Elucidation of the Structures of β1- and β2-Transferrin Using Microprobe-Capture In-Emitter Elution and High-Resolution Mass Spectrometry

BackgroundCerebrospinal fluid (CSF) leak is typically diagnosed by detecting a protein marker {beta}2-transferrin ({beta}2-Tf) in secretion samples. {beta}2-Tf and {beta}1-transferrin ({beta}1-Tf) are glycoforms of human transferrin (Tf). A novel affinity capture technique for sample preparation, called microprobe-capture in-emitter elution (MPIE), was incorporated with high-resolution mass spectrometry (HR-MS) to analyze the Tf glycoforms and elucidate the structures of {beta}1-Tf and {beta}2-Tf. MethodsTo implement MPIE, an analyte is first captured on the surface of a microprobe, and subsequently eluted from the microprobe inside an electrospray emitter. The capture process is monitored in real-time via next-generation biolayer interferometry (BLI). When electrospray is established from the emitter to a mass spectrometer, the analyte is immediately ionized via electrospray ionization (ESI) for HR-MS analysis. Serum, CSF, and secretion samples were analyzed using MPIE-ESI-MS. ResultsBased on the MPIE-ESI-MS results, the structures of {beta}1-Tf and {beta}2-Tf were solved. As Tf glycoforms, {beta}1-Tf and {beta}2-Tf share the amino acid sequence but have varying N-glycans. {beta}1-Tf, the major serum-type Tf, has two G2S2 N-glycans on Asn413 and Asn611. {beta}2-Tf, the major brain-type Tf, has an M5 N-glycan on Asn413 and a G0FB N-glycan on Asn611. ConclusionsThe structures of {beta}1-Tf and {beta}2-Tf were successfully elucidated by MPIE-ESI-MS analysis. The resolving power of the novel MPIE-ESI-MS method was demonstrated in this study. On the other hand, knowing the N-glycan structures on {beta}2-Tf allows for the design of other novel test methods for {beta}2-Tf in the future.