Quantification of low abundance Yersinia pestis markers in dried blood spots by immuno-capture and quantitative high-resolution targeted mass spectrometry

Abstract

Plague, caused by the bacterium Yersinia pestis, is still present in several countries worldwide. Besides, Y. pestis has been designated as Tier 1 agent, the highest rank of bioterrorism agents. In this context, reliable diagnostic methods are of great importance. Here, we have developed an original workflow based upon dried blood spot for simplified sampling of clinical specimens, and specific immuno-mass spectrometry monitoring of Y. pestis biomarkers. Targeted proteins were selectively enriched from dried blood spot extracts by multiplex immunocapture using antibody-coated magnetic beads. After accelerated on-beads digestion, proteotypic peptides were monitored by multiplex LC-MS/MS through the parallel reaction monitoring mode. The DBS-IC-MS assay was designed to quantify both F1 and LcrV antigens, although 10-fold lower sensitivity was observed with LcrV. The assay was successfully validated for F1 with a lower limit of quantification at 5 ng·mL⁻¹ in spiked blood, corresponding to only 0.1 ng on spots. In vivo quantification of F1 in blood and organ samples was demonstrated in the mouse model of pneumonic plague. The new assay could help to simplify the laboratory confirmation of positive point of care F1 dipstick.

Keywords

Biomarker dried blood spot immunocapture mass spectrometry pestis targeted proteomic validation

Introduction

Endemic foci of plague are still active in various countries in Asia,¹ Africa² and the Americas.^3,4 Furthermore, despite the apparent disappearance of the disease in some previously infected areas, the infection has the capacity to reawake after several decades of silence and to cause new epidemics. This is exemplified by the occurrence of a human plague outbreak near Oran in western Algeria in 2003, while no cases were reported in the previous 50 years.⁵ Molecular studies demonstrated that this outbreak was not caused by an importation of the disease, but by the reactivation of a local focus that was thought to be extinct.⁶ Over the past 15 years, nearly 40,000 cases of plague have been recorded to the World Health Organization (WHO) in several countries.⁷ In Madagascar, which is currently the most active plague focus worldwide,⁸ local health authorities have to manage several hundreds of human plague patients every year.

Yersinia pestis, the causative agent of plague, is a highly pathogenic bacterium categorized as a Tier 1 agent due to its high pathogenicity and possible person-to-person transmission.⁹ Plague diagnosis, which is crucial whether under the WHO master plan on health or in the biological warfare context,¹⁰ is primarily based on culture of human samples (blood, sputum and/or bubo aspirates) followed by bacterial identification. Nevertheless, diagnosis difficulties can arise due to low bacterial load in the sample or initiation of antibiotic therapy prior to sampling. Furthermore, the time required for bacteriological confirmation is too long when an effective treatment needs to be administered extremely rapidly.¹⁰

Rapid dipsticks detecting F1 antigen have been developed for point of care diagnosis of suspected plague patients.¹¹ F1 antigen is the most commonly used biomarker of plague.¹² The protein, specific to Y. pestis, is expressed at temperature of 37°C and covers the bacterial surface with a gelatinous antiphagocytic pseudocapsule,¹³ composed of high-molecular weight polymers built from a 15.5 kDa F1 monomer.¹⁴ Large amounts of F1 monomers are shed from the bacterial surface into biological fluids and tissues, and are detectable in blood and bubo samples of infected individuals.^15,16 LcrV (low-calcium response V antigen), a 37.2 kDa protein essential for Y. pestis virulence, was also reported to be released into the extracellular space and to accumulate in serum of infected mice,^17,18 constituting a potential marker of plague in combination with F1.

