Sequencing of novel protein from Bacillus pumilus PH-01 using a high-resolution hybrid quadrupole-time-of-flight mass spectrometer

被引:5
|
作者
Chang, YS
Hong, HB
Kim, BH
Hall, SC
Settineri, TA
机构
[1] Pohang Univ Sci & Technol, Sch Environm Engn, Nam Gu, Pohang 790784, South Korea
[2] Appl Biosyst Inc, Foster City, CA USA
关键词
protein identification; sequencing; protein; Bacillus pumilus; hybrid Q-TOF MS; MS/MS;
D O I
10.1016/S1387-3806(01)00463-8
中图分类号
O64 [物理化学(理论化学)、化学物理学]; O56 [分子物理学、原子物理学];
学科分类号
070203 ; 070304 ; 081704 ; 1406 ;
摘要
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. (C) 2001 Elsevier Science B.V.
引用
收藏
页码:47 / 55
页数:9
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