Matrix-Assisted Laser Desorption and Ionization Time-of-Flight Mass Spectrometry Analysis for the Direct Detection of SARS-CoV-2 in Nasopharyngeal Swabs

Abstract

The most common diagnostic method used for coronavirus disease-2019 (COVID-19) is real-time reverse transcription polymerase chain reaction (PCR). However, it requires complex and labor-intensive procedures and involves excessive positive results derived from viral debris. We developed a method for the direct detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in nasopharyngeal swabs, which uses matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-ToF MS) to identify specific peptides from the SARS-CoV-2 nucleocapsid phosphoprotein (NP). SARS-CoV-2 viral particles were separated from biological molecules in nasopharyngeal swabs by an ultrafiltration cartridge. Further purification was performed by an anion exchange resin, and purified NP was digested into peptides using trypsin. The peptides from SARS-CoV-2 that were inoculated into nasopharyngeal swabs were detected by MALDI-ToF MS, and the limit of detection was 10 6.7 viral copies. This value equates to 10 7.9 viral copies per swab and is approximately equivalent to the viral load of contagious patients. Seven NP-derived peptides were selected as the target molecules for the detection of SARS-CoV-2 in clinical specimens. The method detected between two and seven NP-derived peptides in 19 nasopharyngeal swab specimens from contagious COVID-19 patients. These peptides were not detected in four specimens in which SARS-CoV-2 RNA was not detected by PCR. Mutated NP-derived peptides were found in some specimens, and their patterns of amino acid replacement were estimated by accurate mass. Our results provide evidence that the developed MALDI-ToF MS-based method in a combination of straightforward purification steps and a rapid detection step directly detect SARS-CoV-2-specific peptides in nasopharyngeal swabs and can be a reliable high-throughput diagnostic method for COVID-19.

MALDI-TOF Mass Spectrometric Detection of SARS-CoV-2 Using Cellulose Sulfate Ester Enrichment and Hot Acid Treatment

“… 16 Antibody-based affinity, ultrahigh-speed centrifugation, and ultrafiltration methods have been proposed to enrich viral materials. 17 The sulfate ester column-based enrichment method we present in this study has several advantages. Compared with other enrichment methods, our column-based method only requires sample loading, washing, and elution, which can be done feasibly without intensive personnel training.…”

“…Yoshinari et al made a substantial improvement and reported an approach for the direct detection of SARS-CoV-2 in human nasopharyngeal swabs using a virus purification workflow based on filtration and anion exchange chromatography. 17 In this workflow, nonviral proteins were removed by molecular weight cutoff ultrafiltration (MWCO). The virus was lysed with 2-propanol and the anionic nucleocapsid protein (N protein) was preferentially adsorbed on positively charged Sepharose XL.…”

“…The virus was lysed with 2-propanol and the anionic nucleocapsid protein (N protein) was preferentially adsorbed on positively charged Sepharose XL. The enriched N protein was then treated using trypsin digestion, and peptide signatures of the N protein were characterized by MALDI-TOF MS. 17 Yoshinari and coauthors made critical contributions to the unambiguous detection of SARS-CoV-2 using pathogen signatures. However, the virus purification and peptide preparation protocols are labor-intensive and slow, as they include ultra-high-speed centrifugation and trypsin digestion.…”

“…However, the virus purification and peptide preparation protocols are labor-intensive and slow, as they include ultra-high-speed centrifugation and trypsin digestion. 17 …”

“…Approaches using MALDI-TOF MS as a diagnostic tool for COVID-19 have been demonstrated. − On the basis of the current reports, the detection strategy mainly relies on applying machine learning and artificial intelligence algorithms (AI/ML) to mass peaks (features) extracted from MALDI-TOF MS of either blood, saliva, or nasal swab specimens. − Since the mass peaks acquired by MALDI-TOF MS are generated from human specimens, the features that drive the separation between SARS-CoV-2 positive and negative subjects are drawn from both host-response markers and pathogen signatures. As a result, researchers only report masses in MALDI-TOF MS.…”