Alterations in the Salivary Proteome and N-Glycome of Sjögren's Syndrome Patients

Abstract

We used isobaric mass tagging (iTRAQ) and lectin affinity capture mass spectrometry (MS)-based workflows for global analyses of parotid saliva (PS) and whole saliva (WS) samples obtained from patients diagnosed with primary Sjögren's Syndrome (pSS) who were enrolled in the Sjögren's International Collaborative Clinical Alliance (SICCA) as compared with two control groups. The iTRAQ analyses revealed up- and down-regulation of numerous proteins that could be involved in the disease process (e.g., histones) or attempts to mitigate the ensuing damage (e.g., bactericidal/permeability increasing fold containing family (BPIF) members). An immunoblot approach applied to independent sample sets confirmed the pSS associated up-regulation of β2-microglobulin (in PS) and down-regulation of carbonic anhydrase VI (in WS) and BPIFB2 (in PS). Beyond the proteome, we profiled the N-glycosites of pSS and control samples. They were enriched for glycopeptides using lectins Aleuria aurantia and wheat germ agglutinin, which recognize fucose and sialic acid/N-acetyl glucosamine, respectively. MS analyses showed that pSS is associated with increased N-glycosylation of numerous salivary glycoproteins in PS and WS. The observed alterations of the salivary proteome and N-glycome could be used as pSS biomarkers enabling easier and earlier detection of this syndrome while lending potential new insights into the disease process.

Keywords: N-glycosylation; Sjögren’s Syndrome; glycosite; iTRAQ; isobaric mass tagging; lectin affinity capture; parotid saliva; whole saliva.

Figure 1.

Many of the differentially expressed salivary proteins in saliva samples from pSS patients and control individuals were unique to the donor group. Shown is the number of shared and unique proteins among the groups: pSS vs HC, pSS vs SC, and SC vs HC. Data are shown for both saliva types PS (a) and WS (b) that were analyzed.

Figure 2.

Validation of the proteins identified as differentially expressed in the iTRAQ experiments. Immunoblotting was used to verify the iTRAQ results in an independent cohort of PS and WS samples collected from pSS and HC donors (n = 5 per group). Overall, significantly higher amounts of β2-microglobulin were observed in pSS PS salivas as compared with HC samples (a), thus verifying the up-regulation detected in the iTRAQ experiment. Down-regulation of carbonic anhydrase VI in PS (b) and BPI fold-containing family B member 2 in WS was also confirmed. Lanes 1–5: pSS; lanes 6–10: HC. Refer to Figure S-1 for images of the entire immunoblot.

Figure 3.

Ingenuity Pathway Analysis showed that the proteins, which were DE in Sjogren’s Syndrome pSS vs HC for PS and WS samples, were networked with proteins that are involved in this disease and in psoriasis, another autoimmune condition. (1) Salivary DE proteins (red, up-regulated; blue down-regulated) identified in the iTRAQ analyses that are involved with psoriasis; (2) proteins associated with Sjögren’s Syndrome (purple outline); (3) direct (physical ± functional; solid lines) or indirect (functional; dashed lines) interactions; and (4) shapes outlining the protein names denote the family to which they belong (refer to Table S-6 for shape definitions). The results suggested that many of the observed alterations were networked with pivotal immune and signaling pathway that also implicated in the disease etiology of psoriasis.

Figure 4.

pSS is associated with increased N-glycosylation of salivary proteins. Shown is the percent of total N-glycosites observed in pSS, SC, and HC pooled PS and WS samples. Chromatography on AAL (a) or WGA (b) captured the highest number of N-glycosites from pSS (blue) samples as compared with the SC (red) and HC (green) salivas. Overall, the HC group had the lowest number with SCs at an intermediate level. An exception was the WGA-bound fraction from WS in which the number of N-glycosites observed in the HC group was greater than the SC group.