MSACL 2023 Abstract

Immunoprecipitation Top-Down High-Resolution Mass Spectrometry for the Quantification of the Protein Tumor Biomarker Neuron-Specific Enolase Sebastian A. H. van den Wildenberg (1,2,4) Sylvia Roovers Genet (1,2,4) Maarten A. C. Broeren (1,3,4) Joost L. J. van Dongen (1,4) Daan van de Kerkhof (1,2,4) Luc Brunsveld (1,4) Volkher Scharnhorst (1, 2, 4). 1 Laboratory of Chemical Biology, department of Biomedical Engineering, Eindhoven University of Technology, the Netherlands. 2 Clinical Laboratory, Catharina Hospital Eindhoven, the Netherlands. 3 Clinical Laboratory, Máxima Medical Center, Eindhoven/Veldhoven, the Netherlands. 4 Expert Center Clinical Chemistry Eindhoven, the Netherlands. Sebastian van den Wildenberg, MSc (Presenter) Eindhoven University of Technology >> POSTER (PDF)

Presenter Bio: I am a fourth year PhD student, and my research is focused on the development of mass spectrometry assays for clinical applications. I have a broad background in mass spectrometry, a technique that I have been using since my bachelors. My current research focusses on the development of immunoprecipitation assays coupled to mass spectrometry to isolate lung cancer related protein tumor biomarkers from human serum for lung cancer diagnostics. Currently we use both bottom-up assay followed by triple quadrupole MS and top-down assays followed by QToF MS for analysis. Next to this we are working on other mass spectrometry assay for clinical applications in close collaboration with several hospitals in Eindhoven area.

Introduction:
LC-MS methods using bottom-up based and/or middle-down have been popular methods for the quantification of proteins. However, the development of these methods often come with extensive sample preparation that require thorough assay optimization. In addition to this using bottom-up proteomics sequence coverage is often limited and information about Post Translational Modification’s (PTMs) is lost. Using top-down proteomics intact protein are analyzed, without digestion, reducing sample preparation and retaining the maximum amount of information. Top-down proteomic approaches have their own specific challenges, such as limited sensitivity and the availability of internal standards and reference material.

Objectives:
The primary objective of this study is the development of an immunoprecipitation assay, followed by intact top-down protein analysis using LC-QToF-MS for the quantification of the small cell lung cancer (SCLC) tumor biomarker Neuron Specific Enolase (NSE).

Methods:
Immunoprecipitation was performed by coupling Protein-G-labeled magnetic Dynabeads™ to monoclonal antibodies against NSE. The protein-G-antibody complex was crosslinked with BS(PEG)5. Human (NSE-spiked) serum was incubated with antibody coupled beads. After incubation, the beads were collected using a magnet and serum was removed. Collected beads were washed and the captured NSE proteins were eluted from the beads. The eluted proteins were separated using RP-LC and analyzed by QToF-MS.

Results:
The immunoprecipitation method was developed and optimized for the isolation of NSE. Protein elution from the antibody-antigen-complex was conducted using water-acetonitrile (80:20) + 1% formic acid. Using this method recombinant NSE was successfully isolated from spiked human serum. Adequate linearity and sensitivity were achieved in the clinically relevant concentration range of 0 to 100 ng/mL using both QToF-Full Scan MS + Extracted Ion Chromatogram (XIC) and QToF-Selected Reaction Monitoring.

Conclusion and Outlook:
An immunoprecipitation method couple to a LC-QoF-MS method was developed and optimized for the isolation and quantification of recombinant NSE from spiked human serum. Next steps will focus on the isolation of endogenous human NSE from serum and optimization of the chromatography by transferring the method from UPLC to nano-LC. Additionally, QToF-MS- methods and data collection modes that can be used are compared, such as mass deconvolution, single reaction monitoring (precursor > product), pseudo-single reaction monitoring (precursor > precursor), extracted ion chromatograms and combinations.