Proteomics of Pyrococcus furiosus, a Hyperthermophilic Archaeon Refractory to Traditional Methods
| Autor: | Jonathan Bundy, Amy M. Grunden, Joel Sevinsky, James L. Stephenson, Alice M. Lee |
|---|---|
| Rok vydání: | 2009 |
| Předmět: |
Proteomics
Proteome Protein Conformation Archaeal Proteins Peptide Mapping Biochemistry Lc ms ms Trypsin Isoelectric Point Microwaves Shotgun proteomics Organism Refractory (planetary science) biology Chemistry Hydrolysis General Chemistry Hydrogen-Ion Concentration biology.organism_classification Peptide Fragments Hyperthermophile Pyrococcus furiosus Archaea |
| Zdroj: | Journal of Proteome Research. 8:3844-3851 |
| ISSN: | 1535-3907 1535-3893 |
| DOI: | 10.1021/pr801119h |
| Popis: | Pyrococcus furiosus is one of the most extensively studied hyperthermophilic archaea. Proteins from this hyperthemophile organism are extremely thermostable and are highly resistant to chemical denaturants, organic solvents and proteolytic digestion. This thermostability makes it difficult to apply traditional methods of enzymatically digesting a complex mixture of proteins, commonly a first step in peptide generation in most shotgun proteomics methods. Here, we have developed a simple shotgun proteomics approach for the global identification of the P. furiosus proteome. This methodology uses a detergent-based microwave assisted acid hydrolysis (MAAH) step coupled with an overnight trypsin digest to obtain peptides. Subsequent peptide fractionation by isoelectric focusing in immobilized pH gradients (IPG-IEF), followed by chromatographic separation with reverse phase nano-HPLC and electrospray ionization tandem mass spectrometry (ESI-MS/MS) of peptides enabled the identification of over 900 proteins representing over 44% of the proteome. In most functional classes, over 50% of the predicted proteins were identified, including a number of membrane proteins. This new sample preparation technique will enable extensive proteomics data to be obtained for this organism, thereby enabling the reconstruction of metabolic pathways and promoting a systems biology based understanding of this important extremophile. |
| Databáze: | OpenAIRE |
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