Comprehensive defensin assay for saliva

Abstract

Defensins are highly basic cationic peptides that are important components of the innate and adaptive immune response pathways. In addition, these peptides are involved in CD8+ T cell response to HIV-1, increased pulmonary infection risk among cystic fibrosis patients, upregulated levels of HNP-5 for patients with ulcerative colitis and Crohn's disease, and monitoring HNP-3 levels as a tumor classification scheme for cutaneous T cell lymphomas, and have promise in the pharmaceutical field as a new class of antibiotics. Here we present a parallel assay for the alpha (HNP1-3) and beta (HBD1-2) classes of defensins in saliva that are naturally observed in the concentration range of 1 ng/mL to 10 microg/mL. The method utilizes solid phase extraction of saliva samples combined with liquid chromatography-tandem mass spectrometry to identify and quantitate defensin targets. The approach involves limited sample manipulation and is easily amenable to automation. The saliva samples analyzed are derived from a large cohort study focused on examining the role of polymorphisms in genes of innate and adaptive immunity in modulating the response to vaccination for two gastrointestinal tract infections: typhoid and cholera. The alpha-defensin levels observed range from 1 to 10 microg/mL and correlate well with known active concentrations against a wide variety of pathogens. The observed concentration range for beta-defensins was between the detection limit and 33 ng/mL and had a sensitivity level that was comparable to immunoassay-based detection. This method is easily adapted for use in a clinical immunology setting and can be modified for other biological matrixes. This assay will facilitate examination of the production, secretion, and regulation of defensin peptides in a direct fashion to coordinate levels of these compounds with gender, age, response to vaccination, gene copy number, and oral health.

Figure 1

Mass spectrometric fragmentation patterns of the five defensin targets for the saliva assay. Due to the highly basic nature of these peptides, fragmentation is limited to only a few dissociation routes which results in improved signal-to-noise for absolute quantitation. The product ions used for quantitative purposes are highlighted in the box. a) HNP-1 b) HNP-2 c) HNP-3 d) HBD-1 e) HBD-2.

Figure 1

Mass spectrometric fragmentation patterns of the five defensin targets for the saliva assay. Due to the highly basic nature of these peptides, fragmentation is limited to only a few dissociation routes which results in improved signal-to-noise for absolute quantitation. The product ions used for quantitative purposes are highlighted in the box. a) HNP-1 b) HNP-2 c) HNP-3 d) HBD-1 e) HBD-2.

Figure 1

Mass spectrometric fragmentation patterns of the five defensin targets for the saliva assay. Due to the highly basic nature of these peptides, fragmentation is limited to only a few dissociation routes which results in improved signal-to-noise for absolute quantitation. The product ions used for quantitative purposes are highlighted in the box. a) HNP-1 b) HNP-2 c) HNP-3 d) HBD-1 e) HBD-2.

Figure 1

Mass spectrometric fragmentation patterns of the five defensin targets for the saliva assay. Due to the highly basic nature of these peptides, fragmentation is limited to only a few dissociation routes which results in improved signal-to-noise for absolute quantitation. The product ions used for quantitative purposes are highlighted in the box. a) HNP-1 b) HNP-2 c) HNP-3 d) HBD-1 e) HBD-2.

Figure 1

Mass spectrometric fragmentation patterns of the five defensin targets for the saliva assay. Due to the highly basic nature of these peptides, fragmentation is limited to only a few dissociation routes which results in improved signal-to-noise for absolute quantitation. The product ions used for quantitative purposes are highlighted in the box. a) HNP-1 b) HNP-2 c) HNP-3 d) HBD-1 e) HBD-2.

Figure 2

a) Response of HNP-2 as a function of the concentration of the carrier protein lysozyme. b) Individual calibration curves for HNP-2 with (○) and without (□) a 1 mg/mL of the carrier protein lysozyme present c) Calibration curves for HNP-2 using lysozyme treated vials (□), treated vials after 48 hours (△), and untreated vials (○).

Figure 2

a) Response of HNP-2 as a function of the concentration of the carrier protein lysozyme. b) Individual calibration curves for HNP-2 with (○) and without (□) a 1 mg/mL of the carrier protein lysozyme present c) Calibration curves for HNP-2 using lysozyme treated vials (□), treated vials after 48 hours (△), and untreated vials (○).

Figure 2

a) Response of HNP-2 as a function of the concentration of the carrier protein lysozyme. b) Individual calibration curves for HNP-2 with (○) and without (□) a 1 mg/mL of the carrier protein lysozyme present c) Calibration curves for HNP-2 using lysozyme treated vials (□), treated vials after 48 hours (△), and untreated vials (○).

Figure 3

a) Chromatographic profile of the product ion intensity for synthetic HNP-1, HNP-2, and HNP-3. The inset shows the tandem mass spectrum of the peptide at two different points across the peak for HNP-2. b) Chromatographic profile of the product ion intensity for naturally occurring (saliva sample) HNP-1, HNP-2, and HNP-3. c) Chromatographic profile of the product ion intensity for synthetic HBD-1, and HBD-2. The inset shows the tandem mass spectrum of the peptide at two different points across the peak for HBD-2. d) Chromatographic profile of the product ion intensity for naturally occurring (saliva sample) HBD-1, and HBD-2.

Figure 3

a) Chromatographic profile of the product ion intensity for synthetic HNP-1, HNP-2, and HNP-3. The inset shows the tandem mass spectrum of the peptide at two different points across the peak for HNP-2. b) Chromatographic profile of the product ion intensity for naturally occurring (saliva sample) HNP-1, HNP-2, and HNP-3. c) Chromatographic profile of the product ion intensity for synthetic HBD-1, and HBD-2. The inset shows the tandem mass spectrum of the peptide at two different points across the peak for HBD-2. d) Chromatographic profile of the product ion intensity for naturally occurring (saliva sample) HBD-1, and HBD-2.

Figure 3

a) Chromatographic profile of the product ion intensity for synthetic HNP-1, HNP-2, and HNP-3. The inset shows the tandem mass spectrum of the peptide at two different points across the peak for HNP-2. b) Chromatographic profile of the product ion intensity for naturally occurring (saliva sample) HNP-1, HNP-2, and HNP-3. c) Chromatographic profile of the product ion intensity for synthetic HBD-1, and HBD-2. The inset shows the tandem mass spectrum of the peptide at two different points across the peak for HBD-2. d) Chromatographic profile of the product ion intensity for naturally occurring (saliva sample) HBD-1, and HBD-2.

Figure 3

a) Chromatographic profile of the product ion intensity for synthetic HNP-1, HNP-2, and HNP-3. The inset shows the tandem mass spectrum of the peptide at two different points across the peak for HNP-2. b) Chromatographic profile of the product ion intensity for naturally occurring (saliva sample) HNP-1, HNP-2, and HNP-3. c) Chromatographic profile of the product ion intensity for synthetic HBD-1, and HBD-2. The inset shows the tandem mass spectrum of the peptide at two different points across the peak for HBD-2. d) Chromatographic profile of the product ion intensity for naturally occurring (saliva sample) HBD-1, and HBD-2.

Figure 4

a) Calibration curves for HNP-1 (■), HNP-2 (●), and HNP-3 (▲). In the inset is shown an expanded view of the low concentration region. b) Calibration curves for HBD-1 (●) and HBD-2 (■). The inset shows an expanded view of the low concentration region.

Figure 4

a) Calibration curves for HNP-1 (■), HNP-2 (●), and HNP-3 (▲). In the inset is shown an expanded view of the low concentration region. b) Calibration curves for HBD-1 (●) and HBD-2 (■). The inset shows an expanded view of the low concentration region.