Dataset Information

Targeted Detection of SARS-CoV-2 Nucleocapsid Sequence Variants by Mass Spectrometric Analysis of Tryptic Peptides.

ABSTRACT: COVID-19 vaccines are becoming more widely available, but accurate and rapid testing remains a crucial tool for slowing the spread of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) virus. Although the quantitative reverse transcription-polymerase chain reaction (qRT-PCR) remains the most prevalent testing methodology, numerous tests have been developed that are predicated on detection of the SARS-CoV-2 nucleocapsid protein, including liquid chromatography-tandem mass spectrometry (LC-MS/MS) and immunoassay-based approaches. The continuing emergence of SARS-CoV-2 variants has complicated these approaches, as both qRT-PCR and antigen detection methods can be prone to missing viral variants. In this study, we describe several COVID-19 cases where we were unable to detect the expected peptide targets from clinical nasopharyngeal swabs. Whole genome sequencing revealed that single nucleotide polymorphisms in the gene encoding the viral nucleocapsid protein led to sequence variants that were not monitored in the targeted assay. Minor modifications to the LC-MS/MS method ensured detection of the variants of the target peptide. Additional nucleocapsid variants could be detected by performing the bottom-up proteomic analysis of whole viral genome-sequenced samples. This study demonstrates the importance of considering variants of SARS-CoV-2 in the assay design and highlights the flexibility of mass spectrometry-based approaches to detect variants as they evolve.

SUBMITTER: Maus A

PROVIDER: S-EPMC8610007 | biostudies-literature | 2022 Jan

REPOSITORIES: biostudies-literature

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Targeted Detection of SARS-CoV-2 Nucleocapsid Sequence Variants by Mass Spectrometric Analysis of Tryptic Peptides.

Maus Anthony A Renuse Santosh S Kemp Jennifer J Madugundu Anil K AK Vanderboom Patrick M PM Blommel Joseph J Jerde Calvin C Dasari Surendra S Kipp Benjamin R BR Singh Ravinder J RJ Grebe Stefan K SK Pandey Akhilesh A

Journal of proteome research 20211115 1

COVID-19 vaccines are becoming more widely available, but accurate and rapid testing remains a crucial tool for slowing the spread of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) virus. Although the quantitative reverse transcription-polymerase chain reaction (qRT-PCR) remains the most prevalent testing methodology, numerous tests have been developed that are predicated on detection of the SARS-CoV-2 nucleocapsid protein, including liquid chromatography-tandem mass spectromet ...[more]

PMID: 34779632

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Project description:Chondroitin sulfate proteoglycans (CSPGs) are extracellular matrix components composed of linear glycosaminoglycan (GAG) side chains attached to a core protein. CSPGs play a vital role in neurodevelopment, signal transduction, cellular proliferation and differentiation and tumor metastasis through interaction with growth factors and signaling proteins. These pleiotropic functions of proteoglycans are regulated spatiotemporally by the GAG chains attached to the core protein. There are over 70 chondroitin sulfate-linked proteoglycans reported in cells, cerebrospinal fluid and urine. A core glycan linker of 3-6 monosaccharides attached to specific serine residues can be extended by 20-200 disaccharide repeating units making intact CSPGs very large and impractical to analyze. The current paradigm of CSPG analysis involves digesting the GAG chains by chondroitinase enzymes and analyzing either the protein part, the disaccharide repeats, or both by mass spectrometry. This method, however, provides no information about the site of attachment or the composition of linker oligosaccharides and the degree of sulfation and/or phosphorylation. Further, the analysis by mass spectrometry and subsequent identification of novel CSPGs is hampered by technical challenges in their isolation, less optimal ionization and data analysis. Unknown identity of the linker oligosaccharide also makes it more difficult to identify the glycan composition using database searching approaches. Following chondroitinase digestion of long GAG chains linked to tryptic peptides, we identified intact GAG-linked peptides in clinically relevant samples including plasma, urine and dermal fibroblasts. These intact glycopeptides including their core linker glycans were identified by mass spectrometry using optimized stepped higher energy collision dissociation and electron-transfer/higher energy collision dissociation combined with hybrid database search/de novo glycan composition search. We identified 25 CSPGs including three novel CSPGs that have not been described earlier. Our findings demonstrate the utility of combining enrichment strategies and optimized high-resolution mass spectrometry analysis including alternative fragmentation methods for the characterization of CSPGs.Supplementary informationThe online version contains supplementary material available at 10.1007/s42485-022-00092-3.

Dataset Information

Targeted Detection of SARS-CoV-2 Nucleocapsid Sequence Variants by Mass Spectrometric Analysis of Tryptic Peptides.

Publications

Targeted Detection of SARS-CoV-2 Nucleocapsid Sequence Variants by Mass Spectrometric Analysis of Tryptic Peptides.

Similar Datasets

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