Mass spectrometry in clinical protein biomarker discovery
Year: 2014
Type: dissertation
Abstract: The field of biological sciences has expanded enormously within the last few decades. Developments in techniques and instrumentation have allowed biologist to explore biological mechanisms in an unprecedented detail. One of the most evolved disciplines is the field of proteomics. In general, proteins function in many different biological roles. They serve as structural molecules, in signaling routes mediating information in the cell, in intra- and extracellular transport and trafficking as well as in numerous other cellular functions. The area of protein research entails the study of all things relating to proteins and their functions. These include cellular protein composition, expression changes, protein structure, post-translational modifications and protein-protein interactions. Mass spectrometry (MS) has become one of the key technologies in proteomic research. The relative ease of sample handling and automated MS machinery has made proteomic analysis relatively straightforward. Mass spectrometers work by measuring the weight of intact proteins or protein-derived peptides. Proteomic MS identification is usually done by fragmenting the proteins or peptides in the mass spectrometer and using the resulting mass spectral information in identification of peptide sequence. There are two main strategies of peptide sequence identification: database dependent and de novo identification. Database dependent algorithms utilize known sequence information stored in databases to decipher the peptide amino acid sequence of the MS-observed spectra and use that information to predict the protein from which the peptide is derived from. On the other hand de novo methods try to construct the peptide sequence solely based on the fragmentation patterns of the peptide. The completeness of sequence databases of many species and the speed and efficiency of the search engines have made the database dependent search as the main method in peptide and protein identification. The modern high resolution mass spectrometers along with ultra-performance liquid chromatography have enabled the detection of thousands of protein in one single MS run. This, together with advances in MS-based protein quantification has extended the use of mass spectrometers in discovery type biomarker search. Mass spectrometers are able to produce a large amount of data on numerous proteins that can be used to detect and quantify differences in patient and control samples. This in turn can be used as starting point for more focused validation studies on the acquired data and ultimately lead to useful clinical biomarkers. The focus of this study was to utilize and learn mass spectrometric methodologies and to analyze different proteomic processes in sample types. We analyzed the protein-protein interactions in Baker´s yeast PSA1 protein in various points of batch cultivation using database dependent and de novo protein identification methods. We showed that the interactome of PSA1 is very dynamic depending on the phase of the cultivation. We also showed the limitations and benefits of de novo identification and the combined use of both search strategies in improving the confidence of the identifications. In another study using affinity purification and mass spectrometry we identified Fibrillin-2 as the binding partner of lung cancer associated Gremlin-1 protein. This finding elucidates functions and mechanisms of Gremlin-1 and Fibrillin-2 in malignant tissues. In two mass spectrometry-based protein quantification studies we characterized the protein concentration changes in human plasma during liver transplantation surgery as well as the effect of excess sialic acid production in HEK293 model cell line. In the liver transplantation plasma project we identified protein concentration changes in liver in response to the trauma caused by the surgery using label-based iTRAQ method. We showed consumption and secretion of several coagulation related proteins within the liver suggesting activation of coagulation cascade in the very early phases of the craft reperfusion. In the study of excess sialic acid production we first verified the amounts of sialic acid using mass spectrometry-based multiple reaction monitoring method. We were able to induce the production of sialic acid to almost 70-fold compared to control cells. We also monitored the protein abundance changes in sialic acid producing cells using label free proteins quantification method identifying 105 changed proteins. We analyzed those proteins with several functional enrichment tools revealing modifications in cellular protein transport, metabolic and signaling pathways and in remodeling of cellular adherens junctions. Such large scale MS-analyses using ontology-based tools can significantly aid in deciphering the effect of perturbations to complex systems but also reveal novel functional targets for biomarker discovery. The results obtained from targeted interaction experiments as well as large scale quantification studies can be used as basis for more rigorous investigations on the various subjects in search for potential biomarkers for clinical use. The techniques and methods used in the studies also demonstrate the many uses of mass spectrometric techniques in several fields of proteomic and biological research.
Source: Työväentutkimus Vuosikirja
Author: Ville Parviainen
Language: English
Subfield: Spectroscopy
Field: Chemistry
Domain: Physical Sciences
Open Access status: green
OpenAlex ID: W1168079072