Abstract
Pyridoxal 5'-phosphate (PLP) cooperates with a variety of enzymes in all organisms for many important biological processes. The development of mass spectrometry-based methodology for high-throughput modification analyses could provide an alternative way for PLP identification. The present study aims to identify PLP modification. More PLP site-determining information was obtained by introducing multistage activation (MSA)-assisted collision-induced dissociation (CID). We then utilized immobilized metal ion affinity chromatography (IMAC) with Ti4+ to enrich the PLP peptides. In addition, alkaline phosphatase (ALP) was used to remove the phosphoryl group and further confirm the PLP modification site. MSA was able to greatly enhance the identification and localization of PLP modification. We applied this strategy to analyze PLP-modified proteins in Escherichia coli samples and accurately determine PLP site K270 in tryptophanase. MSA-assisted CID was used to provide better identification of PLP-modified peptides. Furthermore, tryptophanase with PLP modification at K270 in E. coli was identified with Ti4+ -IMAC enrichment followed by ALP treatment. This method provides a promising alternative for investigating biological functions of PLP-modified proteins.
Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
