Rationale: 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.
Methods: 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.
Results: 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.
Conclusions: 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.
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