Homology-Based Functional Proteomics By Mass Spectrometry and Advanced Informatic Methods

• Main Author: Liska, Adam J.
• Other Authors: Technische Universität Dresden, Mathematik und Naturwissenschaften, Biologie, Max-Planck-Instituts für molekulare Zellbiologie und Genetik
• Format: Doctoral Thesis
• Language: English
• Published: Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden 2003
• Subjects: Biochemie; Massenspektrometrie; Proteomik; BLAST; Database Searching; Dunaliella salina; EST; Mass Spectrometry; Microtubule-Associated Proteins; Peptides; Protein Sequence Database; Proteins; Proteomics; Salt Stress Induced Proteins; Sequence-Similarity; Xenopus laevis; ddc:570; rvk:WC 4460; rvk:WD 5085; Datenbank; MAPs; Peptide; Proteine / s.Aminosäurensequenz; Proteomanalyse
• Online Access: http://nbn-resolving.de/urn:nbn:de:swb:14-1071757497859-43887; http://nbn-resolving.de/urn:nbn:de:swb:14-1071757497859-43887; http://www.qucosa.de/fileadmin/data/qucosa/documents/1092/1071757497859-4388.pdf

Functional characterization of biochemically-isolated proteins is a central task in the biochemical and genetic description of the biology of cells and tissues. Protein identification by mass spectrometry consists of associating an isolated protein with a specific gene or protein sequence in silico, thus inferring its specific biochemical function based upon previous characterizations of that protein or a similar protein having that sequence identity. By performing this analysis on a large scale in conjunction with biochemical experiments, novel biological knowledge can be developed. The study presented here focuses on mass spectrometry-based proteomics of organisms with unsequenced genomes and corresponding developments in biological sequence database searching with mass spectrometry data. Conventional methods to identify proteins by mass spectrometry analysis have employed proteolytic digestion, fragmentation of resultant peptides, and the correlation of acquired tandem mass spectra with database sequences, relying upon exact matching algorithms; i.e. the analyzed peptide had to previously exist in a database in silico to be identified. One existing sequence-similarity protein identification method was applied (MS BLAST, Shevchenko 2001) and one alternative novel method was developed (MultiTag), for searching protein and EST databases, to enable the recognition of proteins that are generally unrecognizable by conventional softwares but share significant sequence similarity with database entries (~60-90%). These techniques and available database sequences enabled the characterization of the Xenopus laevis microtubule-associated proteome and the Dunaliella salina soluble salt-induced proteome, both organisms with unsequenced genomes and minimal database sequence resources. These sequence-similarity methods extended protein identification capabilities by more than two-fold compared to conventional methods, making existing methods virtually superfluous. The proteomics of Dunaliella salina demonstrated the utility of MS BLAST as an indispensable method for characterization of proteins in organisms with unsequenced genomes, and produced insight into Dunaliella?s inherent resilience to high salinity. The Xenopus study was the first proteomics project to simultaneously use all three central methods of representation for peptide tandem mass spectra for protein identification: sequence tags, amino acids sequences, and mass lists; and it is the largest proteomics study in Xenopus laevis yet completed, which indicated a potential relationship between the mitotic spindle of dividing cells and the protein synthesis machinery. At the beginning of these experiments, the identification of proteins was conceptualized as using ?conventional? versus ?sequence-similarity? techniques, but through the course of experiments, a conceptual shift in understanding occurred along with the techniques developed and employed to encompass variations in mass spectrometry instrumentation, alternative mass spectrum representation forms, and the complexities of database resources, producing a more systematic description and utilization of available resources for the characterization of proteomes by mass spectrometry and advanced informatic approaches. The experiments demonstrated that proteomics technologies are only as powerful in the field of biology as the biochemical experiments are precise and meaningful.