Comparison of different separation technologies for proteome analyses: Isoform resolution as a prerequisite for the definition of protein biomarkers on the level of posttranslational modifications
| Autor: | Christian Hunzinger, Werner Stegmann, Slobodan Poznanovic, Gerhard P. Schwall, André Schrattenholz |
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| Rok vydání: | 2006 |
| Předmět: |
Proteome
Resolution (mass spectrometry) Ovalbumin Protein digestion Molecular Sequence Data Peptide Proteomics Biochemistry Analytical Chemistry Protein purification Protein Isoforms Electrophoresis Gel Two-Dimensional Trypsin Amino Acid Sequence Phosphorylation Chromatography High Pressure Liquid chemistry.chemical_classification Chromatography Chemistry Organic Chemistry Protein primary structure General Medicine Matrix-assisted laser desorption/ionization Spectrometry Mass Matrix-Assisted Laser Desorption-Ionization Electrophoresis Polyacrylamide Gel Isoelectric Focusing Protein Processing Post-Translational Biomarkers Chromatography Liquid |
| Zdroj: | Journal of Chromatography A. 1123:170-181 |
| ISSN: | 0021-9673 |
| DOI: | 10.1016/j.chroma.2006.06.002 |
| Popis: | In this article we evaluate methods used to reveal the molecular complexity, which is generated in biological samples by posttranslational modifications (PTM) of proteins. We show how distinct molecular differences on the level of phosphorylation sites in a single protein (ovalbumin) can be resolved with different success using 1D and 2D gel-electrophoresis and reversed-phase liquid chromatography (LC) with monolithic polystyrol-divinylbenzol (PS-DVB) columns for protein separation, and matrix-assisted laser desorption ionisation-time of flight mass spectrometry (MALDI-TOF MS) for protein identification. Phosphorylation site analysis was performed using enzymatic dephosphorylation in combination with differential peptide mass mapping. Liquid chromatography-MALDI-TOF MS coupling with subsequent on-target tryptic protein digestion turned out to be the fastest method tested but yielded low resolution for the analysis of PTM, whereas 2D gel-electrophoresis, due to its unique capability of resolving highly complex isoform pattern, turned out to be the most suitable method for this purpose. The evaluated methods complement one another and in connection with efficient technologies for differential and quantitative analysis, these approaches have the potential to reveal novel molecular details of protein biomarkers. |
| Databáze: | OpenAIRE |
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