Abstract
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.
Highlights
Recent advances in data-independent acquisition (DIA), especially owing to the high-performance mass spectrometers and improved data analysis strategies, have enabled unbiased measurement of proteomes.[1−4] A major benefit of DIA compared to data-dependent acquisition (DDA)-based approaches is the reproducible measurements across runs, which allows application to large sample cohorts.[5]
Because most of the identified proteins related to endoplasmic reticulum (ER) or Golgi have not been reported in SARS-CoV-2 studies, we evaluated the protein−protein interaction (PPI) network of each process to enumerate molecules playing a pivotal role in each process
We have demonstrated that comprehensive proteomic profiling of clinical samples using the diaPASEF approach could help decipher host response in response to SARS-CoV-2 infection
Summary
Recent advances in data-independent acquisition (DIA), especially owing to the high-performance mass spectrometers and improved data analysis strategies, have enabled unbiased measurement of proteomes.[1−4] A major benefit of DIA compared to data-dependent acquisition (DDA)-based approaches is the reproducible measurements across runs, which allows application to large sample cohorts.[5]. The upper respiratory tract including the nasopharyngeal mucosa can be sampled using nasopharyngeal swabs (NP swabs), which are the standard sampling for diagnostic testing for COVID-19 In this regard, while several mass spectrometry-based studies have employed NP swabs for detection of viral peptides,[21−23] host proteome profiling of NP swabs has lagged behind[14] it offers an attractive opportunity for studying the impact of SARS-CoV-2 on epithelial as well as immune cells. Proteome profiles of 45 SARS-CoV-2 positive and 45 negative NP swab samples were acquired with diaPASEF mode. The isolation width was set as 25 m/z and precursor ions of 400−1,200 m/z and ion mobility of 0.69−1.47 V s cm−2 were monitored with 64 windows.[6] Collision energy was ramped linearly from 20 to 59 eV as was done for the PASEF-DDA experiments.
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