Sequencing of novel protein from Bacillus pumilus PH-01 using a high-resolution hybrid quadrupole-time-of-flight mass spectrometer
| Autor: | Tina Settineri, Steven C. Hall, Yoon-Seok Chang, Hyobong Hong, Byung-Hoon Kim |
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| Rok vydání: | 2001 |
| Předmět: |
Chromatography
Protein mass spectrometry Edman degradation Chemistry Peptide sequence tag De novo peptide sequencing Condensed Matter Physics Mass spectrometry Tandem mass spectrometry Peptide mass fingerprinting Physical and Theoretical Chemistry Peptide-mass fingerprint Instrumentation Spectroscopy |
| Zdroj: | International Journal of Mass Spectrometry. 209:47-55 |
| ISSN: | 1387-3806 |
| Popis: | Protein identification can be accomplished with an enzymatic digestion of the protein followed by mass spectrometric analysis of the peptide mass fingerprint followed by database searching. However, if a protein is not in a database, sequence information must be obtained to characterize and identify it. This can be done either by classical Edman sequencing or/and by tandem mass spectrometry. To determine the sequence of an unknown protein from Bacillus pumilus PH-01, which adsorbs environmental pollutants such as polychlorinated dibenzo-p-dioxins (PCDDs), dibenzofurans (PCDFs), and biphenyls (PCBs), both sequencing and de novo peptide sequencing by tandem MS/MS of the peptide fragments were performed. Edman sequencing of the reduced and alkylated protein revealed the majority of the sequence; however, all information on disulfide bonding was lost. Therefore, a tryptic digest of the native protein was performed to obtain both complete sequence information and the connectivity of the disulfide bonds. We performed the de novo sequencing using a hybrid quadrupole-time-of-flight mass spectrometer (Q-TOF MS) instrument. The high mass accuracy and sensitivity of the hybrid Q-TOF MS made low-level sequencing of this novel naturally isolated protein possible. |
| Databáze: | OpenAIRE |
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