Standardization of a protocol for shotgun proteomic analysis of saliva.

Talita Mendes Da Silva Ventura,Isabela Tomazini Sabino,Marília Afonso Rabelo Buzalaf,Aline Salgado Dionizio,Nathalia Regina Ribeiro

doi:10.1590/1678-7757-2017-0561

Talita Mendes Da Silva Ventura, Isabela Tomazini Sabino + Show 3 more

Open Access

https://doi.org/10.1590/1678-7757-2017-0561

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Abstract

Saliva contains numerous proteins and peptides, each of them carries a number of biological functions that are very important in maintaining the oral cavity health and also yields information about both local and systemic diseases. Currently, proteomic analysis is the basis for large-scale identification of these proteins and discovery of new biomarkers for distinct diseases. Objective This study compared methodologies to extract salivary proteins for proteomic analysis.Material and Methods Saliva samples were collected from 10 healthy volunteers. In the first test, the necessity for using an albumin and IgG depletion column was evaluated, employing pooled samples from the 10 volunteers. In the second test, the analysis of the pooled samples was compared with individual analysis of one sample. Salivary proteins were extracted and processed for analysis by LC-ESI-MS/MS.Results In the first test, we identified only 35 proteins using the albumin and IgG depletion column, while we identified 248 proteins without using the column. In the second test, the pooled sample identified 212 proteins, such as carbonic anhydrase 6, cystatin isoforms, histatins 1 and 3, lysozyme C, mucin 7, protein S100A8 and S100A9, and statherin, while individual analysis identified 239 proteins, among which are carbonic anhydrase 6, cystatin isoforms, histatin 1 and 3, lactotransferrin, lyzozyme C, mucin 7, protein S100A8 and S100A9, serotransferrin, and statherin.Conclusions The standardization of protocol for salivary proteomic analysis was satisfactory, since the identification detected typical salivary proteins, among others. The results indicate that using the column for depletion of albumin and IgG is not necessary and that performing individual analysis of saliva samples is possible.

Highlights

Saliva is a biological fluid composed of more than 99% water and less than 1% protein, electrolytes and other low-molecular-weight components
In the first test, when the albumin and immunoglobulin G (IgG) depletion column was used, the total amount of protein recovered from the pooled samples after extraction was 8 μg, while only 35 salivary proteins were identified
This study aimed at standardizing a protocol for proteomic analysis of saliva that is sensitive, easy to perform and of low cost, to be used in future experiments involving quantitative shotgun proteomics

Summary

Introduction

Saliva is a biological fluid composed of more than 99% water and less than 1% protein, electrolytes and other low-molecular-weight components. It originates mainly from three pairs of major salivary glands (parotid, submandibular and sublingual glands), as well as from 300 to 400 minor salivary glands present in the oral cavity. Saliva plays a key role in lubrication, chewing, swallowing and digestion. It protects the oral tissues and provides biomarkers for local and systemic diseases. Saliva contains more than 2000 proteins and peptides that are involved in an infinity of different biological functions in the oral cavity. Saliva still plays a large role in the formation of acquired pellicle, which begins only a few seconds after exposure of the enamel to saliva

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