SISCAPA-based approach to detect SARS-CoV-2: What is it?

Sumedha B S, Bangalore University

The novel human coronavirus, SARS-CoV-2 has created a global disease burden infecting>100 million humans in just over a year. It has become a major challenge to the health system worldwide. Detection is very crucial in this pandemic. Studies have shown that early detection is very necessary for disease control. Currently, the gold standard testing is the RT-PCR method. It is a Reverse transcriptase-polymerase chain reaction. Until now, a variety of tests have been developed to detect the viral antigen.  Several other methods developed are based on the detection of viral proteins, antiviral antibodies, and nucleic acids.  Alternate screening methodologies need to be explored as there are some issues concerning reagents and instruments for RT-PCR-based tests

Mass spectrometry in testing for SARS-CoV-2:

Mass spectrometry is an exceptional analytical tool that can be applied for the analysis of a wide range of analytes such as small molecules and peptides.

The use of multiple reaction monitoring (MRM) for targeted analysis is a feasible alternative to immunoassays. Data analysis and automation for sample preparation increase the throughput.

In this study, researchers produced antibodies against viral peptides. These peptides were derived from the nucleocapsid of the virus.  They developed a semiautomated, sensitive SISCAPA-based approach. Detection of the virus was from nasopharyngeal swab samples of COVID-19 patients. Using this method, they were able to successfully detect viral peptides directly from patient samples. Detections also included those with low viral loads.

The novel approach Methodology:

Nucleocapsid protein sequences and target peptides of SARS-CoV-2, MERS, and the common human coronaviruses were obtained. Multiple sequence alignment of these sequences was carried out using Clustal Omega at EMBL-EBI. SARS-CoV-2 belongs to the family of coronaviruses which includes common cold viruses. Using MSA, peptides unique to SARS-CoV-2 were selected. From these peptides, those having amino acids that could undergo any modifications were removed. Peptides that could be reliably detected by mass spectrometry were selected. The selected peptides were – NPANNAAIVLQLPQGTTLPK ITFGGPSDSTGSNNQNGER, and DGIIWVATEGALNTPK. These were purified using high-performance liquid chromatography.

The polyclonal anti-peptide antibodies were conjugated to custom Mass Spectrometric Immunoassay (MSIA). All the samples were then processed and digested. The digested samples were subjected to binding and elution using Versette PlateMate.

The data analysis was performed using Skyline. The peaks were detected, the peak area and coefficient of variation were calculated.

Significance of the study:

The SISCAPA approach uses immunoprecipitation of peptides as surrogates for protein quantitation. So, the selection of peptides unique to the protein of interest is a crucial step. The nucleocapsid protein is being used for the development of assays for the detection of the SARS-CoV-2 virus in clinical samples. This is because it is the most abundant protein in the SARS-CoV-2 proteome.

However, during viral replication and immunoprecipitation nucleocapsid protein interacts with various cellular proteins. This could decrease the sensitivity of the method due to increased ‘background matrix complexity’. To increase the sensitivity of the targeted assay, enrichment of peptides from digested samples was done using anti-peptides antibodies.

Thus, the targeted workflow developed involved-

• digestion of nasopharyngeal swab samples,
• enrichment of viral peptides and
• parallel reaction monitoring analysis using a mass spectrometer

This approach was applied to 30 negative samples and 41 SARS-CoV-2 positive RT-PCR swab samples.

The method was found to be highly sensitive because, in negative samples, none of the target peptides were detected. The peptides detected showed a positive correlation with viral loads.

This approach can be used as an alternate method to detect SARS-CoV-2. This approach can also be applied for detecting other pathogens directly from clinical samples.

Also read: World Science Day for Peace and Development 2021

Reference:

1. Mangalaparthi, K. K. (2021, October 22). A SISCAPA-based approach for detection of SARS-CoV-2 viral antigens from clinical samples. Clinical Proteomics. https://clinicalproteomicsjournal.biomedcentral.com/articles/10.1186/s12014-021-09331-z#citeas

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