Despite tremendous advances in mass spectrometry instrumentation and mass spectrometry-based methodologies, global protein profiling of organellar, cellular, tissue and body fluid proteomes in different organisms remains a challenging task due to the complexity of the samples and the wide dynamic range of protein concentrations. In addition, large amounts of produced data make result exploitation difficult. To overcome these issues, further advances in sample preparation, mass spectrometry instrumentation as well as data processing and data analysis are required. The presented study focuses as first on the improvement of the proteolytic digestion of proteins in in-gel based proteomic approach (Gel-LCMS). To this end commonly used bovine trypsin (BT) was modified with oligosaccharides in order to overcome its main disadvantages, such as weak thermostability and fast autolysis at basic pH. Glycosylated trypsin derivates maintained their cleavage specifity and showed better thermostability, autolysis resistance and less autolytic background than unmodified BT. In line with the “accelerated digestion protocol” (ADP) previously established in our laboratory modified enzymes were tested in in-gel digestion of proteins. Kinetics of in-gel digestion was studied by MALDI TOF mass spectrometry using 18O-labeled peptides as internal standards as well as by label-free quantification approach, which utilizes intensities of peptide ions detected by nanoLC-MS/MS. In the performed kinetic study the effect of temperature, enzyme concentration and digestion time on the yield of digestion products was characterized. The obtained results showed that in-gel digestion of proteins by glycosylated trypsin conjugates was less efficient compared to the conventional digestion (CD) and achieved maximal 50 to 70% of CD yield, suggesting that the attached sugar molecules limit free diffusion of the modified trypsins into the polyacrylamide gel pores. Nevertheless, these thermostable and autolysis resistant enzymes can be regarded as promising candidates for gel-free shotgun approach. To address the reliability issue of proteomic data I further focused on protein identifications with borderline statistical confidence produced by database searching. These hits are typically produced by matching a few marginal quality MS/MS spectra to database peptide sequences and represent a significant bottleneck in proteomics. A method was developed for rapid validation of borderline hits, which takes advantage of the independent interpretation of the acquired tandem mass spectra by de novo sequencing software PepNovo followed by mass-spectrometry driven BLAST (MS BLAST) sequence similarity searching that utilize all partially accurate, degenerate and redundant proposed peptide sequences. It was demonstrated that a combination of MASCOT software, de novo sequencing software PepNovo and MS BLAST, bundled by a simple scripted interface, enabled rapid and efficient validation of a large number of borderline hits, produced by matching of one or two MS/MS spectra with marginal statistical significance.
| Identifer | oai:union.ndltd.org:DRESDEN/oai:qucosa:de:qucosa:23888 |
| Date | 14 May 2009 |
| Creators | Wielsch, Natalie |
| Contributors | Shevchenko, Andrej, Göttfert, Michael, Sebela, Marek, Thiele, Christoph |
| Publisher | Technische Universität Dresden |
| Source Sets | Hochschulschriftenserver (HSSS) der SLUB Dresden |
| Language | English |
| Detected Language | English |
| Type | doc-type:doctoralThesis, info:eu-repo/semantics/doctoralThesis, doc-type:Text |
| Rights | info:eu-repo/semantics/openAccess |
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