Cerebrospinal fluid (CSF) leak can occur as a result of laceration, blunt trauma, or surgery. It is potentially a life-threatening condition if left untreated (1). CSF leak is typically diagnosed by detecting a protein marker beta2-transferrin (beta2-Tf) in secretion samples. beta2-Tf, a proteoform of human transferrin (Tf) (2), mainly present in CSF, is barely detectable in other body fluids (3). beta2-Tf, together with the typical Tf proteoform in serum beta1-Tf, were named after their electrophoretic mobility in gel electrophoresis. The clinical utility and diagnostic value of beta2-Tf in CSF leak have been demonstrated (4). However, the structures of beta1-Tf and beta2-Tf have not been elucidated. 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 study and elucidate the structures of beta1-Tf and beta2-Tf (5).
MPIE can directly couple a label-free optical sensing technology (next-generation biolayer interferometry, BLI) with MS, resolving the challenge of lack of process monitoring in the conventional affinity capture techniques such as bead-based immunoprecipitation. 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 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. By this means, BLI and HR-MS are directly coupled in the form of MPIE-ESI-MS, which is readily deployed to analyze the Tf glycoforms and elucidate the structures of beta1-Tf and beta2-Tf. The study can pave a way for the development of novel clinical assays for beta2-Tf. The Tf glycoforms in CSF samples, serum samples, and secretion samples from patients suspected of CSF leak were analyzed using MPIE-ESI-MS. The Tf glycoforms separated by gel electrophoresis were also analyzed.
Based on the MPIE-ESI-MS results of serum, CSF, and secretion samples, the structures of beta1-Tf and beta2-Tf were elucidated. As Tf glycoforms, beta1-Tf and beta2-Tf share the amino acid sequence but have varying N-glycans. beta1-Tf, the major serum-type Tf, has two G2S2 N-glycans on Asn413 and Asn611, while beta2-Tf, the major brain-type Tf, has an M5 N-glycan on Asn413 and a G0FB N-glycan on Asn611. The analytical sensitivity of MPIE-ESI-MS for CSF samples was tested to evaluate its potential clinical use, and it was demonstrated that it was able to detect beta2-Tf in the 1:9 pooled CSF : water mixture (10-fold diluted). When testing clinical specimens, the MPIE-ESI-MS method has good performance in testing secretion samples that are not blood-contaminated, especially those with ambiguous agarose gel immunofixation electrophoresis (the conventional method) test results.
Through the elucidation of the structures of beta1-Tf and beta2-Tf, the resolving power of MPIE-ESI-MS was demonstrated. Besides basic research, MPIE-ESI-MS can potentially be used in clinical laboratory testing. Moreover, with the known N-glycan structures in beta1-Tf and beta2-Tf, other types of new assays can be designed to detect beta2-Tf in the future. The MPIE-ESI-HR-MS method demonstrated exceedingly good sensitivity to successfully connect a label-free technology and MS, and it has substantial value for biomedical research and clinical diagnostics.
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