Advances in Sample Preparation (Mar 2022)
Comparison of anti-peptide and anti-protein antibody-based purification techniques for detection of SARS-CoV-2 by targeted LC-MS/MS
Abstract
ABSTRACT: The COVID-19 pandemic has necessitated exploration of alternative testing methods for detection of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) to ensure clinical laboratories can continue to provide critical testing results. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is established in many clinical laboratories due its high specificity and sensitivity, making it a logical alternative methodology. However, matching the sensitivity of quantitative reverse transcription-polymerase chain reaction (qRT-PCR) remains challenging, which forced utilization of antibody-based enrichment prior to targeted LC-MS/MS analysis. When utilizing antibody purification techniques, investigators must decide whether to enrich the target protein or peptides, but there are few studies comparing the two approaches to assist in this decision-making process. In this work, we present a comparison of intact protein and peptide antibody-based purification for LC-MS/MS based detection of SARS-CoV-2. We have found that protein purification yields more intense LC-MS/MS signals, but is also less specific, yielding higher noise and more background when compared to peptide purification techniques. Therefore, when using traditional data analysis techniques, the enrichment technique that provides superior sensitivity varies for individual peptides and no definitive overall conclusion can be made. These observations are corroborated when using a novel machine learning approach to determine positive/negative test results, which yielded superior sensitivity when using protein purification, but better specificity and area under the ROC curve when performing peptide purification.
