Background: Cerebrospinal fluid (CSF) leak is typically diagnosed by detecting a protein marker β2-transferrin (β2-Tf) in secretion samples. β2-Tf and β1-transferrin (β1-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 β1-Tf and β2-Tf. Methods: To 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. Results: Based on the MPIE-ESI-MS results, the structures of β1-Tf and β2-Tf were solved. As Tf glycoforms, β1-Tf and β2-Tf share the amino acid sequence but have varying N-glycans. β1-Tf, the major serum-type Tf, has two G2S2 N-glycans on Asn413 and Asn611. β2-Tf, the major brain-type Tf, has an M5 N-glycan on Asn413 and a G0FB N-glycan on Asn611. Conclusions: Through the elucidation of the structures of β1-Tf and β2-Tf, the resolving power of the novel MPIE-ESI-MS method was demonstrated. Moreover, knowing the N-glycan structures on β2-Tf allows for the design of other novel test methods for β2-Tf in the future.

Samuel Yang is also an employee of Gator Bio, which is the manufacturer of the Gator Plus analyzer used in this research.

This study did not receive any funding.

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Yes

The details of the IRB/oversight body that provided approval or exemption for the research described are given below:

IRB protocol 66075 approved by Stanford University Panel on Medical Human Subjects

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All data produced in the present study are available upon reasonable request to the authors.