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32. Top-down proteomics on a microfluidic platform

Author

Zhang, Yuewen, Wright, Maya A., Challa, Pavan K., Saar, Kadi-Liis, Devenish, Sean, Peter, Quentin, and Knowles, Tuomas P. J.

Subjects
Physics - Biological Physics, Quantitative Biology - Biomolecules
Abstract

Protein identification and profiling is critical for the advancement of cell and molecular biology as well as medical diagnostics. Although mass spectrometry and protein microarrays are commonly used for protein identification, both methods require extensive experimental steps and long data analysis times. Here we present a microfluidic top down proteomics platform giving multidimensional read outs of the essential amino acids of proteins. We obtain hydrodynamic radius and fluorescence signals relating to the content of tryptophans, tyrosines and lysines of proteins using a combination of diffusional sizing of proteins, label-free detection and on-chip labelling of proteins with a latent fluorophore in the solution phase. We thereby achieve identification of proteins on a single microfluidic chip by separating and mapping proteins in multidimensional space based on their characteristic physical parameters. Our results have significant implications in the development of easy and rapid platforms to use for native protein identification in clinical and laboratory settings., Comment: 17 pages, 3 figures

Published
2019

41. Investigating protein properties via microfluidic techniques

Author

Zhang, Yuewen and Knowles, Tuomas

Subjects
microfluidics, protein-protein interactions, protein physical properties
Abstract

Proteins are one of the most abundant biomacromolecules and play important roles in bioactivities within organisms, including key functions in DNA replication, immune system response, metabolic reactions and molecular transportation. Proteins fold into their unique compact three-dimensional structures to precisely perform their functions. Knowledge of proteins on their structures and interactions with other molecules or biomacromolecules is fundamental to understand the mechanism of many diseases. Thus, in this thesis, the focus is on studying the physical properties of proteins, and protein interactions using microfluidic techniques. Chapter 1 provides a brief introduction to protein stability and identification as well as their interactions. Then, conventional techniques for studying protein systems are reviewed. Moreover, the principles, designs and applications of microfluidic techniques are introduced. Chapter 2 demonstrates that a microfluidic diffusional sizing device can be employed to study protein interactions with small molecules by measuring the variation of hydrody- namic radius of bovine serum albumin (BSA) in aqueous solution as a function of pH. By simulating the behaviour of folded and unfolded BSA, the relative population of BSA in dif- ferent states can be calculated. In addition, the key residues that regulate the BSA unfolding process are predicted. Furthermore, I utilize a modified microfluidic diffusional sizing device to measure the hydrodynamic radius of Escherichia coli 70S ribosome and study its interactions with antibiotics, such as chloramphenicol. In Chapter 3, a microfluidic device for protein identification is designed to measure the physical parameters of proteins, including the fluorescence intensities of specific amino acids (tryptophan, tyrosine, lysine) and the hydrodynamic radius of proteins. Thus, proteins can be correctly identified in a single microfluidic device. The results have significant implications in development of effective and efficient techniques for precise protein identification in their native state. In Chapter 4, the interactions between a membrane protein (aquaporins) and a small regulator protein (calmodulin) are investigated by detecting the changes of their electrophoretic mobility and diffusivity in microfluidic devices. Then the effective charges of aquaporin0 and its complex with calmodulin are calculated. Finally, the selective binding between calmodulin and different types of aquaporins is investigated based on their relative binding affinities.

Published
2019
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49. The interplay of dopaminergic genotype and parent–child relationship in relation to intra‐individual response time variability in preschoolers: A replication study.

Author

Zhang, Yuewen and Wang, Zhenhong

Subjects
*ATTENTION control, *PRESCHOOL children, *COGNITIVE ability, *STIMULUS & response (Psychology), *COGNITIVE development
Abstract

Intra‐individual response time variability (IIRTV) during cognitive performance is increasingly recognized as an important indicator of attentional control (AC) and related brain region function. However, what determinants contribute to preschoolers' IIRTV received little attention. The present study explored the interaction of dopaminergic polygenic composite score (DPCS) and the parent–child relationship in relation to preschoolers' IIRTV. In the initial sample, 452 preschoolers (M age = 5.17, SD = 0.92) participated in the study. The modified Flanker task was used to evaluate children's IIRTV and their parents were requested to complete the Parent–Child Relationship Scale to assess the parent–child relationship (closeness/conflict). DNA data were extracted from children's saliva samples, and a DPCS was created by the number of COMT, DAT1, and DRD2 alleles associated with lower dopamine levels. Results showed that DPCS significantly interacted with the parent–child closeness to impact preschoolers' IIRTV. Specifically, preschoolers with higher DPCS exhibited lower IIRTV under higher levels of the parent–child closeness, and greater IIRTV under lower levels of the parent–child closeness compared to those with lower DPCS, which supported the differential susceptibility theory (DST). A direct replication attempt with 280 preschoolers (M age = 4.80, SD = 0.86) was conducted to investigate whether the results were in accordance with our exploratory outcomes. The interactive effect of DPCS and the parent–child closeness on IIRTV was confirmed. Additionally, the significant interactive effect of DPCS and the parent–child conflict on IIRTV was found in the replication study. The findings indicate that preschoolers' IIRTV, as an indicator of AC and related brain region function, is influenced by the interactions of dopaminergic genotypes and the parent–child relationship. Research Highlights: We investigated the Gene × Environment mechanism to underline the intra‐individual response time variability as an indicator of attentional control (AC) in Chinese preschoolers.Dopaminergic polygenic composite score (COMT, DAT1, and DRD2) interacted with the parent–child relationship to predict preschoolers' intra‐individual reaction time variability.A direct replication attempt has been conducted, and the results were in accordance with our exploratory outcomes, which increased the credibility of the present findings.The findings highlight the importance of considering precursors, including polygenic and environmental factors, which contribute to the development of early cognitive performance such as AC. [ABSTRACT FROM AUTHOR]

Published
2024
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