Cargando…

Untargeted analysis of the airway proteomes of children with respiratory infections using mass spectrometry based proteomics

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 seaml...

Descripción completa

Detalles Bibliográficos
Autores principales: Sande, Charles J., Mutunga, Martin, Muteti, Jacqueline, Berkley, James A., Nokes, D. James, Njunge, James
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group UK 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6138648/
https://www.ncbi.nlm.nih.gov/pubmed/30217988
http://dx.doi.org/10.1038/s41598-018-32072-3
_version_ 1783355369714614272
author Sande, Charles J.
Mutunga, Martin
Muteti, Jacqueline
Berkley, James A.
Nokes, D. James
Njunge, James
author_facet Sande, Charles J.
Mutunga, Martin
Muteti, Jacqueline
Berkley, James A.
Nokes, D. James
Njunge, James
author_sort Sande, Charles J.
collection PubMed
description 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.
format Online
Article
Text
id pubmed-6138648
institution National Center for Biotechnology Information
language English
publishDate 2018
publisher Nature Publishing Group UK
record_format MEDLINE/PubMed
spelling pubmed-61386482018-09-15 Untargeted analysis of the airway proteomes of children with respiratory infections using mass spectrometry based proteomics Sande, Charles J. Mutunga, Martin Muteti, Jacqueline Berkley, James A. Nokes, D. James Njunge, James Sci Rep Article 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. Nature Publishing Group UK 2018-09-14 /pmc/articles/PMC6138648/ /pubmed/30217988 http://dx.doi.org/10.1038/s41598-018-32072-3 Text en © The Author(s) 2018 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
spellingShingle Article
Sande, Charles J.
Mutunga, Martin
Muteti, Jacqueline
Berkley, James A.
Nokes, D. James
Njunge, James
Untargeted analysis of the airway proteomes of children with respiratory infections using mass spectrometry based proteomics
title Untargeted analysis of the airway proteomes of children with respiratory infections using mass spectrometry based proteomics
title_full Untargeted analysis of the airway proteomes of children with respiratory infections using mass spectrometry based proteomics
title_fullStr Untargeted analysis of the airway proteomes of children with respiratory infections using mass spectrometry based proteomics
title_full_unstemmed Untargeted analysis of the airway proteomes of children with respiratory infections using mass spectrometry based proteomics
title_short Untargeted analysis of the airway proteomes of children with respiratory infections using mass spectrometry based proteomics
title_sort untargeted analysis of the airway proteomes of children with respiratory infections using mass spectrometry based proteomics
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6138648/
https://www.ncbi.nlm.nih.gov/pubmed/30217988
http://dx.doi.org/10.1038/s41598-018-32072-3
work_keys_str_mv AT sandecharlesj untargetedanalysisoftheairwayproteomesofchildrenwithrespiratoryinfectionsusingmassspectrometrybasedproteomics
AT mutungamartin untargetedanalysisoftheairwayproteomesofchildrenwithrespiratoryinfectionsusingmassspectrometrybasedproteomics
AT mutetijacqueline untargetedanalysisoftheairwayproteomesofchildrenwithrespiratoryinfectionsusingmassspectrometrybasedproteomics
AT berkleyjamesa untargetedanalysisoftheairwayproteomesofchildrenwithrespiratoryinfectionsusingmassspectrometrybasedproteomics
AT nokesdjames untargetedanalysisoftheairwayproteomesofchildrenwithrespiratoryinfectionsusingmassspectrometrybasedproteomics
AT njungejames untargetedanalysisoftheairwayproteomesofchildrenwithrespiratoryinfectionsusingmassspectrometrybasedproteomics