Figure 2.

Mining Human PLP-DEs with PL1 in HEK293

(A) Targeted metabolomics analysis of PL and PLP levels in HEK293 cells. Concentrations of both vitamers decreased over time in media with PL1 (n = 4 biological replicates). No endogenous B₆ vitamers were present at the time point of labeling after seven passages.

(B) Labeling strategy using PL1 and PL as a control. Cells were passaged in media containing either PL1 or PL. Following cell lysis, the internal aldimine of PLP-DEs was reduced with sodium borohydride (NaBH₄) and proteins were subjected to CuAAC to attach biotin. After avidin enrichment, proteins were digested with trypsin for subsequent liquid chromatography-tandem mass spectrometry analysis (P = OPO₃²⁻).

(C) Volcano plot representing the t test results of PL1 labeling in HEK293 compared with the PL control revealed significant enrichment of many PLP-DEs (blue) (n = 6 biological replicates). Cut-off values were defined as an enrichment factor of log₂ = 1 (2-fold enrichment) and −log₁₀ (p value) of 1.3 (solid lines).

See also Table S1.