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Full text release has been delayed at the author's request until May 05, 2025

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Development of Isobaric Peptide Probes for Multiplex Disease Detection using Mass Spectrometry-Based Immunoassay

Zerrudo, Stephanee Joy Biangco
http://orcid.org/0009-0003-4149-3278
http://rave.ohiolink.edu/etdc/view?acc_num=osu1713195692087418

Abstract Details

2024, Master of Science, Ohio State University, Chemistry.
Multiplexed biomarker detection has proven to be important in offering a more effective and accurate disease diagnosis compared with single biomarker detection. Mass spectrometry (MS), an analytical technique that provides sensitive and specific analyte detection, has long been employed for signal transduction of immunoassays toward disease detection. Unlike with direct detection of biomarkers, the use of small molecule mass reporters allows the use of simpler and less expensive portable mass spectrometers. Our research group has previously used quaternary ammonium species as mass reporters for MS-based immunoassays performed on inexpensive paper substrates. However, the number of unique masses that could be generated with the quaternary ammonium species was limited by the commercial availability of the starting materials, which in turn limited the multiplexing ability of our MS-based immunoassay platform. The aim of this thesis was to develop isobaric peptide probes that employ small peptides as mass reporters. The isobaric peptide probes were developed by designing and characterizing peptide mass reporters to investigate their fragmentation patterns in tandem MS (MS/MS) and their sensitivity via nano-electrospray ionization (nESI). Results showed that peptides containing two arginines (Arg) that sequester mobile protons and a cleavage site of aspartic acid (Asp) and proline (Pro) provide two dominant b- and y-type ions in MS/MS. The position of the Asp-Pro bond determines the mass of the corresponding b- and y-type ions. By changing the position of the Asp-Pro amino acids with a particular peptide, we can generate different isobaric peptide probes that fragment to give distinct diagnostic ions. The limit of detection (LOD) and quantification (LOQ) of the optimized peptide design (AcIRNPTIDPINR MW 1350.5 Da) in nESI MS/MS were 0.35 ng/mL (0.26 nM) and 1.03 ng/mL (0.77 nM), respectively. In preparation for the use of the isobaric peptides in immunoassay, we synthesized a cleavable peptide probe by coupling the peptide to a conjugation unit through Steglich esterification. The ester bond formed between the peptide and conjugate unit is cleavable at pH 12. This pH-sensitive cleavage releases a +2-charge state ion at m/z 676 in the full mass spectra. Expected diagnostic fragment ions are detected in MS/MS when this peptide ion is subjected to collision-induced dissociation. This signifies the potential of using isobaric peptides in MS-based immunoassays for multiplexed disease detection.
Abraham Badu-Tawiah (Advisor)
Heather Allen (Committee Member)
Vicki Wysokci (Committee Member)
48 p.
Analytical Chemistry; Chemistry
mass spectrometry; mass spectrometry-based immunoassay; peptide; multiplexing; peptide probe; fragmentation of peptides

Recommended Citations

Citations

  • Zerrudo, S. J. B. (2024). Development of Isobaric Peptide Probes for Multiplex Disease Detection using Mass Spectrometry-Based Immunoassay [Master's thesis, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1713195692087418

    APA Style (7th edition)

  • Zerrudo, Stephanee Joy. Development of Isobaric Peptide Probes for Multiplex Disease Detection using Mass Spectrometry-Based Immunoassay. 2024. Ohio State University, Master's thesis. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1713195692087418.

    MLA Style (8th edition)

  • Zerrudo, Stephanee Joy. "Development of Isobaric Peptide Probes for Multiplex Disease Detection using Mass Spectrometry-Based Immunoassay." Master's thesis, Ohio State University, 2024. http://rave.ohiolink.edu/etdc/view?acc_num=osu1713195692087418

    Chicago Manual of Style (17th edition)

osu1713195692087418
© 2024, all rights reserved.
This open access ETD is published by The Ohio State University and OhioLINK.