Novel assay for identification of semicarbazide-sensitive amine oxidase by a priority-based strategy in mass spectrometry

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 protein 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 C₁₈ analytical column and detected by ESI-MS-MS. In the direct data-dependent method (also called traditional method), the semicarbazide-sensitive amine oxidase (SSAO) cannot be identified by LC-ESI-MS-MS. Compared with the traditional 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, 120 μg, 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, respectively. The MS/MS spectra of two unique peptides of SSAO were confirmed by manual identification. 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. Copyright.