Quantitative analysis of age specific variation in the abundance of human female parotid salivary proteins

Abstract

Human saliva is a protein-rich, easily accessible source of potential local and systemic biomarkers to monitor changes that occur under pathological conditions; however, little is known about the changes in abundance associated with normal aging. In this study, we performed a comprehensive proteomic profiling of pooled saliva collected from the parotid glands of healthy female subjects, divided into two age groups 1 and 2 (20-30 and 55-65 years old, respectively). Hydrophobic charge interaction chromatography was used to separate high- from low-abundance proteins prior to characterization of the parotid saliva using multidimensional protein identification technology (MudPIT). Collectively, 532 proteins were identified in the two age groups. Of these proteins, 266 were identified exclusively in one age group, while 266 proteins were common to both groups. The majority of the proteins identified in the two age groups belonged to the defense and immune response category. Of note, several defense related proteins (e.g., lysozyme, lactoferrin and histatin-1) were significantly more abundant in group 2 as determined by G-test. Selected representative mass spectrometric findings were validated by Western blot analysis. Our study reports the first quantitative analysis of differentially regulated proteins in ductal saliva collected from young and older female subjects. This study supports the use of high-throughput proteomics as a robust discovery tool. Such results provide a foundation for future studies to identify specific salivary proteins which may be linked to age-related diseases specific to women.

Figure 1. Fractionation and SDS-PAGE analysis of parotid saliva

(A) Parotid saliva collected from seven subjects (group 1; 20–30 years of age) was pooled and subjected to hydrophobic charge interaction chromatography (HCIC). Saliva (76 mg protein) was applied to HCIC column (arrow 1), and then eluted with a 50mM Tris HCl pH 8.1 to 50 mM sodium acetate pH 4 (100:0) (arrow 2), followed by 50 mM Tris HCl pH 8.1: 50mM sodium acetate pH (0:100) (arrow 3). The unbound saliva was collected as 5 ml fractions in the flow through (FT, fractions 2–16) and elutions (E, fractions 17–61) were collected as 1ml fractions. Protein was detected at 214 nm. (B) SDS-PAGE analysis of pooled starting saliva (Lane ST), size marker (lane M), flow through fractions (FT, 3–16) and elution fraction (E, 18–53). Amylases and histatins are indicated by the arrows (AMY and HIS) at ~55 kDa and ~8kDa, respectively. Gels were run under reducing conditions, followed by Simple Blue staining. Lane M is SeeBlue (Invitrogen) prestained molecular mass marker ranging from 3.5 to 260 kDa.

Figure 2. Fractionation and SDS-PAGE analysis of parotid saliva

(A) Parotid saliva collected from seven subjects within 55–65 yrs was pooled and the salivary protein (56mg) was treated as described in Fig. 1 legend. The unbound saliva was collected as 5 ml fractions for flow through (FT) (2–15 and elutions (E) (16–60) were collected as 1ml fractions. Peaks were detected at 214 nm. (B) SDS-PAGE analysis of the flow through (FT, 3–15) and elution (E, 21–53). Chromatography for group 2 was performed identically as group 1.

Figure 3. Overlap of the proteins identified from pooled parotid saliva between the different age groups

Venn diagram showing the differentially expressed proteins identified per group and the number of proteins overlapping between the different groups.

Figure 4. Annotation of the number of identified parotid proteins by function

Protein annotation was obtained using UniProtKB/Swiss-Prot databases. Percentages of the total number of proteins identified from pooled parotid saliva from each age group are assigned to functional categories. Classification of proteins identified from 20–30 yrs (A), and 55–65 yrs (B) female subjects.

Figure 5. Comparison of the functional categories and number of proteins identified within each functional category in the pooled parotid saliva

(A) Pair-wise comparison of the number of differentially expressed proteins in the four most abundant functional categories between groups 1 and 2. (B) Pair-wise comparison of the number of differentially expressed proteins in seven functional categories between groups 1 and 2.

Figure 6. Validation of two representative salivary proteins by Western blot analysis

Immunoblots of pooled parotid saliva from groups 1 and 2 probed antibodies that detect (A) Carbonic anhydrase VI and (B) amylases.