Assessing Relative Conformational Stability of Native and Tagged Myoglobin Via Collision-Induced Unfolding

Document Type

Thesis

Degree Name

Master of Science (MS)

Department

Chemistry

Date of Award

9-1-2026

Abstract

Protein tagging is commonly used in biochemical studies to simplify purification and handling, but its effect on protein conformational stability is not always well understood. Travelling Wave Ion Mobility–Mass Spectrometry (TWIMS) has become a useful technique for studying protein structure through collision cross section (CCS) measurements, while Collision-Induced Unfolding (CIU) allows observation of how proteins respond to increasing energy and undergo structural changes. Despite these advances, there is still limited information on how peptide tags influence protein stability and how the intrinsic properties of the peptide contribute to these effects. The aim of this study is to investigate how peptide tagging affects the relative conformational stability of the Myoglobin protein and to examine the structural behaviour of the peptide used as the tag. Ion Mobility–Mass Spectrometry was carried out in both MS and MS/MS modes to obtain mass spectral data and select the native charge state of Myoglobin. The selected ions were then subjected to increasing trap collision energy to induce unfolding using the CIU technique. CCS calibration was performed to convert drift time values into CCS. These calibrated values were used to generate CIU heat maps (CCS vs collision energy), which enabled comparison of the unfolding behaviour of native and peptide-tagged Myoglobin. In addition, the peptide was analyzed separately by forming peptide–metal complexes. CCS values obtained after calibration were used in molecular modelling to investigate the possible structure and conformational preferences of the peptide in its metal-bound state. From the CIU heat maps, differences in unfolding transitions and CCS changes was analyzed to determine the effect of the tag on conformational stability of myoglobin. In addition, CCS data obtained for the peptide–metal complexes were used in molecular modelling to predict the likely structure and conformational behaviour of the peptide. Overall, this study demonstrates how Ion Mobility–Mass Spectrometry, together with CIU, CCS calibration, and molecular modelling, can be used to better understand protein stability and the structural impact of peptide tagging.

Advisor

Laurence Angel

Subject Categories

Chemistry | Physical Sciences and Mathematics

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