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Untargeted analysis of the airway proteomes of children with respiratory infections using mass spectrometry based proteomics
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Sande, Charles J., Mutunga, Martin, Muteti, Jacqueline, Berkley, James A., Nokes, D. James and Njunge, James (2018) Untargeted analysis of the airway proteomes of children with respiratory infections using mass spectrometry based proteomics. Scientific Reports, 8 (1). 13814 . doi:10.1038/s41598-018-32072-3 ISSN 2045-2322.
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WRAP-untargeted-analysis-airway-proteomes-children-mass-proteomics-Nokes-2018.pdf - Published Version - Requires a PDF viewer. Available under License Creative Commons Attribution 4.0. Download (3027Kb) | Preview |
Official URL: http://dx.doi.org/10.1038/s41598-018-32072-3
Abstract
The upper airway – which consists mainly of the naso- and oro-pharynx - is the first point of contact between the respiratory system and microbial organisms that are ubiquitous in the environment. It has evolved highly specialised functions to address these constant threats whilst facilitating seamless respiratory exchange with the lower respiratory tract. Dysregulation of its critical homeostatic and defence functions can lead to ingress of pathogens into the lower respiratory tract, potentially leading to serious illness. Systems-wide proteomic tools may facilitate a better understanding of mechanisms in the upper airways in health and disease. In this study, we aimed to develop a mass spectrometry based proteomics method for characterizing the upper airways proteome. Naso- and oropharyngeal swab samples used in all our experiments had been eluted in the Universal Transport Media (UTM) containing significantly high levels of bovine serum albumin. Our proteomic experiments tested the optimal approach to characterize airway proteome on swab samples eluted in UTM based on the number of proteins identified without BSA depletion (Total proteome: Protocol A) and with its depletion using a commercial kit; Allprep, Qiagen (cellular proteome: Protocol B, Ci, and Cii). Observations and lessons drawn from protocol A, fed into the design and implementation of protocol B, and from B to protocol Ci and finally Cii. Label free proteome quantification was used in Protocol A (n = 6) and B (n = 4) while commercial TMT 10plex reagents were used for protocols Ci and ii (n = 83). Protocols Ci and ii were carried out under similar conditions except for the elution gradient: 3 h and 6 h respectively. Swab samples tested in this study were from infants and children with and without upper respiratory tract infections from Kilifi County Hospital on the Kenyan Coast. Protocol A had the least number of proteins identified (215) while B produced the highest number of protein identifications (2396). When Protocol B was modified through sample multiplexing with TMT to enable higher throughput (Protocol Ci), the number of protein identified reduced to 1432. Modification of protocol Ci by increasing the peptide elution time generated Protocol Cii that substantially increased the number of proteins identified to 1875. The coefficient of variation among the TMT runs in Protocol Cii was <20%. There was substantial overlap in the identity of proteins using the four protocols. Our method was were able to identify marker proteins characteristically expressed in the upper airway. We found high expression levels of signature nasopharyngeal and oral proteins, including BPIFA1/2 and AMY1A, as well as a high abundance of proteins related to innate and adaptive immune function in the upper airway. We have developed a sensitive systems-level proteomic assay for the systematic quantification of naso-oro-pharyngeal proteins. The assay will advance mechanistic studies of respiratory pathology, by providing an untargeted and hypothesis-free approach of examining the airway proteome.
Item Type: | Journal Article | ||||||
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Subjects: | Q Science > QP Physiology | ||||||
Divisions: | Faculty of Science, Engineering and Medicine > Science > Life Sciences (2010- ) | ||||||
Library of Congress Subject Headings (LCSH): | Respiratory infections in children, Airway (Medicine), Pharynx, Proteomics, Mass spectrometry | ||||||
Journal or Publication Title: | Scientific Reports | ||||||
Publisher: | Nature Publishing Group | ||||||
ISSN: | 2045-2322 | ||||||
Official Date: | 14 September 2018 | ||||||
Dates: |
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Volume: | 8 | ||||||
Number: | 1 | ||||||
Article Number: | 13814 | ||||||
DOI: | 10.1038/s41598-018-32072-3 | ||||||
Status: | Peer Reviewed | ||||||
Publication Status: | Published | ||||||
Access rights to Published version: | Open Access (Creative Commons) | ||||||
Date of first compliant deposit: | 18 September 2018 | ||||||
Date of first compliant Open Access: | 19 September 2018 | ||||||
RIOXX Funder/Project Grant: |
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