Karl Sköld

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.

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.

Neuropeptidomics: expanding proteomics downwards.

Biological function is mainly carried out by a dynamic population of proteins and peptides which may be used as markers for disease diagnosis, prognosis and as a guide for effective treatment. The study of proteins is called proteomics and it is generally performed by two-dimensional gel electrophoresis and mass spectrometric methods. However, gel-based proteomics is methodologically restricted from the low mass region, which includes important endogenous peptides. The study of endogenous peptides, peptidomics, is complicated by protein fragments produced post-mortem during conventional sample handling. Nanoflow liquid chromatography and MS, together with improved methods for sample preparation, have been used to semi-quantitatively monitor endogenous peptides in brain tissue. When rapidly heat-denatured brain tissue was analysed, these methods enabled simultaneous detection of hundreds of peptides and the identification of several endogenous peptides not previously described in the literature. In an application of the MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) model for Parkinsons disease, the expression of the small protein PEP-19 was compared with controls. The levels were found to be significantly decreased in the striatum of MPTP-treated animals.

Striatal Proteomic Analysis Suggests that First L-Dopa Dose Equates to Chronic Exposure

L-3,4-dihydroxypheylalanine (L-dopa)-induced dyskinesia represent a debilitating complication of therapy for Parkinsons disease (PD) that result from a progressive sensitization through repeated L-dopa exposures. The MPTP macaque model was used to study the proteome in dopamine-depleted striatum with and without subsequent acute and chronic L-dopa treatment using two-dimensional difference in-gel electrophoresis (2D-DIGE) and mass spectrometry. The present data suggest that the dopamine-depleted striatum is so sensitive to de novo L-dopa treatment that the first ever administration alone would be able (i) to induce rapid post-translational modification-based proteomic changes that are specific to this first exposure and (ii), possibly, lead to irreversible protein level changes that would be not further modified by chronic L-dopa treatment. The apparent equivalence between first and chronic L-dopa administration suggests that priming would be the direct consequence of dopamine loss, the first L-dopa administrations only exacerbating the sensitization process but not inducing it.

Neuropeptidomics Strategies for Specific and Sensitive Identification of Endogenous Peptides

A new approach using targeted sequence collections has been developed for identifying endogenous peptides. This approach enables a fast, specific, and sensitive identification of endogenous peptides. Three different sequence collections were constituted in this study to mimic the peptidomic samples: SwePep precursors, SwePep peptides, and SwePep predicted. The searches for neuropeptides performed against these three sequence collections were compared with searches performed against the entire mouse proteome, which is commonly used to identify neuropeptides. These four sequence collections were searched with both Mascot and X! Tandem. Evaluation of the sequence collections was achieved using a set of manually identified and previously verified peptides. By using the three new sequence collections, which more accurately mimic the sample, 3 times as many peptides were significantly identified, with a false-positive rate below 1%, in comparison with the mouse proteome. The new sequence collections were also used to identify previously uncharacterized peptides from brain tissue; 27 previously uncharacterized peptides and potentially bioactive neuropeptides were identified. These novel peptides are cleaved from the peptide precursors at sites that are characteristic for prohormone convertases, and some of them have post-translational modifications that are characteristic for neuropeptides. The targeted protein sequence collections for different species are publicly available for download from SwePep.

Striatal Alterations of Secretogranin-1, Somatostatin, Prodynorphin, and Cholecystokinin Peptides in an Experimental Mouse Model of Parkinson Disease

The principal causative pathology of Parkinson disease is the progressive degeneration of dopaminergic neurons in the substantia nigra pars compacta projecting to the striatum in the brain. The information regarding the expression of neuropeptides in parkinsonism is very limited. Here we have elucidated striatal neuropeptide mechanisms in experimental parkinsonism using the unilateral 6-hydroxydopamine model to degenerate dopamine neurons. A thoroughly controlled sample preparation technique together with a peptidomics approach and targeted neuropeptide sequence collections enabled sensitive detection, identification, and relative quantitation of a great number of endogenous neuropeptides. Previously not recognized alterations in neuropeptide levels were identified in the unilateral lesioned mice with or without subchronic 3,4-dihydroxy-l-phenylalanine administration, the conventional treatment of Parkinson disease. Several of these peptides originated from the same precursor such as secretogranin-1, somatostatin, prodynorphin, and cholecystokinin. Disease-related biotransformation of precursors into individual peptides was observed in the experimental model of Parkinson disease. Several previously unreported potentially biologically active peptides were also identified from the striatal samples. This study provides further evidence that neuropeptides take part in mediating the central nervous system failure associated with Parkinson disease.

