Reaction Monitoring mass spectrometry assay. CONCLUSIONS: ALS classification based on the CSF biomarker panel proposed in this study could become a valuable predictive tool for early clinical risk stratification. Of the numerous CSF proteins identified, many have putative roles in ALS-related metabolic processes, particularly in chromogranin-mediated secretion signaling pathways. While a stand-alone clinical application of this classifier will only be possible after further validation and a multicenter trial, it could be readily used to complement current ALS diagnostics and might also provide new insights into the pathomechanisms of this disease in the future.

Site-selective lysine modification of native proteins and peptides via kinetically controlled labeling.

The site-selective modification of the proteins RNase A, lysozyme C, and the peptide hormone somatostatin is presented via a kinetically controlled labeling approach. A single lysine residue on the surface of these biomolecules reacts with an activated biotinylation reagent at mild conditions, physiological pH, and at RT in a high yield of over 90%. In addition, fast reaction speed, quick and easy purification, as well as low reaction temperatures are particularly attractive for labeling sensitive peptides and proteins. Furthermore, the multifunctional bioorthogonal bioconjugation reagent (19) has been achieved allowing the site-selective incorporation of a single ethynyl group. The introduced ethynyl group is accessible for, e.g., click chemistry as demonstrated by the reaction of RNase A with azidocoumarin. The approach reported herein is fast, less labor-intensive and minimizes the risk for protein misfolding. Kinetically controlled labeling offers a high potential for addressing a broad range of native proteins and peptides in a site-selective fashion and complements the portfolio of recombinant techniques or chemoenzymatic approaches.

Dietary phytoestrogen supplementation induces sex differences in the myocardial protein pattern of mice: a comparative proteomics study.

Elevated cardiovascular risk in postmenopausal women and beneficial actions of estrogen replacement in animal models have been related to protective effects of estrogens. However, randomized trials of hormone replacement therapy with synthetic estrogens in humans failed confirmation and phytoestrogens, natural plant hormones with agonistic properties for estrogen receptors, could represent potential alternatives. The aim of the present study is to characterize an animal model for alternative hormone replacement with genistein as a natural estrogenic compound. We performed a 2-DE/ESI-LC-MS approach in order to identify protein species varying with genistein receipt and sex in their relative abundance in the healthy murine heart (http://www.mpiib-berlin.mpg.de/2D-PAGE). Oral genistein treatment revealed a substantial effect on the relative abundance of both estrogen receptors. Several enzymes of the fatty acid metabolism and their transcriptional regulators varied differentially in male and in female animals, at the transcript and/or the protein species level. Increased levels of enzyme species involved in the oxidative phosphorylation and generation of ROS were accompanied by decreased amounts of antioxidants in male mice receiving genistein compared with control males, which have been previously associated with various pathological conditions. Exposure of female animals to genistein provoked an increased abundance of two species of LIM domain-binding protein and one species of desmin. These proteins have been associated with cardiac hypertrophy and our data warrant caution for the use of them as molecular markers, since the animals did not exhibit any histological signs of cardiac hypertrophy.

Adaptation of proteomic techniques for the identification and characterization of protein species from murine heart.

Disturbed energy metabolism with impaired fatty acid oxidation, ATP synthesis and changing levels of contractile proteins has been observed during the development and manifestation of cardiovascular diseases, with the latter showing sexual differences in terms of onset, manifestation and progress. Estrogenic compounds, such as estrogens and phytoestrogens, are known to exert beneficial effects on several cardiovascular parameters. However, global studies implying the normal, non-failing myocardium are rare. Thus, identifying and characterizing protein patterns involved in the maintenance of normal heart physiology at the protein species level will help understanding disease conditions. In this study, we performed an adapted 2-DE/MS approach in order to identify and characterize post-translational modified and truncated protein species from murine heart. Female and male animals of different age were receiving the phytoestrogen genistein and comparative analyses were performed to identify sex and genistein treatment-related effects. Selected 2-DE spots that exposed varying abundance between animal groups and identified as identical proteins were subject to multi-protease cleavage to generate an elevated sequence coverage enabling characterization of post-translational modifications and truncation loci via high-resolution MS. Several truncated, phosphorylated and acetylated species were identified for mitochondrial ATP synthase, malate dehydrogenase and trifunctional enzyme subunit alpha. However, confirmation of several of these modifications by manual spectra interpretation failed. Thus, our results warrant caution for the blind trust in software output. For the regulatory light chain of myosin, we identified an N-terminal processed species, which so far has been related to ischemic conditions only. We tried to unravel the information content of protein species separated by high-resolution 2-DE as an alternative to high-throughput proteomics, which mainly is interested in lists of protein names, ignoring the protein species identity.

Metal-coded affinity tag labeling: a demonstration of analytical robustness and suitability for biological applications.

Quantitative peptide and protein analysis is one of the most promising fields in modern life science. Besides stable isotope coded labeling, metal chelate complexes are an alternative tool for quantification. The development of metal-coded affinity tags (MeCAT) was aimed to provide a robust tool for the quantification of peptides and proteins by utilizing lanthanide-harboring metal tags. It was shown that MeCAT is suited for relative quantification of proteins via standard mass spectrometric methods. The approach of tagging biomolecules with MeCAT offers the unique advantage of absolute quantification via inductively coupled plasma mass spectrometry (ICPMS), a well-established technique for assessing concentrations down to low attomole ranges. This work investigates the compatibility of MeCAT labeling to analysis workflows such as nano liquid chromatography/electrospray ionization tandem mass spectrometry (nano-LC/ESI-MS(n)). Focus was given toward the separation behavior of labeled peptides and the dynamic range of detection and peptide charge distribution. Furthermore, the stability of MeCAT under harsh analytical conditions was investigated. With the application of the MeCAT technique to a standard analysis scheme in proteomics, such as the investigation of changes in an Escherichia coli proteome, we successfully addressed the suitability to utilize MeCAT on biological samples. Furthermore, we demonstrated that MeCAT complexes are stable under a variety of conditions and that by applying LC/ESI-MS it is possible to cover a dynamic range of 2 orders of magnitude down to the low femtomole range with an average standard deviation below 15%. Therefore, this technique is suitable to common proteomic workflows and enables relative as well as absolute differential peptide quantification.