Martin Schuerenberg

A Novel Lipidomic Strategy Reveals Plasma Phospholipid Signatures Associated with Respiratory Disease Severity in Cystic Fibrosis Patients

The aim of this study was to search for lipid signatures in blood plasma from cystic fibrosis (CF) patients using a novel MALDI-TOF-ClinProTools™ strategy, initially developed for protein analysis, and thin layer chromatography coupled to MALDI-TOF (TLC-MALDI). Samples from 33 CF patients and 18 healthy children were subjected to organic extraction and column chromatography separation of lipid classes. Extracts were analyzed by MALDI-TOF, ion signatures were compared by the ClinProTools™ software and by parallel statistical analyses. Relevant peaks were identified by LC-MSn. The ensemble of analyses provided 11 and 4 peaks differentially displayed in CF vs healthy and in mild vs severe patients respectively. Ten ions were significantly decreased in all patients, corresponding to 4 lysophosphatidylcholine (18∶0, 18∶2, 20∶3, and 20∶5) and 6 phosphatidylcholine (36∶5, O-38∶0, 38∶4, 38∶5, 38∶6, and P-40∶1) species. One sphingolipid, SM(d18∶0), was significantly increased in all patients. Four PC forms (36∶3, 36∶5, 38∶5, and 38∶6) were consistently downregulated in severe vs mild patients. These observations were confirmed by TLC-MALDI. These results suggest that plasma phospholipid signatures may be able to discriminate mild and severe forms of CF, and show for the first time MALDI-TOF-ClinProTools™ as a suitable methodology for the search of lipid markers in CF.

High throughput, high specificity capture and high resolution detection of beta-amyloid fragments using TOF mass spectrometry

regions of normal human brain and in brain pathologies such as Alzheimer diseased brain. Immunohistochemical staining with anti-Abeta-peptide monoclonal antibody revealed the amyloid plaques, that are intensely stained with this antibody. Interestingly, staining a consecutive serial section with the anti-PATE-M antibodies clearly showed colocalization of PATE-M protein to these plaques . Conclusions: In conclusion: it is known that neuronal nicotinic acetylcholine receptors are found throughout the nervous system and participate in the pathophysiology of Alzheimer’s disease. We have demonstrated that certain PATE-like proteins modulate the activity of nicotinic acetylcholine receptors, moreover, PATE-M protein is generated at reduced levels in Alzheimer-diseased brains and that it colocalizes to Alzheimer-related amyloid plaques, taken together all implicate involvement of the PATE-M protein in the pathophysiology of Alzheimer’s disease.

ImmunoImaging: Tissue-less tissue imaging and simplification of the tissue proteome with a simple sample preparation protocol

Background: MALDI tissue analysis enables correlation of molecular spectra with morphological and clinical features. However, peptide and protein imaging is an untargeted approach that enables discovery, but falls short in providing information about targeted molecules of interest. The crude sample is extremely complex and lower-abundance/low-hydrophilicity species such as Beta-amyloid are difficult to find. We describe here a hyphenated approach that combines immunological sample enrichment and MALDI imaging: ImmunoImaging. MALDI imaging becomes a targeted analysis tool preserving spatial abundance patterns, providing molecular information about the recognized proteins and the distribution of highly related antigens such as the set of truncation variants of the Beta-amyloid protein information that goes undetected with all current targeted tissue analysis methods (FISH, IHC). Methods: Biological species were transferred from fresh tissue onto reactive surfaces with pre-coupled 6E10 mouse-monoclonal anti-Beta-amyloid antibody. The selective profiles were used to recreate a targeted molecular image of the tissue. Animal control brain tissue injected with a mixture of Beta-amyloid calibrants was compared against the same type of control tissue. Animal model brain tissue was also investigated. Samples were investigated on a Bruker Ultraflex III MALDI TOF/TOF instrument. Results: ImmunoImaging is shown to give sensitive molecular detection with retention of spatial orientation of biological species transferred from a fresh tissue onto a selective binding surface. ImmunoImaging allowed targeting of the Beta-amyloid material within a sample simultaneous concentration and clean-up of the peptides of interest. Use of an epitope which targeted multiple peptide species simultaneously allowed the investigation not only of the total amount of Beta-amyloid in a given location, but also simultaneous investigation into the location of all reacting Beta-amyloid peptides. Conclusions: Investigation of animal model samples show direct marker profiling from a fresh tissue as proof-of-principle for spatially resolving Alzheimer’s features within sections of the brain. All Beta-amyloid peptides responding to the antibody epitope may simultaneously be monitored, taking advantage of the epitope cross reactivity. This allows sensitive investigation into whether Beta-amyloid peptides co-occur, or occur in different locations throughout the brain. This is a tool that may lead to further understanding of the interaction of different peptide species at different locations within the brain.

Differential expression of beta-amyloid fragments in crude brain lysate samples using immunoassay TOF mass spectrometry

Background: Beta amyloid fragment deposits in the brain are associated with the onset of neurodegenerative diseases, including Alzheimer’s disease. The use of TOF mass spectrometry in measuring these fragments has the advantage of eliminating cross-reactivity errors associated with other methods such as ELISA, and allows for simultaneous detection of numerous fragments from any sample. Methods: Immuno-specific surfaces were prepared using ProteinChip array functionalized with amino groups. These reactive surfaces were used to immobilize 6E10 monoclonal antibody, specific to N-terminus beta-amyloid fragments. Negative control surfaces were prepared by using non-specific bovine IgG in place of 6E10 on the same reactive surfaces. Artificial CSF was used to prepare calibration material and internal standard. Fragments 1-33, 1-38, 1-40, and 1-42 were spiked across a range of concentration to prepare calibration material. Non-naturally occurring fragment Cys 1-24 was selected to prepare internal standard, as it is recognized by 6E10 Mab. Six human brain frontal cortex samples were analyzed, three from Alzheimer’s diagnosed and three from non Alzheimer’s sources. These samples were lysed in the presence of TUC, DTT, and protease inhibitors. The lysed samples were diluted in artificial CSF and applied directly to both 6E10 and Bovine IgG immobilized surfaces. After wash and desalting steps, alpha-cyano-4-hydroxycinnamic acid was applied and the targets were assayed in a Bruker ultrafleXtreme mass spectrometer. Results: Differential expression of Beta Amyloid fragments was observed. In known Alzheimer’s frontal brain cortex lysate multiple fragments were detected on the 6E10 surface and not on the control Bovine IgG surface. The non-Alzheimers frontal brain cortex lysate samples showed no beta-amyloid fragments on either surface type. Quantitative results were reported using calibration generated in artificial CSF. The use of an internal standard was shown to help minimize variability and improve results. The use of pre-coupled antibody arrays further reduces assay time and increases assay reproducibility. Conclusions: TOF mass spectrometry has been used to successfully develop a method for the detection of beta-amyloid fragments from human brain tissue. Quantitative results are obtained using calibration materials and internal standard. Multiple fragments are simultaneously monitored and differences are detected in diseased vs. normal brains.