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. Systemswide 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.