Current F1 and LcrV immuno-based assays have the limitation of potential cross-reactions with other antigens, possibly leading to false-positive results. Mass spectrometry (MS)-based assays, by allowing specific detection and quantification of proteins in complex biological matrices,¹⁹ are useful complement to immunoassays for confirmation. In particular, liquid chromatography–tandem MS operated in the selected reaction monitoring (SRM) or parallel reaction monitoring (PRM) modes, referred as targeted-proteomic, provides quantitative monitoring of selected proteins with high specificity and reproducibility.²⁰ Our group previously described a targeted proteomic approach for the direct detection of Y. pestis in environmental samples.²¹ Nevertheless, application to biological samples for plague diagnosis was not reported.

The growing interest of MS for clinical applications has led to new technological developments in recent years.^22,23 In this way, solutions for simple handling and easier transport of clinical samples to the laboratory are developing. Targeted proteomic has been combined for the last decade with dried matrix spot (DMS), including dried blood spot (DBS), to facilitate sample gathering, storage and transportation before MS analysis.^24,25 DBS sample format provides several advantages: (i) blood samples can be obtained at any location without the special requirements associated with conventional blood draws, (ii) only small amounts of blood (typically 10–20 µL) per spot are needed for analysis, (iii) the blood spots are dried in air and shipped at room temperature (RT), and (iv) drying the blood prolongs compound stability and minimizes risks of contamination with blood-borne viruses (HIV and hepatitis) during sampling.²⁶ However, the use of DBS-MS during plague disease outbreaks would require major improvement in sensitivity to reach detection limits in accordance with the low amount of Y. pestis antigens released in blood at the early stage of the disease.¹¹

In this work, we investigated the application of DBS-MS for the quantification of Y. pestis proteins released in biological fluids during infection, focusing on F1 and LcrV antigens. The analytical protocol is based on multiplex immunocapture (IC) to selectively purify and concentrate the low abundant markers and targeted high-resolution tandem MS. All analytical steps were optimized to reach in vivo quantification of the plague markers. A lower limit of quantification (LLOQ) at 5 ng·mL⁻¹ was achieved for F1 and in vivo quantification was successfully demonstrated on dried blood and tissue spots from infected mice.

Experimental section

Bacterial strains, culture conditions and safety

The fully virulent Y. pestis strain CO92 used in this study was handled in a Biosafety Level 3 laboratory. Bacteria were grown at 28°C on Luria–Bertani agar plates supplemented with 0.002% (w/v) hemin (LBH) for 48 h before use. Bacteria were suspended in sterile saline solution (0.9% NaCl), and bacterial concentrations were evaluated by OD measurement at 600 nm, followed by plating of 10 fold dilutions of bacterial suspensions on LBH plates.

Biological samples and animal experiments

Uninfected blank whole blood and plasma of healthy six-week-old female OF1 mice were purchased from Charles River (Wilmington, MA, USA). Biological fluids were collected into K₂-EDTA-coated plastic tubes and aliquoted into 2 mL microcentrifuge tubes. Hematocrit between 40% and 50% was measured for each blood sample. Uninfected spleens were taken from healthy sacrificed OF1 mice.

Mouse model of pneumonic plague

Six-week-old OF1 female mice (Charles River Laboratory) were housed in an animal biosafety level 3 facility (Institut Pasteur) accredited by the French Ministry of Agriculture (accreditation B 75 15–01), in compliance with French and European regulations on care and protection of Laboratory Animals (EC Directive 86/609, French Law 2001–486 issued on June 6, 2001). Mouse infections were carried out following the approved protocol CETEA 2014-0025. For infection, mice were anesthetized by intraperitoneal injection of xylasine/ketamin, and 2 × 10⁴ cfu of Y. pestis CO92 in a total volume of 20 µL were administered intranasally to mimic pneumonic plague. Six animals were sacrificed when they exhibited clinical signs of severe infection, and their spleen, blood and lungs were collected. Bacterial loads were determined by plating blood samples and spleen and lungs homogenates on LBH plates.

