Needle lost in the haystack: multiple reaction monitoring fails to detect Treponema pallidum candidate protein biomarkers in plasma and urine samples from individuals with syphilis

Abstract

Background:Current syphilis diagnostic strategies are lacking a sensitive manner of directly detectingTreponema pallidumantigens. A diagnostic test that could directly detectT. pallidumantigens in individuals with syphilis would be of considerable clinical utility, especially for the diagnosis of reinfections and for post-treatment serological follow-up.Methods:In this study, 11 candidateT. pallidumbiomarker proteins were chosen according to their physiochemical characteristics,T. pallidumspecificity and predicted abundance. Thirty isotopically labelled proteotypic surrogate peptides (hPTPs) were synthesized and incorporated into a scheduled multiple reaction monitoring assay. Protein extracts from undepleted/unenriched plasma (N = 18) and urine (N = 4) samples from 18 individuals with syphilis in various clinical stages were tryptically digested, spiked with the hPTP mixture and analysed with a triple quadruple mass spectrometer.Results:No endogenous PTPs corresponding to the eleven candidate biomarkers were detected in any samples analysed. To estimate the Limit of Detection (LOD) of a comparably sensitive mass spectrometer (LTQ-Orbitrap), two dilution series of rabbit cultured purifiedT. pallidumwere prepared in PBS. Polyclonal anti-T. pallidumantibodies coupled to magnetic Dynabeads were used to enrich one sample series; no LOD improvement was found compared to the unenriched series. The estimated LOD of MS instruments is 300T. pallidum/ml in PBS.Conclusions:Biomarker protein detection likely failed due to the low (femtomoles/liter) predicted concentration ofT. pallidumproteins. Alternative sample preparation strategies may improve the detectability ofT. pallidumproteins in biofluids.