Mass spectrometric and immunological proteome analysis and method development
We developed a workflow and an automated workstation for proteome fractionation, analysis and biomarker search in body fluids in collaboration with Analytik Jena. This method combines three orthogonal native chromatographic separation steps (size exclusion, followed by anion exchange and lectin affinity chromatography). As a result more than 4100 sub-fractions per sample are produced within 384-well microplates. These sub-fractions are analyzed by mass spectrometry, immunoassays, enzymatically, or by spectrophotometric readout. Besides automation and high precision, this method has several advantages: high proteome coverage, flexible dynamic range with respect to molecular weight and sample amount, optional enzymatic and immunological analytics additional to mass spectrometry. Without exception, all intrinsic components and information are preserved after fractionation, including natural complex formation, fragmentation, and biological activities. Such information is beneficial for comprehensive profiling, for biomarker search, as well as for efficient evaluation. Applications show versatility with profiling plasma proteomes of humans, cattle, goat and mouse, and human cerebrospinal fluid (CSF). E.g., with several human samples about 5000 and 3500 non-redundant proteins could be identified in blood plasma and CSF, respectively. Applying this method to biomarker search yields candidate markers that have already been validated in sepsis, Alport syndrome, and psoriasis. Further applications to acute respiratory infection and to the neurodegenerative disease ALS identifies huge numbers of altered proteins in CSF, respectively, which may present promising candidates for evaluation.
Background, applied tools, and some recent results are shown in a presentation (HUPO2013, Yokohama), a Video (Automated proteome fractionation), and in a open access book chapter (https://www.novapublishers.com/catalog/product_info.php?products_id=41176).
Development of tools, methods, and strategies for sample preparation
To improve reproducibility and throughput as well as applicability various tools are under development for automatized fractionation (column arrays, robots), for sample preparation (micro reactors for cell culture, medium exchange, desalting, tryptic digest, concentration), and comprehensive data analysis to improve reproducibility, throughput and practicability. Here we collaborate with the Central Scientific Workshop of the University Hospital and several regional companies. For process control and all-round analytics, a new software tool was developed accompanying the whole fractionation process.
All available tools are applied to proteomic biomarker search. To this, up to four samples from patients and controls are parallel fractionated; all homologeous fractions are compared spectrophotometrically, probably selected, and further analyzed. This workflow has been applied to biomarker search for various nephropathies, Psoriatric arthritis, neurodegenerative diseases, and several infectious diseases. We are looking forward to suggestions and new scientific questions from clinical partners.