Chemicals and reagents

Purified F1 protein (Institut Pasteur) and recombinant LcrV (CEA/LERI) were quantified using the BCA kit mini assay protocol in NanoDrop 1000 Spectrophotometer. Ammonium bicarbonate (ABC), sodium deoxycholate (DOC), iodoacetic acid (IAA), 1,4-dithiothretiol (DTT), formic acid (FA), trifluoroacetic acid (TFA) and acetonitrile were purchased from Sigma-Aldrich (St. Louis, MO, USA). Rapigest SF was from Waters (Milford, MA, USA).

Internal standards

Six isotope-labeled AQUA peptides with labeled amino acids were synthesized by Life Technologies (Carlsbad, CA, USA) for F1 antigen: F1A′, ITLTYK[¹³C₆; ¹⁵N₂], F1B, DFDISPK[¹³C₆; ¹⁵N₂], F1C, YTDA[¹³C₃; ¹⁵N]VTVTV[¹³C₅; ¹⁵N]SNQ and LcrV: LV1, FNSAIEALNR[¹³C₆; ¹⁵N₄], LV2, IYSVIQAEINK[¹³C₆; ¹⁵N₂] and LV3, ILAYFLPEDAILK[¹³C₆; ¹⁵N₂]. Labeled peptides were provided in solution, measured by amino acid analysis (AQUA Ultimate grade).

Preparation of DMS

Twenty microliters of biological fluids were spotted onto DBS collection cards (903 Protein Saver Card, Whatman) using an electronic laboratory pipette. Forty microliters of a 1% TFA solution was immediately added after matrix spotting for inactivation of DMS from infected mice. Samples were dried overnight at RT. The entire DMS spot was then carefully excised and placed in a 1.5 mL microcentrifuge tube. Each DMS sample was extracted with 500 µL of DOC (0.1%, w/v dissolved in 50 mM ABC) at 40°C with vortexing at 1000 r/min for 1 h (Thermomixer, Eppendorf). Extracts from infected mice were transferred into Ultrafree MC filter tubes of 0.22 µm pore size (Millipore, Billerica, MA) and spun at 10,000 g for 5 min for total inactivation. Inactivation of bacteria was confirmed by verification of the absence of viable bacteria after culture.²¹

Multiplexed IC and enzymatic digestion

M-280 tosylactivated magnetic beads (Life Technologies, Carlsbad, CA, USA) were prepared according to the supplier protocol. Two batches of IgG-coupled beads were prepared simultaneously: one with the F1-specific commercial monoclonal antibody YPF19 (Abcam, Cambridge), and the other one with the LcrV-specific monoclonal antibody λ24, produced by the Laboratoire d’Etudes et de Recherches en Immunoanalyse as previously described.²⁷

Two hundred micrograms of antibody were covalently linked to the beads to obtain 500 µL of IgG-coupled bead solutions. Five microliters of each bead solution (F1 and LcrV-antibody coated beads) were added to 400 µL of DMS extracts diluted in 0.1% DOC. Samples were incubated for 1 h at RT with gentle shaking, beads-coated with IgG protein were then retained on the magnetic support and washed three times with PBS to remove weak non-specific binding. The complexes were then resuspended in 20 µL of 20% ACN in 50 mM ABC buffer. Each sample was sequentially denatured 10 min at 90°C, reduced with 10 mM dithiothreitol (DTT) at 60°C for 30 min, and alkylated with 20 mM iodoacetamide (IAA) solution at RT for 30 min. Finally, enzymatic digestion was performed by the addition of 2 µL of 0.5 mg.mL⁻¹ trypsin with incubation at 37°C for 2 h. After digestion, magnetic beads were removed and trypsin digested samples collected. Twenty microliters of H₂O/ACN/FA (95:5:0.1, v/v/v) and 5 µL of labeled AQUA peptides (25 ng·mL⁻¹ in H₂O/ACN/FA (95:5:0.1, v/v/v)) were added for a final concentration of 2.5 ng·mL⁻¹.

