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

Study participants

Between January 2014 and August 2015, 120 patients attending the Institute of Tropical Medicine Antwerp clinic, over the age of 17 years, and in whom a new diagnosis of syphilis was made and had not received antibiotics in the preceding thirty days, were recruited into the cohort study. Thirty HIV-positive controls, in whom the diagnosis of syphilis was excluded via serological and PCR testing, were also recruited. The diagnosis and staging of syphilis was according to the Centers for Disease Control and Prevention classification⁴³, and treatment was administered according to European guidelines⁴⁴. All patient sera were tested for syphilis using a RPR test (BD Macro-Vue RPR card test, Becton, Dickinson and Co., Sparks, MD, United States of America (USA)) and an antibody detection Treponema pallidum Particle Agglutination test (SERODIA-TPPA Fujirebio Inc., Tokyo, Japan). A PCR test targeting T. pallidum polA was also performed on serum⁴⁵ and whole blood samples were tested for multiple gene targets⁴⁶, as previously described. Selection criteria of participants from the cohort study for the MRM assay analysis included a range of syphilis clinical stages and prioritized predicted high bacterial loads, as demonstrated by positive PCR tests and/or high RPR titres. Patients with early stage syphilis (primary, secondary, early latent) that were plasma and/or whole blood PCR positive for T. pallidum were expected to have the highest bacterial load^12,13.

Plasma and urine sample processing

Plasma was collected immediately before Benzathine Penicillin G intramuscular injection using 7.5 ml EDTA-coated blood collection tubes (Sarstedt Monovette, Nümbrecht, Germany). We refer to these samples as the pre-penicillin samples. A selection of randomly selected patients participated in an additional blood draw three hours after penicillin treatment since studies have shown penicillin to be fast acting on T. pallidum, leading to consequent cell lysis and antigen release⁴⁷. These samples are termed the post-penicillin samples. Plasma was chosen for the MRM assay according to HUPO guidelines⁴⁸. Protease inhibitors were not added to the plasma samples since previous studies did not demonstrate a significant higher protein yield with treated samples⁴⁹ and peptides could inadvertently be modified⁵⁰. Plasma were subjected to dual centrifugation in an Eppendorf 22331 centrifuge (Hamburg, Germany) in an effort to minimize cellular contamination: whole blood was centrifuged at 2000 g for 10 minutes at ambient temperature, followed by transfer of the plasma fraction to a 50 ml falcon tube and centrifugation at 2400 g for 15 minutes. All plasma were processed and aliquoted into cryovials for storage at -80 °C in a long-term freezer unit (Eppendorf U725-G Innova New Brunswick, Hamburg, Germany) until further testing. Mid-stream random-void urine samples were collected and processed following HUPO guidelines⁵¹, including centrifugation for 10 minutes at 2000 g at ambient temperature in order to remove insoluble contents such as cells and casts. Urine was aliquoted into 15 ml falcon tubes and stored at -80 °C until further testing. All plasma and urine samples were processed within three hours of collection and were only subjected to one freeze thaw cycle.

T. pallidum protein biomarker selection

In a previous descriptive study we used non-gel based complementary MS techniques to characterize the proteome of in vivo rabbit cultured T. pallidum¹⁷. Candidate T. pallidum biomarker proteins for the MRM assay were chosen based on the following specific criteria: relative protein abundance (based on semi-quantitative spectral counting techniques¹⁷), Clusters of Orthologous Groups (COG) functional categorization, microarray transcriptome data¹⁸, protein size, physicochemical properties (i.e. previously detected by MS), predicted subcellular localization¹⁷ and literature review. Each of the candidate biomarkers were digested in silico by subjecting the FASTA-formatted sequences to tryptic digestion, assuming 100 % digestion efficiency. Proteotypic peptides (PTPs) corresponding to these proteins were determined using ESPPredictor⁵² and pBLAST⁵³; analysis of the protein and PTPs was performed to determine possible homology with other bacterial species and human proteins. After PTP selection was finalized, isotopically labelled synthetic peptide standards (hPTPs) corresponding to the selected PTPs were synthesized (Heavy Peptide™ AQUA Basic with > 95 % purity; Thermo Fisher Scientific, Ulm, Germany).

