Development of Cleavable Peptide Probes for Mass Spectrometry Based Immunoassays

Development of Cleavable Peptide Probes for Mass Spectrometry Based Immunoassays

Bibliographic Details
Main Author: Velez Burgos, Tatiana, Velez-Burgos
Language:English
Published: The Ohio State University / OhioLINK 2017
Subjects:
Analytical Chemistry
Chemistry
mass spectrometry
peptides
paper based immunoassay
mass spectrometry immunoassay
Online Access:http://rave.ohiolink.edu/etdc/view?acc_num=osu1500041301949337
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spelling ndltd-OhioLink-oai-etd.ohiolink.edu-osu15000413019493372021-08-03T07:03:22Z Development of Cleavable Peptide Probes for Mass Spectrometry Based Immunoassays Velez Burgos, Tatiana, Velez-Burgos Analytical Chemistry Chemistry mass spectrometry peptides paper based immunoassay mass spectrometry immunoassay Molecular diagnostics based on antibodies and complementary DNAs is themethod of choice for high-throughput protein biomarker detection. Most of thesemethods rely on colorimetric detection via enzymatic reactions or fluorescent probes.However, there are some restrictions regarding probe stability and multiplexedcapabilities. Herein, we propose a novel mass spectrometry-based diagnostic platform forrapid and multiplexed detection of protein disease biomarkers. The core of this approachis the use of available cleavable isobaric peptides as mass reporter in immunoassays. Allpeptides probes are rationally designed to have the same molecular weight but dissociateto give specific fragment ions when subjected to collision-induced dissociation (CID),providing well-defined spectra resolution in a single experiment for multiple biomarkers.The project has three main aspects: (i) optimization of conditions for peptide iongeneration and fragmentation to obtain highly sensitive mass reporters with characteristicfragment ions, (ii) design and incorporation of the selected mass reporter peptides into acleavable probe unit, and (iii) subsequently use the cleavable probe for immunoreaction,on wax-printed paper substrates. The cleavable probe unit will feature three functionalcharacteristics: (a) sulfur-containing (-NCS) groups for coupling to antibodies specific tothe protein biomarker of interest, (b) easily ionizable peptide molecules for sensitive MSdetection, and (c) a cleavable linkage allowing release of the peptide. To assess the peptide suitability as mass reporters, nano-electrospray ionization (nESI) and paper-sprayionization were used to generate ions for mass spectrometry (MS) characterization.Three peptides were designed and synthesized: AKRRG, RRGKA, and GARKK,all having molecular weight of 586 Da. nESI-MS analysis of these peptides showed apredominant peak at m/z 294 for doubly protonated [M+2H]2+ ions and minor signalsat m/z 196 and 587 as triply [M+H]3+ and singly charged [M+H]+ species, respectively.All peptides can be sensitively detected at 3 nM concentration levels, in tandem MS(MS/MS) mode. Most importantly, fragment ions from the three isobaric peptidesproduced in MS/MS are different, providing a simple way to differentiate them as masstags for different diseases. For example, CID of [M+H]+ species from GARRK produceda signature fragment ion at m/z 441 via elimination of lysine (K) residue. Similarly, CIDof [M+2H]2+ ions from GARRK yielded signature fragment ion at m/z 230. CID data forall peptides tested are discussed in details. Linear calibrations curves are easily generatedfor [M+2H]2+ ions in 3–100 nM concentration range using the characteristic fragmentions.Moreover, we observed that fragmentation efficiencies of the [M+2H]2+ ions fromthe selected peptides were different. Ion mobility experiments, however, revealed that thethree [M+2H]2+ peptide ions have comparable cross sections. This result suggests thatability to derive unique fragment ions from isobaric peptides with different efficienciesmay be related to the mobile proton mechanism where restrictions in the movement ofcharge affect ease of fragmentation. Bio-conjugation of the GARRK peptide-probe isachieved. A peptide-probe was successfully obtained and future applications on paper-based microfluidic device will allow quantification through paper-spray massspectrometry. 2017-10-30 English text The Ohio State University / OhioLINK http://rave.ohiolink.edu/etdc/view?acc_num=osu1500041301949337 http://rave.ohiolink.edu/etdc/view?acc_num=osu1500041301949337 unrestricted This thesis or dissertation is protected by copyright: all rights reserved. It may not be copied or redistributed beyond the terms of applicable copyright laws.
collection NDLTD
language English
sources NDLTD
topic Analytical Chemistry
Chemistry
mass spectrometry
peptides
paper based immunoassay
mass spectrometry immunoassay
spellingShingle Analytical Chemistry
Chemistry
mass spectrometry
peptides
paper based immunoassay
mass spectrometry immunoassay
Velez Burgos, Tatiana, Velez-Burgos
Development of Cleavable Peptide Probes for Mass Spectrometry Based Immunoassays
author Velez Burgos, Tatiana, Velez-Burgos
author_facet Velez Burgos, Tatiana, Velez-Burgos
author_sort Velez Burgos, Tatiana, Velez-Burgos
title Development of Cleavable Peptide Probes for Mass Spectrometry Based Immunoassays
title_short Development of Cleavable Peptide Probes for Mass Spectrometry Based Immunoassays
title_full Development of Cleavable Peptide Probes for Mass Spectrometry Based Immunoassays
title_fullStr Development of Cleavable Peptide Probes for Mass Spectrometry Based Immunoassays
title_full_unstemmed Development of Cleavable Peptide Probes for Mass Spectrometry Based Immunoassays
title_sort development of cleavable peptide probes for mass spectrometry based immunoassays
publisher The Ohio State University / OhioLINK
publishDate 2017
url http://rave.ohiolink.edu/etdc/view?acc_num=osu1500041301949337
work_keys_str_mv AT velezburgostatianavelezburgos developmentofcleavablepeptideprobesformassspectrometrybasedimmunoassays
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