Ultra high-performance liquid chromatography and high-resolution MS analysis

LC/HRMS (PRM mode) was performed on an Ultimate 3000 UHPLC system coupled to a quadrupole-Orbitrap mass spectrometer (Q-Exactive, Thermo Fisher Scientific, Bremen, Germany) operated under time-scheduled sequential PRM acquisition. Peptides were injected (20 µL) onto an Aeris C18 column (100 Å, 1.7 µm, 2.1 × 150 mm, Phenomenex, Torrance, CA, USA) and eluted with a non-linear gradient: after an isocratic step for 1 min at 5% ACN with 0.1% FA, two step gradient from 5% to 60% ACN was run over 10 min at a flow rate of 500 µL/min. The column oven temperature was set to 50°C. Eluted peptides were introduced into the Q-Exactive instrument, operating in positive ion mode, by electrospray ionization (ESI) with an ion spray voltage of 3.5 kV. Precursor ions from native and labeled peptides were selected separately in the quadrupole (Table S1) with an isolation window of 1.5 m/z. They were fragmented in the HCD cell using nitrogen as collision gas and an optimized normalized collision energy (Table S1). All fragment ions were transferred to the Orbitrap. Resolution was set at 17,000 or 35,000 at m/z 200 (full width at half maximum) depending on the number of peptides eluting in a timeframe, automatic gain control to 1e6 and maximum injection time to 125 ms. PRM data were processed with Xcalibur 2.2 software (Quan Browser, Thermo Fisher Scientific, Bremen, Germany).

Results and discussion

Plague diagnosis is performed on biological samples such as bubo aspirates, blood or sputum. The challenging detection of low abundance plague markers in small volumes of these complex biological matrices extracted from dried spots requires a selective sample pretreatment strategy prior to MS analysis. DBS or DMS (i.e. other matrices than blood) followed by IC extraction was evaluated for multiplex detection of two Y. pestis biomarkers, F1 and LcrV antigens. All analytical steps were optimized for in vivo quantification of the plague markers with high sensitivity. Inactivation of the pathogenic spots for safe handling was also investigated.

Development of the multiplex DBS-IC-MS assay

Selection of proteotypic peptides for F1 and LcrV antigens, and targeted-MS development

Proteotypic peptides were selected in order to get 6–25 amino acid length, a good chromatographic characteristic, and high ionization efficiency. Tryptic digests of recombinant F1 and LcrV were used for the selection of best responding peptides. All tryptic peptides containing methionine, cysteine or tryptophan residues that could undergo oxidation during sample handling were avoided. Peptide specificity was confirmed by performing Blast similarity searches against the Uniprot database (taxon: Mus musculus). Finally, three proteotypic peptides of each protein were selected to insure assay specificity, and their isotopically labeled counterparts were synthetized to be used as internal standards and to optimize MS and MS/MS conditions (Table S1). AQUA peptides were selected over full-length stable-isotope-labeled version of the proteins, e.g. PSAQ standards,²⁸ to maintain cost-effectiveness. The PRM mode was used for peptide detection. In this targeted analysis mode, several peptides fragments ions are simultaneously detected at high resolution and high mass accuracy for detection at high specificity in complex matrices.²⁹ During data processing, the peak area of the selected fragments ions (Table S1) was summed to increase the signal-to-noise ratio of monitored peptides.³⁰ Optimized parameters for each peptide are reported in Table S1. Signal of the two best responder peptides (F1A/F1B and LV2/LV3 for F1 and LcrV, respectively) was used for the quantitative analysis.