Plasma and urine sample preparation for MRM assay analysis

Protein concentrations of urine and plasma samples were determined based on the area under curve at 214 nm using a RP-C4 column (Vydac 214TP5415; 4.6×150 mm, particle size 5 μm; Alltech Associates Inc., Lokeren, Belgium) coupled to an Alliance e2695 HPLC system equipped with a 996 PDA detector (Waters Corporation, Milford, MA, USA). For each sample, 250 µg of protein was precipitated by adding six volumes of ice cold LC-MS grade acetone (Biosolve, Valkenswaard, the Netherlands) followed by overnight incubation in freezer unit (Liebherr, Bulle, Switzerland) at -20 °C. In all cases, lo-bind Eppendorf tubes (Eppendorf, Hamburg, Germany) were used to ensure high recovery rates of proteins and peptides. Protein pellets were re-suspended in 50 mM Tris-HCl/6 M urea/5 mM dithiothreitol /10 % beta-mercaptoethanol (25 µL/100 µg protein) at pH 8.7. For the denaturation and reduction process all samples were incubated at 65 °C in a hot water bath for 1 hour. Subsequently, proteins in all fractions were diluted in 50 mM Tris-HCl/ 1 mM CaCl₂ (75 µL/100 µg protein) and alkylated by adding 200 mM iodoacetamide (10 µL/100 µg protein) during 1 hour at ambient temperature and protected from light. Proteomics-grade modified trypsin (Promega, Madison, WI, US) was added at a 30:1 protein-to-enzyme ratio. After incubation at 37 °C in a hot water bath for 18 hours the digestion was stopped by freezing the samples. Protein digests were desalted by SPE using GracePure SPE C18-Max (50 mg) (W. R. Grace & Co., Columbia, MD, US) RP cartridges and a vacuum manifold. SPE cartridges were conditioned with 100 % methanol and equilibrated with 100 % LC/MS grade H₂O and 0.1 % formic acid (FA). After loading the complete acidified (0.1 % FA) tryptic digest, peptides were washed with 10 % methanol and eluted with 40 % methanol/ 40 % acetonitrile (ACN) and 0.1 % FA. Eluted peptides were lyophilized and frozen at -20 °C until further analysis. Immediately before analysis, lyophilized digests were resuspended in 5 % ACN/0.1 % FA and spiked with a mixture of all hPTPs.

MRM assay optimization and mass spectrometric analysis

Optimization of each PTP was performed on a triple quadruple mass spectrometer (Waters Xevo TQ, Waters Corporation, Milford, MA, US) in order to obtain the most intense transitions. The capillary voltage was tuned to approximately 2 kV with a source temperature of 150 °C. Desolvation temperature was set at 400 °C with a nitrogen gas flow of 800 L/h. Cone voltage, collision energy and dwell times were optimized for each of the PTPs. All PTPs were dissolved in mobile phase A (MP-A), containing 5 % ACN (LC/MS grade) and 0.1 % FA. For each of the peptides individually, the Limit of Detection (LOD) was determined by performing a dilution series in MP-A. Based on these concentrations, a mixture of all hPTPs was made. A balanced hPTP mixture has been shown to increase quantification accuracy and reproducibility compared to an equimolar mixture in previous studies³⁵. To check for possible suppressive effects of the plasma matrix, the hPTP mixture was spiked into plasma from a control study subject. A balanced mixture of hPTP (concentrations detailed in Supplementary File 1) was spiked into 50 µg of plasma digest. Chromatographic separation of the plasma and urine samples was performed on a RP-C18 UPLC column (Waters, CSH 150 × 2.1 mm, 1.7 µm at 35 °C) connected to an Acquity UPLC system (Waters Corporation, Milford, MA, USA). In order to separate all peptides as best as possible, an optimized linear gradient of Mobile Phase B (MP-B) (0.1 % FA in 100 % ACN) was applied: 5 % MP-B during 1 min and from 5 to 35 % MP-B in 5 min, followed by a steep increase to 100 % MP-B in 1 min, all at a flow rate of 300 µL/min. Based on the specific retention times of each peptide, three scheduled MRM runs of 10 minutes were generated, each of them containing 20 MS1 channels (10 endogenous (T. pallidum) PTPs without isotopic label and 10 channels with a synthetic hPTP equivalent). At least three transitions (ion pairs) were selected for each peptide of interest. For each scheduled MRM analysis, 50 µg of peptides (injection loop of 5 µL) per plasma/urine sample were loaded onto the analytical column. In addition to an extensive needle wash after each injection, a blank run was performed between two subsequent clinical samples to prevent carry-over effects. Data acquisition was controlled by MassLynx version 4.1, while targeted datasets were analysed by TargetLynx, which is part of MassLynx (Waters Corporation, Milford, MA, USA). All Xevo TQ MS raw spectral files are available at PeptideAtlas⁵⁴ with the identifier PASS00978.

