Comparative salivary proteome of hepatitis B- and C-infected patients

Abstract

Hepatitis B and C virus (HBV and HCV) infections are an important cause of cirrhosis and hepatocellular carcinoma. The natural history has a prominent latent phase, and infected patients may remain undiagnosed; this situation may lead to the continuing spread of these infections in the community. Compelling reasons exist for using saliva as a diagnostic fluid because it meets the demands of being an inexpensive, noninvasive and easy-to-use diagnostic method. Indeed, comparative analysis of the salivary proteome using mass spectrometry is a promising new strategy for identifying biomarkers. Our goal is to apply an Orbitrap-based quantitative approach to explore the salivary proteome profile in HBV- and HCV-infected patients. In the present study, whole saliva was obtained from 20 healthy, (control) 20 HBV-infected and 20 HCV-infected subjects. Two distinct pools containing saliva from 10 subjects of each group were obtained. The samples were ultracentrifuged and fractionated, and all fractions were hydrolyzed (trypsin) and injected into an LTQ-VELOS ORBITRAP. The identification and analyses of peptides were performed using Proteome Discoverer1.3 and ScaffoldQ + v.3.3.1. From a total of 362 distinct proteins identified, 344 proteins were identified in the HBV, 326 in the HCV and 303 in the control groups. Some blood proteins, such as flavin reductase (which converts biliverdin to bilirubin), were detected only in the HCV group. The data showed a reduced presence of complement C3, ceruloplasmin, alpha(1)-acid glycoprotein and alpha(2)-acid glycoprotein in the hepatitis-infected patients. Peptides of serotransferrin and haptoglobin were less detected in the HCV group. This study provides an integrated perspective of the salivary proteome, which should be further explored in future studies targeting specific disease markers for HBV and HCV infection.

Figure 1. Outline of the procedures for the identification of whole-saliva proteins by prefractionation and shotgun proteomics.

Two Microcon ultracentrifuge filters, YM-10K and YM-3K, were used to prefractionate whole saliva into three fractions. Fraction 2 contained proteins within 3–10 kDa, and fraction 3 contained proteins above 10 kDa. The proteins were digested into peptides with trypsin for subsequent protein identification by LCMS/MS and database searching. All steps were performed in duplicate with two biological samples.

Figure 2. Venn diagram of proteins identified by LC-MS/MS exclusive to patients infected with hepatitis viruses B and C (HBV and HCV) versus the control group and those found in all these groups.

The tables list proteins exclusive to the HBV, HCV and control groups.