Protein denaturation and digestion

Robust protein quantitation by bottom-up proteomic requires reproducible and close to completeness digestion. The conventional protocol for trypsin digestion consists in an overnight incubation at 37°C with a ratio of protein to trypsin between 10:1 and 100:1 by mass. In order to enhance and accelerate trypsin digestion of the plague biomarkers, denaturation conditions and digestion kinetic were investigated on an equimolar mix of F1 and LcrV antigens. The acid labile surfactant RapiGest SF^31,32 and organic-aqueous solutions, i.e. 20 and 80% acetonitrile, were tested to achieve a rapid and complete digestion of proteins.²⁹ In our case, the highest peptide recovery was observed for F1 antigen with a 10 min denaturation at 90°C either in 0.05% RapiGest SF or 20% acetonitrile (Figure 1). The 20% acetonitrile protocol was finally selected as it was the most efficient for LcrV (Figure 1). In addition, omission of Rapigest obviates the surfactant degradation step, reducing the sample preparation time. Trypsin activity was drastically reduced at a higher percentage of 80% acetonitrile (Figure 1), as previously observed.³³ Following a 2-h digestion time in 20% acetonitrile, signal of proteotypic peptides culminated (Figure 1), indicating that the maximum digestion recovery was reached. Of note, signal of LV3 and to a lesser extent F1A tended to level off after 2 h of incubation, due to probable peptide degradation.³⁴

Sample preparation for detection of the low abundance markers in matrix

Y. pestis biomarkers extraction from the dried spots was adapted from previous works,³⁵ by means of incubation in 0.1% DOC. Only few strategies are reported for protein enrichment from the DBS extract, despite the high protein complexity of blood matrix, which potentially impacts MS performance. Immunoaffinity enrichment, or IC, is the gold standard to achieve high sensitivity.^27,32 The protocol was developed using functionalized magnetic beads and previously described conditions by our group.³⁰ The simultaneous IC of the two markers was implemented by addition of a mixture of beads separately coated with antibodies against F1 or LcrV antigens. To determine whether such multiplex IC had any detrimental impact on recovery and assay sensitivity, fresh mouse blood was spiked with F1 and LcrV on DBS cards and analyzed either by single or by multiplex IC procedure. Similar MS signals were obtained (Figure 2), illustrating the absence of any detrimental impact of the simultaneous protocol.

Preliminary investigation of assay performance in mice whole blood spiked with recombinant F1 and LcrV illustrated a lowest detectable concentration at 5 ng·mL⁻¹ with a linear increase up to 2000 ng·mL⁻¹ for F1 antigen (Table 1). Regarding LcrV, sensitivity was dramatically impacted with a limit of detection above 50 ng·mL⁻¹. The result can be attributed either to low affinity of the selected monoclonal antibody for the recombinant LcrV protein in capture conditions, or from a binding of recombinant LcrV to endogenous proteins or on polypropylene tubes. Consequently, only F1 quantification was considered in the validation experiment.

Table 1.

Curve performance for F1 antigen by DMS-IC-MS in mouse fresh blood, plasma and spleen crush.

Antigen F1	Fresh blood			Plasma			Homogenate of spleen
standard conc. (ng·mL⁻¹)	*[p] estimated (ng·mL⁻¹)	Accuracy %	Linearity r²	*[p] estimated (ng·mL⁻¹)	Accuracy %	Linearity r²	*[p] estimated (ng·mL⁻¹)	Accuracy %	Linearity r²
5	4.31	86	0.9952	5.45	109	0.9969	3.92	78	0.9982
10	10.92	109		11.07	111		7.81	78
25	25.59	102		22.57	90		27.60	110
50	45.31	91		51.46	103		63.86	128
100	115.23	115		82.16	82		91.24	91
250	275.45	110		243.80	98		280.24	112
500	496.62	99		561.26	112		514.27	103
1.000	900.88	90		917.55	92		1046.75	105
2.000	2099.62	105		2022.05	101		1927.27	96

[p]: biomarker protein concentration.

DMS-IC-MS: dried matrix spot-immunocapture-mass spectrometry.

Method performances for F1 antigen

The final assay procedure is illustrated in Figure 3. Quantification is provided by external F1 calibration standards prepared in DBS or DMS and processed similarly to QCs samples, combined with internal standardization by AQUA peptides. Most critical parameters of assay validation were evaluated, including matrix effects, LLOQ, recovery, linearity, precision and stability in mouse whole blood (fresh or frozen) and plasma before application to samples from infected mice. Spleen homogenate was also tested as artificial matrix of the bubo content.