Magnetic bead antibody-based enrichment of T. pallidum proteins and approximation of the MS LOD for T. pallidum protein detection

T. pallidum protein enrichment was performed using magnetic beads (Dynabeads® M-270, Life Technologies, CA, USA) coated with biotin-conjugated polyclonal T. pallidum- specific antibodies (PA1-73103, Thermo Fisher Scientific, CA, USA) through streptavidin-biotin conjugation. According to the manufacturer’s protocol, 10 µg of antibody was used to bind 1 mg of beads (approximately 5 × 10⁷ beads).

In vivo rabbit cultured purified T. pallidum DAL-1 strain extracts^55,56 were kindly provided by the group of David Šmajs from the Masaryk University, Czech Republic. The original concentration of the T. pallidum extract was approximately 10⁶ bacteria/ml as quantified under darkfield microscopy using a Olympus BX41 (Olympus Corporation, Tokyo, Japan) equipped with darkfield microscope condenser DCW 1.4-1.2; magnification 10×40. Samples were stored in 1 ml phosphate buffered saline (PBS) and only subjected to one freeze-thaw cycle. Two dilution series of T. pallidum were prepared, each time starting in 1 ml of PBS and finally equating to eight approximate bacterial concentrations: 10⁴, 10³, 300, 100, 33, 10, 3 and 0 bacteria/ml.

For one dilution series, each of the eight fractions were incubated with a constant amount (~10⁵) of magnetic beads coated with polyclonal anti-T. pallidum antibodies. After incubation for two hours at 4° C and magnetic separation, the supernatant was discarded and beads were washed three times with PBS. To lyse the antibody bound bacteria, 1 ml of PBS was added to each bead sample, these were sonicated on ice using a Sonics Vibra Cell VC130 (Sonics and Materials Inc., Newtown, CT, USA) (two times 30 seconds with an amplitude of 50 %). The bead fraction was retained (retentant) after sonication by using magnetic separation. Released proteins were precipitated adding ice-cold acetone and incubated overnight at -20 °C. Tryptic digestion was performed, following the aforementioned procedure, on both the precipitated proteins (supernatant) and directly “on-bead” (retentate), to test for possible unreleased proteins during sonication. For the second dilution series (unenriched), 1 ml was directly drawn from each of the eight samples. The samples from this series were also sonicated on ice (two times 30 seconds with an amplitude of 50 %) to lyse the bacteria. Released proteins were then acetone precipitated and subsequently digested, in conformance with the other parallel series procedure.

Liquid chromatography-electrospray ionization-LTQ-Orbitrap mass spectrometry analysis of enriched and non-enriched serially diluted T. pallidum samples

Peptide mixtures were separated by RPLC on a Waters nano-UPLC system using a nanoACQUITY BEH C18 Trap column (100 Å, 5 μm, 180 μm × 20 mm) connected to a nanoACQUITY BEH C18 analytical Column (130 Å, 1.7 μm, 100 μm × 100 mm) (Waters Corporation, Milford, MA, USA). Peptides were dissolved in MP-A, containing 2 % ACN and 0.1 % FA and spiked with 20 fmol [Glu1]-fibrinopeptide B, which serves as an internal calibrant. A linear gradient of MP-B (0.1 % FA in 98 % ACN) from 2 to 45 % MP-B in 45 min, followed by a steep increase to 95 % MP-B in 2 min at a flow rate of 400 nl/min. The nano-LC was coupled online with a LTQ Orbitrap Velos (Thermo Scientific, San Jose, CA, US) mass spectrometer using a PicoTip Emitter (New Objective, Woburn, MA, US) linked to a nanospray ion source. The mass spectrometer was set up in a data dependent acquisition MS/MS mode where a full scan spectrum (350–2500 m/z, resolution of 60.000) was followed by a maximum of ten CID tandem mass spectra (100 to 2000 m/z). Peptide ions were selected as the twenty most intense peaks of the MS scan. CID scans were acquired in the LTQ ion trap part of the mass spectrometer with normalized collision energy of 32 %.

