DIA-Based Proteome Profiling of Nasopharyngeal Swabs from COVID-19 Patients

Since
the recent outbreak of COVID-19, there have been intense
efforts to understand viral pathogenesis and host immune response
to combat SARS-CoV-2. It has become evident that different host alterations
can be identified in SARS-CoV-2 infection based on whether infected
cells, animal models or clinical samples are studied. Although nasopharyngeal
swabs are routinely collected for SARS-CoV-2 detection by RT-PCR testing,
host alterations in the nasopharynx at the proteomic level have not
been systematically investigated. Thus, we sought to characterize
the host response through global proteome profiling of nasopharyngeal
swab specimens. A mass spectrometer combining trapped ion mobility
spectrometry (TIMS) and high-resolution QTOF mass spectrometer with
parallel accumulation-serial fragmentation (PASEF) was deployed for
unbiased proteome profiling. First, deep proteome profiling of pooled
nasopharyngeal swab samples was performed in the PASEF enabled DDA
mode, which identified 7723 proteins that were then used to generate
a spectral library. This approach provided peptide level evidence
of five missing proteins for which MS/MS spectrum and mobilograms
were validated with synthetic peptides. Subsequently, quantitative
proteomic profiling was carried out for 90 individual nasopharyngeal
swab samples (45 positive and 45 negative) in DIA combined with PASEF,
termed as diaPASEF mode, which resulted in a total of 5023 protein
identifications. Of these, 577 proteins were found to be upregulated
in SARS-CoV-2 positive samples. Functional analysis of these upregulated
proteins revealed alterations in several biological processes including
innate immune response, viral protein assembly, and exocytosis. To
the best of our knowledge, this study is the first to deploy diaPASEF
for quantitative proteomic profiling of clinical samples and shows
the feasibility of adopting such an approach to understand mechanisms
and pathways altered in diseases.