Salivary proteomic and genomic biomarkers for primary Sjögren's syndrome

Abstract

Objective: To identify a panel of protein and messenger RNA (mRNA) biomarkers in human whole saliva (WS) that may be used in the detection of primary Sjögren's syndrome (SS).

Methods: Mass spectrometry and expression microarray profiling were used to identify candidate protein and mRNA biomarkers of primary SS in WS samples. Validation of the discovered mRNA and protein biomarkers was also demonstrated using real-time quantitative polymerase chain reaction and immunoblotting techniques.

Results: Sixteen WS proteins were found to be down-regulated and 25 WS proteins were found to be up-regulated in primary SS patients compared with matched healthy control subjects. These proteins reflected the damage of glandular cells and inflammation of the oral cavity system in patients with primary SS. In addition, 16 WS peptides (10 up-regulated and 6 down-regulated in primary SS) were found at significantly different levels (P < 0.05) in primary SS patients and controls. Using stringent criteria (3-fold change; P < 0.0005), 27 mRNA in saliva samples were found to be significantly up-regulated in the primary SS patients. Strikingly, 19 of 27 genes that were found to be overexpressed were interferon-inducible or were related to lymphocyte filtration and antigen presentation known to be involved in the pathogenesis of primary SS.

Conclusion: Our preliminary study has indicated that WS from patients with primary SS contains molecular signatures that reflect damaged glandular cells and an activated immune response in this autoimmune disease. These candidate proteomic and genomic biomarkers may improve the clinical detection of primary SS once they have been further validated. We also found that WS contains more informative proteins, peptides, and mRNA, as compared with gland-specific saliva, that can be used in generating candidate biomarkers for the detection of primary SS.

Figure 1

Comparative analysis of proteins in whole saliva (WS) samples from patients with primary Sjögren’s syndrome (pSS) and age-, sex-, and ethnicity-matched control subjects, as determined by 2-dimensional gel electrophoresis (2-DE) and liquid chromatography–quadrupole time-of-flight mass spectrometry (LC-Q-TOF-MS). Shown are the 2-DE patterns of proteins in pooled WS from 10 control subjects and 10 primary SS patients. A total of 100 µg of total proteins from each pooled sample was used for the 2-D gel separation. The differentially expressed proteins (spots 1–42; see Table 1 for the complete list) were identified using in-gel tryptic digestion and LC-Q-TOF-MS.

Figure 2

Analysis of α-enolase by electrospray ionization tandem mass spectrometry (ESI-MS/MS) and immunoblotting. A, ESI-MS/MS spectrum of the tryptic peptide TIAPALVSK (mass/charge [m/z] 450.3 atomic mass units [amu]) from α-enolase. This protein was found to be overexpressed in whole saliva from patients with primary Sjögren’s syndrome (pSS), as determined by 2-dimensional gel electrophoresis. B, Immunoblotting of whole saliva from 10 patients with primary SS and 10 age-, sex-, and ethnicity-matched control subjects for α-enolase and actin. An equal amount of proteins from each sample was used for the immunoblots.

Figure 3

Principal components analysis of the gene expression data in patients with primary Sjögren’s syndrome (SS) and in age-, sex-, and ethnicity-matched control subjects. Results of the principal components (PC1 and PC2) analysis suggest that the gene expression data we obtained segregated the 8 control subjects (blue symbols) from the 10 primary SS patients (red symbols).

Figure 4

Heat map of 27 mRNA that were significantly up-regulated in patients with primary Sjögren’s syndrome (SS) as compared with the age-, sex-, and ethnicity-matched control subjects, as determined by microarray profiling analysis. Control and SS patient numbers are shown at the bottom.