Development of a thiol-ene microfluidic chip for CE-MS of peptides and proteins
- 1Protein Analysis Group, Department of Pharmacy, University of Copenhagen, 2100 Copenhagen Ø, Denmark
- 2National Centre for Nano Fabrication and Characterization, DTU Nanolab, Technical University of Denmark, 2800 Kgs., Lyngby, Denmark
- 3Pharmaceutical Analytical Chemistry Group, Department of Pharmacy, University of Copenhagen, 2100 Copenhagen Ø, Denmark
Abstract
Microfluidic capillary electrophoresis coupled with mass spectrometry (MCE-MS) has emerged as a promising platform for miniaturizing biomolecular analysis due to its low sample consumption, rapid separations, and potential for integration of multiple analytical functions on a single device. However, peptide and protein analysis with MCE-MS devices can be limited by electrospray ionization stability and surface adsorption. Here, we have developed a thiol-ene-based MCE device with an integrated electrospray emitter, fabricated using a double-replication molding approach. We improved the performance of the MCE-MS microchip by optimizing electrospray ionization stability and systematically evaluating surface modification strategies to minimize non-specific adsorption and improve separation repeatability. Different MCE channel surface coatings, including linear polyacrylamide and poly(ethylene glycol) methyl ether methacrylate (PEGMA), were investigated for their influence on peptide separations. Both coatings improved separation performance compared to unmodified devices, providing enhanced migration repeatability and reduced non-specific adsorption. PEGMA coatings provided the most balanced overall performance, combining comprehensive analyte compatibility with repeatable migration time and improved peak symmetry with narrower peak width. The optimized coated microchip was utilized for peptide mapping MCE-MS/MS analysis of pepsin-digested hemoglobin. PEGMA-coated chips enabled peptide identification within a separation window of approximately 60 s, achieving sequence coverages of 92.2% and 78.8% for the α- and β-chains, respectively. In addition, intact protein separations of insulin, β-lactoglobulin, and tropomyosin were demonstrated under acidic MCE-MS conditions, with repeatable migration times. These results highlight the potential of thiol-ene-based MCE-MS as a versatile platform for peptide and protein analysis.
Graphical Abstract:
Supplementary Information: The online version contains supplementary material available at https://doi.org/10.1007/s00216-026-06778-7.
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BibTeX
@article{rouhi2026development,
title = {{Development of a thiol-ene microfluidic chip for CE-MS of peptides and proteins}},
author = {Omid Rouhi and Jordan T. Aerts and Kamille C. Staack and Delaram R. Mikalsen and Stephan S. Keller and Mohammad Ramezannezhad and Nickolaj J. Petersen and Jörg P. Kutter and Kasper D. Rand},
journal = {Analytical and Bioanalytical Chemistry},
year = {2026},
month = sep,
doi = {10.1007/s00216-026-06778-7},
url = {https://doi.org/10.1007/s00216-026-06778-7},
note = {CC BY 4.0},
}
RIS
TY - JOUR
TI - Development of a thiol-ene microfluidic chip for CE-MS of peptides and proteins
AU - Omid Rouhi
AU - Jordan T. Aerts
AU - Kamille C. Staack
AU - Delaram R. Mikalsen
AU - Stephan S. Keller
AU - Mohammad Ramezannezhad
AU - Nickolaj J. Petersen
AU - Jörg P. Kutter
AU - Kasper D. Rand
PY - 2026
DA - 2026/09/10
JO - Analytical and Bioanalytical Chemistry
DO - 10.1007/s00216-026-06778-7
UR - https://doi.org/10.1007/s00216-026-06778-7
AB - Microfluidic capillary electrophoresis coupled with mass spectrometry (MCE-MS) has emerged as a promising platform for miniaturizing biomolecular analysis due to its low sample consumption, rapid separations, and potential for integration of multiple analytical functions on a single device. However, peptide and protein analysis with MCE-MS devices can be limited by electrospray ionization stability and surface adsorption. Here, we have developed a thiol-ene-based MCE device with an integrated electrospray emitter, fabricated using a double-replication molding approach. We improved the performance of the MCE-MS microchip by optimizing electrospray ionization stability and systematically evaluating surface modification strategies to minimize non-specific adsorption and improve separation repeatability. Different MCE channel surface coatings, including linear polyacrylamide and poly(ethylene glycol) methyl ether methacrylate (PEGMA), were investigated for their influence on peptide separations. Both coatings improved separation performance compared to unmodified devices, providing enhanced migration repeatability and reduced non-specific adsorption. PEGMA coatings provided the most balanced overall performance, combining comprehensive analyte compatibility with repeatable migration time and improved peak symmetry with narrower peak width. The optimized coated microchip was utilized for peptide mapping MCE-MS/MS analysis of pepsin-digested hemoglobin. PEGMA-coated chips enabled peptide identification within a separation window of approximately 60 s, achieving sequence coverages of 92.2% and 78.8% for the α- and β-chains, respectively. In addition, intact protein separations of insulin, β-lactoglobulin, and tropomyosin were demonstrated under acidic MCE-MS conditions, with repeatable migration times. These results highlight the potential of thiol-ene-based MCE-MS as a versatile platform for peptide and protein analysis. Graphical Abstract: Supplementary Information: The online version contains supplementary material available at https://doi.org/10.1007/s00216-026-06778-7.
SN - 1618-2642
SN - 1618-2650
ER -