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.