Study of β 1 -transferrin and β 2 -transferrin using microprobe-capture in-emitter elution and high-resolution mass spectrometry

Cerebrospinal fluid (CSF) leak can be diagnosed in clinical laboratories by detecting a diagnostic marker β 2 -transferrin (β 2 -Tf) in secretion samples. β 2 -Tf and the typical transferrin (Tf) proteoform in serum, β 1 -transferrin (β 1 -Tf), are Tf glycoforms. An innovative affinity capture technique for sample preparation, called microprobe-capture in-emitter elution (MPIE), was incorporated with high-resolution mass spectrometry (HR-MS) to study the Tf glycoforms and the primary structures of β 1 -Tf and β 2 -Tf. 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. Based on the MPIE-ESI-MS results, the primary structures of β 1 -Tf and β 2 -Tf were elucidated. As Tf glycoforms, β 1 -Tf and β 2 -Tf share the amino acid sequence but contain varying N-glycans: (1) β 1 -Tf, the major serum-type Tf, has two G2S2 N-glycans on Asn413 and Asn611; and (2) β 2 -Tf, the major brain-type Tf, has an M5 N-glycan on Asn413 and a G0FB N-glycan on Asn611. The resolving power of the innovative MPIE-ESI-MS method was demonstrated in the study of β 2 -Tf as well as β 1 -Tf. Knowing the N-glycan structures on β 2 -Tf allows for the design of more novel test methods for β 2 -Tf in the future.