Matrix effects

Matrix effects were determined using individual fresh whole blood from mice (n = 6). Matrix effect was determined by comparing the ratio peptide/IS from blood samples with a reference (injection solvent). As shown in Figure S2, matrix effect was lower than 20% when F1 was spiked at LQC level or at higher concentrations. This result illustrates the selectivity of the immunoaffinity extraction from complex whole blood matrix.

Linearity, recovery, precision and accuracy

Consecutive analytical batches were analyzed over separate days to assess linearity, precision and accuracy of DBS/DMS-Multiplex IC in fresh whole blood (n = 3 days), mouse plasma (n = 1 day) and spleen homogenates (n = 3 days). Each batch contained nine calibration standards (n = 3 at each level). The method was linear for F1 from 5 to 2000 ng·mL⁻¹ in all matrices with both quantitative peptides (Table 1). DBS/DMS calibration standards showed an acceptable accuracy over the wide linear dynamic range (78–115%), even at the LLOQ value, i.e. 5 ng·mL⁻¹. All calibration data are summarized in Table 1.

Recovery was evaluated by spiking the same protein amount in DBS/DMS and after the IC step. Comparing the results, F1 recovery was determined at 76% and 45% in fresh blood and spleen homogenates, respectively (Table S2). The values correspond to the amount of F1 recovered from spiked blood or spleen, after DBS/DMS extraction and IC. The results indicate a good global recovery of F1 with our DBS-IC protocol. Assay precision was evaluated by replicate analyses of QCs and was found acceptable with CV below 20% in fresh blood, plasma and spleen homogenates. No real differences were observed between each matrix (Table 2).

Table 2.

Precision and accuracy for F1 antigen by DMS-IC-MS in mouse fresh blood, plasma and spleen crush.

QCs of F1 antigen
	Fresh blood			Plasma			Homogenate of spleen
Theoretical [QC] ng·mL⁻¹	LQC	MQC	HQC	LQC	MQC	HQC	LQC	MQC	HQC
Theoretical [QC] ng·mL⁻¹	15	150	1500	15	150	1500	15	150	1500
1	14.93	139.37	1584.20	15.45	143.79	1259.84	15.39	185.49	1280.93
2	13.90	137.70	1248.48	15.53	144.41	1029.07	12.72	180.62	1225.44
3	12.85	153.58	1842.67	15.05	140.28	1150.42	12.42	170.32	1243.30
mean	13.89	143.53	1558.45	15.34	142.83	1146.44	13.51	178.81	1280.93
CV%	7.50	6.08	19.12	1.66	1.56	10.07	12.09	4.33	2.27
Accuracy%	92.6	95.7	103.9	102.3	95.2	76.4	90.1	119.2	83.3

Note: Three levels of quality controls were performed for evaluation: low, medium and high QC values at 15, 150 and 1500 ng·mL⁻¹, respectively (n = 3/concentration).

DMS-IC-MS: dried matrix spot-immunocapture-mass spectrometry.

We also evaluated the linearity and accuracy of measurements in frozen blood samples. Frozen blood is preferable for preparation of calibration standards in routine assays, avoiding daily animal bleedings, whereas DBS sampling of test samples is usually performed on a finger stick fresh capillary blood sample. Freezing the whole blood causes red blood cell lysis and consequently changes the properties of the sample. We evaluated the linearity and accuracy of measurements performed on frozen blood samples. A non-linear profile of the calibration curve (n = 2) was obtained with frozen blood (Figure S1), which could result from a higher competition for binding to the antibody after red blood cell lysis and the subsequent saturation of binding sites at 2000 ng·mL⁻¹. Restriction of the F1 calibration range allowed a linear fitting from 5 to 1000 ng·mL⁻¹ with acceptable accuracy between 93 and 119%. In addition, three QC were prepared at different levels in fresh blood, and concentrations were back calculated from the calibration curve in frozen blood (Figure S1). Accuracies were observed in the range 98.5 to 102 %, proving the successful measurement of fresh blood samples with frozen blood calibration curves.