Obtained spectra were screened against the T. pallidum reference and resequenced databases (UniProt ID proteome UP000014259¹⁵ and UP000000811⁵⁷ using the MASCOT search engine (Matrix Science; version 2.1.03) based on the digestion enzyme trypsin. Carbamidomethylation of cysteines was listed as a fixed modification, while methionine oxidation was set as a variable modification. A maximum of one missed cleavage was tolerated. Mass tolerance was set to 10 ppm for the precursors and 0.8 Da for the fragment ions. False discovery rate was set at 5 %. Scaffold Q+ (version 4.6.2, Proteome Software Inc., Portland, OR, US) was used to validate MS/MS-based peptide and protein identifications. Protein identifications were accepted if they could be established at greater than 95.0 % probability according to the protein prophet algorithm⁵⁸.

All LTQ-Orbitrap MS/MS raw spectral data is available at PeptideAtlas⁵⁴ with the identifier PASS00978.

Study subject inclusion

Eighteen syphilis-infected study participants were selected for the MRM assay analyses (Table 1). All participants were male and identified as MSM. A third of the participants (6/18; 33 %) were HIV positive. Five (28 %) presented with primary, eleven secondary (61 %), and two early latent (11 %) stage disease. Thirteen participants were confirmed T. pallidum-positive by serum and/or whole blood PCR testing. Four participants had indeterminate PCR results, meaning their sample was weakly positive. A second confirmatory PCR was not performed on these samples. One patient was negative for both whole blood and serum PCR. All participants tested positive with both the RPR and TPPA tests. The median RPR value was 1/64 (Interquartile range (IQR): 1/16- 1/128). In total, 22 samples were analysed, including N = 12 pre-penicillin treatment plasma, N = 6 post-penicillin treatment plasma and N = 4 pre-penicillin treatment urine samples.

T. pallidum protein biomarker selection

Eleven T. pallidum proteins were selected as candidate biomarkers (Table 2). Most selected biomarkers had high normalized spectral abundance factor (NSAF) scores according to our previous study¹⁷ (median 4.02; IQR: 1.97-6.97) and high microarray signal ratios¹⁸ (median 3.05; IQR: 0.74-6.8). The median protein molecular weight was 39 kDa (IQR: 28-81). Two proteins were predicted to be located in the flagellum (TP_0249 and TP_0792), two in the ribosome (TP_0250b and TP_0244) and the subcellular localization of five proteins was unknown. Protein TP_0326, a BamA orthologue, has been experimentally shown^59–61 to be localized in the outer membrane. A typical target for PCR assays is polA, coding protein TP_0105⁶². One protein, Peptidyl-prolyl cis-trans isomerase (TP_0862) was found in a previous proteomics study where it demonstrated moderate reactivity during immunoblot experiments with human and rabbit T. pallidum infected serum¹⁶. Protein TprG (TP_0317) is part of the paralogous tpr gene family that encodes candidate virulence factors⁶³ and is partially homologous to Tpr E/J. According to pBLAST analysis, all chosen biomarker proteins and corresponding PTPs did not demonstrate high homology with other pathogens, non-pathogenic commensal bacterial or human proteins (data not shown). One to three corresponding well-suited PTPs were selected for each biomarker, for a total of 30 PTPs. Details pertaining to these are provided in Table 2.

Multiple reaction monitoring assay optimization

The LOD for each peptide was determined individually by performing a dilution series of MP-A whereby the median LOD was 68.5 (IQR 14.2-176.7) picomoles. Once the peptide mixture composition was optimized based on the LOD, 2 µL of this mixture (Supplementary File 1) was spiked into 50 µg plasma from a control patient whereby no significant variations in the signal of the hPTP transitions could be detected, indicating that there was no evidence of transition interference from the plasma. After optimizing each of the PTPs, three different sets of transitions were combined in an MRM assay based on their chromatographic retention time, as detailed in Supplementary File 1. The experiments contained a total of 141 targeted ion pairs (transitions) corresponding to 30 PTPs from eleven T. pallidum proteins. Ten of the eleven proteins were represented by two or more (h)PTPs (Table 2/ Supplementary File 1). In total, three scheduled MRM assays of 10 minutes, each containing 20 peptides (10 endogenous (T. pallidum) peptides and 10 hPTP standards) were developed. These assays were evaluated based on a balanced mixture of all 30 hPTPs standards. Unfortunately, although each of the 30 spiked hPTPs could be detected, none of the selected endogenous T. pallidum peptides could be identified in any of the MRM assays (Figure 1; Supplementary File 2*).

