Surface Functionalized Nanoprobe-based Mass Spectrometry for Site-specific Enrichment and Identification of S-nitrosylated Peptid
Date Issued
2010
Date
2010
Author(s)
Chou, Kao-Yu
Abstract
S-nitrosylation is a reversible posttranslational modification, covalently interact with nitric oxide (NO) on cysteine thiol group of proteins, involving in a wide range of cellular pathways and physiological processes, such as immune response and redox regulation. Due to the labile nature of S-NO bond and low abundance of endogenously S-nitrosylated proteins in vivo, unambiguous identification of S-nitrosylated proteins and S-nitrosylation sites remains methodologically challenging. Based on the study of Zhang et al., we used 4 different reactive groups of thioester- and ester-phosphine compounds, S-propyl 2-(diphenylphosphino) benzothioate (termed as L364) and others, to enrich S-nitrosylated proteins and peptides. A standard peptide, synthetic ESGSLSPEHGPVVVHC215SAGIGR of protein tyrosine phosphatase 1 B (PTP1B), which has been published for S-nitrosylation in vivo, was S-nitrosylated in vitro. Based on the traceless Staudinger ligation-type mechanism, the disulfide bond was formed when phosphine-ester ligand site-specifically reacted with S-NO bond on S-nitrosylated peptides. LC-MS/MS analysis showed that free S-nitrosylated peptide was significantly decreased after reacting with all ligands, compared to the signal of initial S-nitrosylated PTP1B peptides. Simultaneously, the L364-modified PTP1B peptide was identified by Mascot database search. Due to the better reaction efficiency of L364, the L364 was chose to conjugate on magnetic nanoparticle (L364@MNP) using anhydride linkage for site-specifically enrichment of S-nitrosylated proteins and peptides. Consistent with the appropriate reaction of ligand, S-nitrosylated PTP1B peptide was enriched by L364@MNP. Limit of detection (LOD) experiment showed that about 10 nM of S-nitrosylated PTP1B peptide was enriched and identified by MALDI-TOF. Moreover, the S-nitrosylated PTP1B peptide mixed in tryptic BSA peptide mixture was also site-specifically captured by anhydride-L364@MNP and identified by MALDI-TOF MS. In addition, S-nitrosylated ESGSLSPEHGPVVVHC215SAGIGR from tryptic digested PTP1B protein was also directly and site-specifically enriched by anhydride-L364@MNP. Therefore, anhydride-L364@MNP coupling mass spectrometry analysis can provide a direct and site-specific enrichment for identification of S-nitrosylated proteins and peptides.
Subjects
S-nitrosylation
Nanoprobe
Mass Spectrometry
Type
thesis
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