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Department:
Bioresource Engineering
Research:
The Temporal Analysis of Yeast Proteome Using Shotgun Proteomics
Synopsis:
The objective of my research is to study the temporal yeast proteome (Saccharomyces cerevisiae and Pichia stipitis) during ethanol fermentation using shotgun proteomics (so called Multidimensional Protein Identification Technology (MudPIT)). Shotgun proteomics is a mass spectrometry technique used to detect the global protein expression of the proteome in an organism. MudPIT, a shotgun proteomic technique, integrates a strong cation exchange resin, a reverse phase resin in a biphasic column and a tandem mass spectrometry to identify the partial sequence of peptides. Protein samples from the fermentation are digested typically with trypsins and systematically separated depending on the charge and hydrophobicity in the liquid chromatography phase. The tandem mass spectrometry fragments and measure the mass of the peptide with high accuracy. The SEQUEST algorithm correlates the peptide fragmentation spectra with amino acid sequences in a database, and DTASelect creates a scoring correlation system for peptide fragment spectra and observed protein from the genetic database. S. cerevisiae proteins have been identified using MudPIT analysis. The high-throughput analyses of the proteome identified proteins with hydrophobic transmembrane domains, transcription factors and protein kinases that are in low abundance. Extreme isoelectric point (pI) and molecular weight were also identified with the same sensitivity as any other protein. The proteome of the yeast can be studied by analyzing the attributes of the proteins obtained from the mass
spectrometry through shotgun proteomics. The databases associated with protein attributes are the Functional Catalogue, the Gene Ontology, and Clusters of Orthologous groups of proteins.
S. cerevisiae is also one of the best-known microorganisms for the production of ethanol because of its high ethanol yield and tolerance. However, S. cerevisiae cannot utilize the cellobiose and pentose present in the hydrolyzed lignocellulosic biomass.
In order to utilize the pentose and cellobiose, another pentose fermenting yeast found in the gut of the passaid beetles called Pichia stipitis could be utilized. P. stipitis is capable of fermenting cellobiose, xylose and arabinose that are present in lignocellulose. However, traditionally fermentations are monitored through pH, substrate concentrations, biomass and dissolved oxygen. The actual microbial physiological state can only be “inferred” from these parameters. Studying the yeast proteome by using mass spectrometry is expected to provide an in depth knowledge
of ethanol fermentation at the proteome level.
Bioresource Engineering
Research:
The Temporal Analysis of Yeast Proteome Using Shotgun Proteomics
Synopsis:
The objective of my research is to study the temporal yeast proteome (Saccharomyces cerevisiae and Pichia stipitis) during ethanol fermentation using shotgun proteomics (so called Multidimensional Protein Identification Technology (MudPIT)). Shotgun proteomics is a mass spectrometry technique used to detect the global protein expression of the proteome in an organism. MudPIT, a shotgun proteomic technique, integrates a strong cation exchange resin, a reverse phase resin in a biphasic column and a tandem mass spectrometry to identify the partial sequence of peptides. Protein samples from the fermentation are digested typically with trypsins and systematically separated depending on the charge and hydrophobicity in the liquid chromatography phase. The tandem mass spectrometry fragments and measure the mass of the peptide with high accuracy. The SEQUEST algorithm correlates the peptide fragmentation spectra with amino acid sequences in a database, and DTASelect creates a scoring correlation system for peptide fragment spectra and observed protein from the genetic database. S. cerevisiae proteins have been identified using MudPIT analysis. The high-throughput analyses of the proteome identified proteins with hydrophobic transmembrane domains, transcription factors and protein kinases that are in low abundance. Extreme isoelectric point (pI) and molecular weight were also identified with the same sensitivity as any other protein. The proteome of the yeast can be studied by analyzing the attributes of the proteins obtained from the mass
spectrometry through shotgun proteomics. The databases associated with protein attributes are the Functional Catalogue, the Gene Ontology, and Clusters of Orthologous groups of proteins.
S. cerevisiae is also one of the best-known microorganisms for the production of ethanol because of its high ethanol yield and tolerance. However, S. cerevisiae cannot utilize the cellobiose and pentose present in the hydrolyzed lignocellulosic biomass.
In order to utilize the pentose and cellobiose, another pentose fermenting yeast found in the gut of the passaid beetles called Pichia stipitis could be utilized. P. stipitis is capable of fermenting cellobiose, xylose and arabinose that are present in lignocellulose. However, traditionally fermentations are monitored through pH, substrate concentrations, biomass and dissolved oxygen. The actual microbial physiological state can only be “inferred” from these parameters. Studying the yeast proteome by using mass spectrometry is expected to provide an in depth knowledge
of ethanol fermentation at the proteome level.
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