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.

Estimation of mass spectrometry LOD and ineffective T. pallidum protein enrichment using magnetic bead coupled polyclonal anti-T. pallidum antibodies

Two T. pallidum spiking dilution series were prepared in PBS and subjected to LTQ-Orbitrap MS/MS analysis in order to estimate the LOD of MS detection. One of the series was subjected to an additional polyclonal antibody coupled magnetic bead enrichment step, including sonication of the beads and subsequent separate measurement of the lysate and on-bead digestion retentate (Figure 2).

Figure 2. Work-flow diagram describing the estimation of T. pallidum protein MS LOD experiments.

In total, eight different concentrations of T. pallidum (from 10⁴ 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 of T. 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.

Two unique T. pallidum proteins, Cytoplasmic filament protein A (TP_0748) and Lipoprotein antigen Tp47 (TP_0574), were found in the 300 bacteria/ml fraction in the enriched and unenriched samples, respectively (Figure 3; Supplementary File 3). Therefore, the LOD based on a high-resolution LTQ-Orbitrap instrument was approximately 300 bacteria/ml PBS for both the antibody enriched and unenriched samples, meaning there was no significant improvement in LOD using bead enrichment. No proteins were detected in any sample concentrations for the enriched bacterial lysate (supernatant) fraction. Possibly, the sonication conditions were not harsh enough to lyse the bacteria on the beads and lysis was mainly the results of trypsin treatment under denaturing conditions. In total, eight unique T. pallidum proteins were found in both the unenriched and enriched retentate dilution series in one or more of the concentrations analyzed: 60 kDa chaperonin (TP_0030), Flagellar filament outer layer protein flaA1 (TP_0249), Alkyl hydroperoxide reductase (TP_0509), Lipoprotein antigen Tp47 (TP_0574), Galactose ABC superfamily ATP binding cassette transporter, binding protein (TP_0684), Cytoplasmic filament protein A (TP_0748) and the Flagellar filament core proteins flaB1/B3 (TP_0792/TP_0870). Four proteins, Lipoprotein, 15 kDa (TP_0171), 10 kDa chaperonin (TP_1013), Elongation factor Tu (TP_0187) and Tp34 lipoprotein (TP_0971) were only found in the unenriched and enriched series, respectively. Ten unique T. pallidum proteins were found in the highest concentration (10⁴ bacteria/ml) for in the enriched retentate sample (N = 10) and non-enriched sample (N = 10), two proteins detected were unique to either the enriched or unenriched samples (Figure 3). Five unique T. pallidum proteins were found in the 10³ bacteria/ml sample, including N = 4 in the unenriched and N = 4 in the retentate fractions. A peptide (LSGGVAVIK) related to 60 kDa chaperonin (TP_0030) was detected in the low concentration (100/ 33/ 10/ 3 bacteria/ml) and in the negative control samples of the enriched sample series. This was likely a false-positive non-specific peptide secondary to rabbit protein contamination since this short peptide sequence is closely homologous to the Oryctolagus cuniculus (rabbit) 60 kDa heat shock protein, or could have originated from the beads or antibodies. As a result, it has been excluded from the analysis. Three T. pallidum proteins detected in both the enriched and unenriched sample series were also biomarker candidates tested in the MRM assay experiments: Flagellar filament core protein flaB2 (TP_0792), Cytoplasmic filament protein A (TP_0748) and the Flagellar filament outer layer protein flaA1 (TP_0249). Detailed information about the identified proteins, peptides, coverage and search parameters can be found in Supplementary File 3. Rough concentration calculations estimated that our target PTPs would be present in the femtomoles per liter range in human T. pallidum infection (calculations presented in Supplementary File 4).

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.