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Current syphilis diagnostic strategies are lacking a sensitive manner of directly detecting  antigens. A diagnostic test that could directly detect  antigens in individuals with syphilis would be of considerable clinical utility, especially for the diagnosis of reinfections and for post-treatment serological follow-up.  In this study, 11 candidate  biomarker proteins were chosen according to their physiochemical characteristics,  specificity 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.  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 purified  were prepared in PBS. Polyclonal anti-  antibodies 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 300 /ml in PBS.  Biomarker protein detection likely failed due to the low (femtomoles/liter) predicted concentration of  proteins. Alternative sample preparation strategies may improve the detectability of  proteins in biofluids.

f1000research

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