Stability

DBS format is known to ensure stability of analytes during storage and shipment. A stability study in whole blood was performed to support similar statement in our conditions. In this work, we stored DBS samples spiked with 150 ng·mL⁻¹ of F1 antigen at −20°C, 4°C and RT for 15 and 30 days. The stability was determined by comparing the ratio of proteotypic peptides and internal standards to the ratio values obtained on day 1 at RT. As shown in Figure S3, F1 was stable over time, excepting a slight signal decrease after 15 days at RT when comparing with refrigerated or frozen samples. This slight difference was not observed after one month, and was therefore not considered as significant. Stability was therefore demonstrated for 30 days at RT.

In summary, the DBS/DMS-IC-MS protocol was successfully validated for quantification of F1 antigen in mouse whole blood, plasma and spleen extracts with variability below 20% and stability at RT for one month.

Application to samples contaminated with Y. pestis

Inactivation of pathogenic spots

The issue of possible persistence of live bacteria in dried spots is crucial for safe handling of samples contaminated with Y. pestis. DMS/DBS sampling is recognized to provide a greater safety than venipuncture in collection and transport of infectious samples.³⁶ However, we found that the drying step was not sufficient to kill all Y. pestis bacteria present in artificially spiked blood or spleen extracts. Additional treatment of the spots was therefore necessary while maintaining an efficient quantification of the Y. pestis markers. Of several chemical and physical microbial inactivation procedures tested (including alcohol, heat and acids³⁷), 1% aqueous TFA was the only treatment that had no significant impact on method performance. Only a slight decrease of F1 peptide signals was observed, without affecting the limits of detection and the range of linearity (Figure S4). Bacteria inactivation by TFA was then determined on Y. pestis spiked in blood or tissues extract at concentration ranging from 10² to 10⁹ CFU/spot. TFA was first added directly on dried spots, but bacterial culture of excised spots evidenced Y. pestis survival, possibly because dried blood and tissue extracts formed a kind of impermeable film at the surface of the filter that prevented a complete absorption of TFA in the spotted sample. Aqueous TFA was then added immediately following sample spotting, before drying. Under these conditions, complete inactivation was obtained for up to 10⁶ CFU per spot on DBS card. For Y. pestis concentrations above 10⁶ CFU per spot, the TFA treatment had to be combined with 0.22 µm filtration for bacterial inactivation. Total Y. pestis inactivation was then obtained, even at high bacterial doses such as 10⁹ CFU/spot.

Assay on bacterial solutions

F1 concentration was first determined from bacterial spots containing increasing numbers of Y. pestis CFU grown at 37°C. F1 antigen concentrations increased linearly with the number of bacteria (Figure 4), indicating a strong relationship between Y. pestis concentrations and the DMS-IC-MS response. As low as ≈400 CFU/spot of Y. pestis allowed to quantify F1 above LLOQ (i.e. 5 ng·mL⁻¹) in DMS-IC-MS (Figure 4), illustrating an excellent assay sensitivity considering the low initial sample volume (20 µl).

Assay in infected mice

To evaluate the performances of the assay in vivo, mice were infected with the fully virulent strain Y. pestis CO92 by intranasal administration (10 LD₅₀) to mimic pneumonic plague. The animals were sacrificed when they exhibited clinical signs of infection and whole blood, spleen, and lungs were taken for the DBS-IC-MS assay.

