A novel assay for the identification of semicarbazide sensitive amine oxidase in human umbilical artery tissue by a priority based strategy in the mass spectrometry was developed. The pro tein extract was separated b...A novel assay for the identification of semicarbazide sensitive amine oxidase in human umbilical artery tissue by a priority based strategy in the mass spectrometry was developed. The pro tein extract was separated by SDS PAGE, and then an entire band at 96 KDa was excised and digested by trypsin. The digested peptides were separated by capillary ClS analytical column and detected by ESI MS MS. In the direct data dependent method （also called traditional method）, the semicarbaz ide sensitive amine oxidase （SSAO） cannot be identified by LC-ESI-MS-MS. Compared with the tra ditional method, our assay by a priority based strategy in the mass spectrometry can successfully identify the target protein SSAO in the complex biological sample. As 60 μg, 120μg, 240 μg of total protein extract were loaded on the SDS PAGE, the Mascot result showed that SSAO score was 46, 86 and 137, the sequence coverage was 2 % , 5 % and 10 % , and the peptide count was 2, 6 and 10, re spectively. The MS/MS spectra of two unique peptides of SSAO were confirmed by manual identifica tion. The band at 96 KDa included SSAO was validated by the Western blot. The assay significantly improved the score and coverage of target protein and enhanced the identification of reliability and the confidence.展开更多
基金Supported by the National Basic Research Project of China(2007CB14100)
文摘A novel assay for the identification of semicarbazide sensitive amine oxidase in human umbilical artery tissue by a priority based strategy in the mass spectrometry was developed. The pro tein extract was separated by SDS PAGE, and then an entire band at 96 KDa was excised and digested by trypsin. The digested peptides were separated by capillary ClS analytical column and detected by ESI MS MS. In the direct data dependent method （also called traditional method）, the semicarbaz ide sensitive amine oxidase （SSAO） cannot be identified by LC-ESI-MS-MS. Compared with the tra ditional method, our assay by a priority based strategy in the mass spectrometry can successfully identify the target protein SSAO in the complex biological sample. As 60 μg, 120μg, 240 μg of total protein extract were loaded on the SDS PAGE, the Mascot result showed that SSAO score was 46, 86 and 137, the sequence coverage was 2 % , 5 % and 10 % , and the peptide count was 2, 6 and 10, re spectively. The MS/MS spectra of two unique peptides of SSAO were confirmed by manual identifica tion. The band at 96 KDa included SSAO was validated by the Western blot. The assay significantly improved the score and coverage of target protein and enhanced the identification of reliability and the confidence.