Impact of Temperature Dependent Sampling Procedures in Proteomics and Peptidomics – A Characterization of the Liver and Pancreas Post Mortem Degradome

Little is known about the nature of post mortem degradation of proteins and peptides on a global level, the so-called degradome. This is especially true for nonneural tissues. Degradome properties in relation to sampling procedures on different tissues are of great importance for the studies of, for instance, post translational modifications and/or the establishment of clinical biobanks. Here, snap freezing of fresh (<2 min post mortem time) mouse liver and pancreas tissue is compared with rapid heat stabilization with regard to effects on the proteome (using two-dimensional differential in-gel electrophoresis) and peptidome (using label free liquid chromatography). We report several proteins and peptides that exhibit heightened degradation sensitivity, for instance superoxide dismutase in liver, and peptidyl-prolyl cis-trans isomerase and insulin C-peptides in pancreas. Tissue sampling based on snap freezing produces a greater amount of degradation products and lower levels of endogenous peptides than rapid heat stabilization. We also demonstrate that solely snap freezing related degradation can be attenuated by subsequent heat stabilization. We conclude that tissue sampling involving a rapid heat stabilization step is preferable to freezing with regard to proteomic and peptidomic sample quality.

The impact of biosampling procedures on molecular data interpretation

The separation between biological and technical variation without extensive use of technical replicates is often challenging, particularly in the context of different forms of protein and peptide modifications. Biosampling procedures in the research laboratory are easier to conduct within a shorter time frame and under controlled conditions as compared with clinical sampling, with the latter often having issues of reproducibility. But is the research laboratory biosampling really less variable? Biosampling introduces within minutes rapid tissue-specific changes in the cellular microenvironment, thus inducing a range of different pathways associated with cell survival. Biosampling involves hypoxia and, depending on the circumstances, hypothermia, circumstances for which there are evolutionarily conserved defense strategies in the range of species and also are relevant for the range of biomedical conditions. It remains unclear to what extent such adaptive processes are reflected in different biosampling procedures or how important they are for the definition of sample quality. Lately, an increasing number of comparative studies on different biosampling approaches, post-mortem effects and pre-sampling biological state, have investigated such immediate early biosampling effects. Commonalities between biosampling effects and a range of ischemia/reperfusion- and hypometabolism/anoxia-associated biological phenomena indicate that even small variations in post-sampling time intervals are likely to introduce a set of nonrandom and tissue-specific effects of experimental importance (both in vivo and in vitro). This review integrates the information provided by these comparative studies and discusses how an adaptive biological perspective in biosampling procedures may be relevant for sample quality issues.

Biomarkers of disease and post-mortem changes - Heat stabilization, a necessary tool for measurement of protein regulation.

This review focuses on post sampling changes and how the Stabilizor system has been used to control this natural biological process and potential implications on cancer-specific biomarkers due to post sampling changes. Tissue sampling is a major traumatic event that can have drastic effects within a very short timeframe at the molecular level [1] resulting in loss of sample quality due to post-mortem changes. A heat-stabilization technology, using the Stabilizor system, has been developed to quickly and permanently abolish the enzymatic activity that causes these changes post-sampling and so preserve sample quality. The Stabilizor system has been shown to give better sample quality when analyzing a variety of tissues in various proteomic workflows. In this paper we discuss the impact of using heat-stabilized tissue in different proteomic applications. Based on our observations regarding the overlap between commonly changing proteins and proteins found to change post-mortem we also highlight a group of proteins of particular interest in cancer studies.