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

Geert A Van Raemdonck, Kara K Osbak, Xaveer Van Ostade, Chris R Kenyon, Geert A Van Raemdonck, Kara K Osbak, Xaveer Van Ostade, Chris R Kenyon

Abstract

Background: Current syphilis diagnostic strategies are lacking a sensitive manner of directly detecting Treponema pallidum antigens. A diagnostic test that could directly detect T. pallidum antigens 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 candidate T. pallidum biomarker proteins were chosen according to their physiochemical characteristics, T. pallidum 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. 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 purified T. pallidum were prepared in PBS. Polyclonal anti- T. pallidum 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 T. pallidum/ml in PBS. Conclusions: Biomarker protein detection likely failed due to the low (femtomoles/liter) predicted concentration of T. pallidum proteins. Alternative sample preparation strategies may improve the detectability of T. pallidum proteins in biofluids.

Trial registration: ClinicalTrials.gov NCT02059525.

Keywords: MRM; Multiple Reaction Monitoring; Treponema pallidum; antigen test; biomarker discovery; plasma; syphilis; targeted proteomics.

Conflict of interest statement

No competing interests were disclosed.

Figures

Figure 1.. Intensity plots displaying MRM experiments…
Figure 1.. Intensity plots displaying MRM experiments on plasma from an individual with secondary stage syphilis.
(a) synthetic hPTPs, even numbers and (b) endogenous (T. pallidum) PTPs, odd numbers; gradient 1 of 3. For each peptide the number of selected transitions (channels) is reported. The x-axis shows the chromatographic retention time of the corresponding peptide while the y-axis shows the relative intensity of the MS2 signal. Note: Signal fluctuations present in the ‘endogenous’ PTP chromatogram are always the result of just one transition, often coupled with a shift in retention time and differing m/z-values differ from the hPTP run, thus these are considered to be noise.
Figure 2.. Work-flow diagram describing the estimation…
Figure 2.. Work-flow diagram describing the estimation of T. pallidum protein MS LOD experiments.
In total, eight different concentrations ofT. pallidum (from 10 4 to 0 bacteria/ml PBS) were treated in three different ways i)T. pallidum was enriched using magnetic beads coated with polyclonal anti-T. pallidum antibodies and lysed by sonication for release ofT. pallidum proteins in the supernatant. Acetone precipitated proteins were trypsinized; ii) In order to detect any remaining protein on the beads, the beads were also trypsinized (retentant on-bead trypsinization); iii) As a control, non-enriched samples were sonicated and immediately trypsinized. *-proteins selected as candidate biomarkers in this study. All samples were analysed by an LTQ-Orbitrap mass spectrometer.
Figure 3.. T. pallidum proteins detected in…
Figure 3.. T. pallidum proteins detected in LOD magnetic bead coupled polyclonal anti- T. pallidum antibody enrichment experiments (protein and peptide identification threshold of 95 %).
*-proteins selected as candidate biomarkers in this study.

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