Coupling immuno-magnetic capture with LC–MS/MS(MRM) as a sensitive, reliable, and specific assay for SARS-CoV-2 identification from clinical samples

Abstract

Graphical abstract Recently, numerous diagnostic approaches from different disciplines have been developed for SARS-CoV-2 diagnosis to monitor and control the COVID-19 pandemic. These include MS-based assays, which provide analytical information on viral proteins. However, their sensitivity is limited, estimated to be 5 × 10 4 PFU/ml in clinical samples. Here, we present a reliable, specific, and rapid method for the identification of SARS-CoV-2 from nasopharyngeal (NP) specimens, which combines virus capture followed by LC–MS/MS(MRM) analysis of unique peptide markers. The capture of SARS-CoV-2 from the challenging matrix, prior to its tryptic digestion, was accomplished by magnetic beads coated with polyclonal IgG-α-SARS-CoV-2 antibodies, enabling sample concentration while significantly reducing background noise interrupting with LC–MS analysis. A sensitive and specific LC–MS/MS(MRM) analysis method was developed for the identification of selected tryptic peptide markers. The combined assay, which resulted in S/N ratio enhancement, achieved an improved sensitivity of more than 10-fold compared with previously described MS methods. The assay was validated in 29 naive NP specimens, 19 samples were spiked with SARS-CoV-2 and 10 were used as negative controls. Finally, the assay was successfully applied to clinical NP samples ( n = 26) pre-determined as either positive or negative by RT-qPCR. This work describes for the first time a combined approach for immuno-magnetic viral isolation coupled with MS analysis. This method is highly reliable, specific, and sensitive; thus, it may potentially serve as a complementary assay to RT-qPCR, the gold standard test. This methodology can be applied to other viruses as well. Supplementary Information The online version contains supplementary material available at 10.1007/s00216-021-03831-5.

Proteomics‐based mass spectrometry profiling of SARS‐CoV‐2 infection from human nasopharyngeal samples

“…After enrichment and tryptic digestion for viral proteins, analysis was completed on an RP ultra‐performance liquid chromatography (UPLC) combined with Xevo Triple‐Q MS in MRM mode. After rounds of method optimization, they improved the sensitivity and selectivity of the workflow (Schuster et al, 2022 ). Given the complexity of nasal swab samples, enrichment steps to further isolate the SARS‐CoV‐2 proteome is recommended to improve the identification and quantitation of these proteins.…”

SARS-CoV-2 spike antigen quantification by targeted mass spectrometry of a virus-based vaccine

“…Ammonium bicarbonate (NH 4 HCO 3 , Cat Number A6141-500 G) and Octyl-β-D-glucopyranoside (OG, Cat Number O8001-1 G) were purchased from Sigma-Aldrich. Spike protein was synthesized in-house as previously described ( Schuster et al, 2022 , 2021 ). Phosphate‐buffered saline (PBS, pH 7.4, Cat.…”

“…Therefore, it can selectively identify and quantify compounds within complex matrices. To improve the assay’s performances, an additional step has been added, in which the virus is captured and thereby enriched by immuno-magnetic beads, which reduces background interferences ( Schuster et al, 2022 ).…”

“…We therefore applied IMS of VSV-ΔG-spike from the formulation matrix. This can be achieved using immuno-magnetic beads, as described recently in our lab ( Schuster et al, 2022 ). In this study, a rapid and simple protocol for SARS-CoV-2 capture from challenging matrices, prior to its tryptic digestion, was accomplished using magnetic beads coated with polyclonal IgG anti SARS-CoV-2 antibodies (anti spike or anti SARS-CoV-2), enabling sample concentration while significantly reducing background noise that interferes with LC-MS analysis.…”

Evaluation of Variant-Specific Peptides for Detection of SARS-CoV-2 Variants of Concern

“…These approaches use preset channels each with a specified retention time and m / z signals that are searched. To keep the MS analysis time short, typically four peptides are detected. − DIA has been performed in nasal pharyngeal swabs, saliva, and gargle solutions to provide fast and sensitive analyses using an inclusion list of tryptic peptides from SAR-CoV-2 protein. ,,, To compare the limits of detection (LOD)­s, we converted the unit to ng (0.6 ng viral protein material = 1 pfu) and found reported values varied from 2 to 460 ng. The Cov-MS consortium reported detection limits of 2 pg (0.00333 pfu), using an 8 min MRM schedule consisting of 30 transitions for 10 selected peptides with dedicated collision energy and cone voltage .…”