In this mouse model of pneumonic plague, six surviving animals could be used for biomarker measurements. Among them, soluble F1 was not detected in the DBS/DMS samples from the two animals with negative bacterial culture. In the four other animals, circulating bacteria were present in their blood and F1 antigen was detected with both peptides to levels ranging from 30 to 3000 ng·mL⁻¹ (Table 3). They also had high bacterial loads in their spleen (>9 × 10⁶ CFU) and lungs (>10⁵ CFU). In the most heavily infected animal (mouse #p2), the F1 concentration reached 47 µg.mL⁻¹ in the spleen (Table 3). F1 antigen levels in blood and spleen correlated with bacterial loads on DBS spots, as shown in Figure S5. Lungs from the mouse model were also analyzed, although the assay was not validated in this matrix. F1 antigen was detected in lungs of the four animals, with highest concentration at 215 ng·mL⁻¹ in mouse #p2 (Table 3).

Table 3.

Determination of soluble F1 antigen by DMS-IC-MS in blood, spleen and lungs of a mouse model of pneumonic plague.

	Level of F1 in mice matrices (ng·mL⁻¹)
		Blood	Lungs	Spleen
Mouse#p2	CFU/spot	4.72E + 05	3.24E + 04	1.1E + 06
	F1_A′	2557.0	230.3	53,850.0
	F1_B	3296.2	200.3	40,280.0
Mean (A′:B)	F1	2926.6	215.3	47,065.0
Mouse#p3	CFU/spot	1.8E + 05	4.52E + 04	6.8E + 05
	F1_A′	914.4	163.4	25,870.0
	F1_B	850.0	145.7	22,385.0
Mean (A′:B)	F1	882.2	154.6	24,127.5
Mouse#p5	CFU/spot	1.04E + 04	5.94E + 03	4.56E + 04
	F1_A′	36.8	27.4	710.0
	F1_B	35.8	15.8	442.0
Mean (A′:B)	F1	36.3	21.6	576.0
Mouse#p7	CFU/spot	3.12E + 04	6.00E + 03	1.08E + 05
	F1_A′	95.5	30.0	6380.0
	F1_B	107.2	18.5	4861.0
Mean (A′:B)	F1	101.3	24.2	5620.5

ND: non detected; DMS-IC-MS: dried matrix spot-immunocapture-mass spectrometry.

Conclusion

In this study, F1 antigen quantification was fully validated and enabled determinations in whole blood and tissue spots from infected mice developing pneumonic plague. IC was combined with DBS/DMS sampling for enhanced assay sensitivity, resulting in limit quantification similar to F1 dipsticks,¹⁶ at 5 ng·mL⁻¹, corresponding to only 0.1 ng on spots. Assay specificity is given by the selective binding to the antibody and the MS quantification of F1 sequences. Stability of F1 was demonstrated in DBS at RT, thus avoiding any freezing and facilitating field sampling. In addition, shipment of dried spots severely reduces the risk of dispersion and accidental contaminations inherent to liquid samples. The assay should therefore facilitate in the future the laboratory confirmation of positive F1 dipsticks. Further optimization of this protocol could include: (i) evaluation of additional treatments to kill the totality of the bacteria spotted, (ii) application and validation for human blood samples, (iii) implementation of a more sensitive nano- or capillary-liquid chromatography to lower the limit of quantification with the objective of an earlier F1 quantification in DBS spots during the infectious process, and (iv) identification of other Y. pestis-specific protein, e.g. pesticin, murin toxin, that could be used as additional plague markers in a multiplex method. Quantification of LcrV was not enough sensitive because of sticking to plastic tubes and/or endogenous proteins; nevertheless, we demonstrated the multiplex ability of the approach.

Supplemental Material

Supplemental material for Quantification of low abundance Yersinia pestis markers in dried blood spots by immuno-capture and quantitative high-resolution targeted mass spectrometry

Supplemental Material for Quantification of low abundance Yersinia pestis markers in dried blood spots by immuno-capture and quantitative high-resolution targeted mass spectrometry by Aline Rifflet, Sofia Filali, Jérôme Chenau, Stéphanie Simon, François Fenaille, Christophe Junot, Elisabeth Carniel and François Becher in European Journal of Mass Spectrometry

Footnotes

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This project was supported by the French Joint Ministerial Program of R&D against CBRNE risk.

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