Impact of prefractionation using Gradiflow on two-dimensional gel electrophoresis and protein identification by matrix assisted laser desorption/ionization-time of flight-mass spectrometry
| Autor: | David L. Wong, Nicole Chow, Alfred L. Gaertner, Beth Fryksdale, Brian S. Miller, Lisa Pang |
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| Rok vydání: | 2003 |
| Předmět: |
Trichoderma
Chromatography Protein mass spectrometry Resolution (mass spectrometry) Chemistry Clinical Biochemistry Static Electricity Analytical chemistry Proteins Mass spectrometry Biochemistry Capillary electrophoresis–mass spectrometry Sample preparation in mass spectrometry Analytical Chemistry Surface-enhanced laser desorption/ionization Fungal Proteins Molecular Weight Matrix-assisted laser desorption/ionization Peptide mass fingerprinting Cellulase Spectrometry Mass Matrix-Assisted Laser Desorption-Ionization Electrophoresis Gel Two-Dimensional |
| Zdroj: | Electrophoresis. 24(19-20) |
| ISSN: | 0173-0835 |
| Popis: | Prefractionation of protein samples prior to two-dimensional electrophoresis (2-DE) has the potential to increase the dynamic detection range for proteomic analysis. We evaluated a membrane-based electrophoretic separation technique (Gradiflow) for its ability to fractionate an exoproteome sample from the filamentous fungus Trichoderma reesei. The sample was separated on the basis of size and charge. Buffer optimization was found to be necessary for successful size fractionation. Fractionation by charge was used to resolve the sample into four fractions that were subjected to analysis by two-dimensional electrophoresis (2-DE). Enhanced detection of low-abundance proteins with selective removal of high-abundance species was achieved. Fractionated and unfractionated samples were examined for differences in the ability to identify proteins following 2-DE using trypsin in-gel digestion followed by peptide mass fingerprinting using matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS). Fractionated samples showed marked improvement in protein identification ability and sequence coverage. This study demonstrates the utility of the Gradiflow for fractionation, resulting in an enhancement of resolution and characterization of a moderately complex proteome. |
| Databáze: | OpenAIRE |
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