= Emerging. More than 5 years before clinical availability. (24.37%, 2023)
= Expected to be clinically available in 1 to 4 years. (39.50%, 2023)
= Clinically available now. (36.13%, 2023)
MSACL 2023 : Luo

MSACL 2023 Abstract

Self-Classified Topic Area(s): Proteomics > Assays Leveraging MS

Podium Presentation in Steinbeck 3 on Thursday at 16:50 (Chair: Christopher Chouinard / William Perry)

Using Microprobe-Capture In-Emitter Elution and High-Resolution Mass Spectrometry for Characterization and Clinical Testing of β2-Transferrin

Ruben Y. Luo (1,2), Christopher Pfaffroth (2), Samuel Yang (1), Kevin Hoang (2), James L. Zehnder (1,2), Run-Zhang Shi (1,2)
(1) Department of Pathology, Stanford University, Stanford, CA (2) Clinical Laboratories, Stanford Health Care, Palo Alto, CA

Ruben Y. Luo, PhD, DABCC (Presenter)
Stanford University

Presenter Bio: Ruben Y. Luo, PhD, DABCC, FAACC is currently an Assistant Professor of Pathology at Stanford University and the Associate Director of Clinical Chemistry Laboratory at Stanford Health Care. He received PhD in chemistry from Stanford University, and had several years of working experience in the clinical diagnostic industry. He completed clinical chemistry fellowship at the University of California San Francisco. Dr. Luo is dedicated to the innovation of clinical diagnostic technologies, and his research focuses on applying top-down mass spectrometry and label-free immunoassays to the characterization and precise measurement of clinical diagnostic markers. He has been invited to present in international conferences consecutively for years, e.g., American Association for Clinical Chemistry (AACC), American Society for Mass Spectrometry (ASMS), Mass Spectrometry & Advances in Clinical Lab (MSACL). His research awards include 2022 AACC George Grannis Award for Excellence in Research and Scientific Publication, 2021 NACCCA Outstanding Research Award, 2020 ASCP “40 Under Forty” Honoree, etc.



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.

1. McCudden CR, Senior BA, Hainsworth S, Oliveira W, Silverman LM, Bruns DE, et al. Evaluation of high resolution gel beta2-transferrin for detection of cerebrospinal fluid leak. Clinical Chemistry and Laboratory Medicine (CCLM). 2013;51:311–5.
2. Papadea C, Schlosser RJ. Rapid Method for beta2-Transferrin in Cerebrospinal Fluid Leakage Using an Automated Immunofixation Electrophoresis System. Clinical Chemistry. 2005;51:464–70.
3. Gorogh T, Rudolph P, Meyer JE, Werner JA, Lippert BM, Maune S. Separation of beta2-Transferrin by Denaturing Gel Electrophoresis to Detect Cerebrospinal Fluid in Ear and Nasal Fluids. Clinical Chemistry. 2005;51:1704–10.
4. Zaret DL, Morrison N, Gulbranson R, Keren DF. Immunofixation to Quantify beta2-Transferrin in Cerebrospinal Fluid to Detect Leakage of Cerebrospinal Fluid from Skull Injury. Clinical Chemistry. 1992;38:1909–12.
5. Luo RY, Yang S. Using Microprobe-Capture In-Emitter Elution to Directly Couple Label-Free Optical Sensing Technology with Mass Spectrometry for Top-Down Protein Analysis. Chemistry; 2022 Oct.

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