Goran Mitulović Goran Mitulović

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.72309

#### Abstract

Following the sequencing of the human genome, the mapping of the human proteome is the next task to being completed in order to gain knowledge on how proteins are involved in disease genesis, growth, therapy, and healing. As contrary to the genome, which is relatively static, the human proteome is significantly more complex and highly dynamic. Whilst the majority of the research is being focused on analyzing either the proteome of tumor tissues and tumor cells or the proteome of serum and plasma, little attention has been awarded to the analysis of proteomes in saliva or urine. The proteome in saliva can help providing important information on processes involving health issues in dentistry, head and neck cancers, gastric cancers or neurology, to name just a few. However, this is changing and the proteomics research community is increasingly focusing on deciphering the salivary proteome. So far, more than 3000 proteins have been identified in different studies and more is to come with new instrumentation and methods available. Some of the proteomics methods applied for analysis of salivary proteins will be discussed in this chapter.

DOI: 10.5772/intechopen.72309

Keywords: salival, proteomics, diagnostics, biomarkers, chromatography, mass spectrometry

#### 1. Introduction

The raise of proteomics and the continuous development and improvement of analytical instruments such as high-performance liquid chromatography (HPLC) and mass spectrometry (MS) have substantially fuelled the development of instrumental methods for analysis of proteins for both research and clinical questions [1–6]. Proteomics is not only addressing the efficient separation of peptides upon, mostly, tryptic digestion of proteins and their sensitive detection using mass spectrometry. Proteomics is a technology enabling significantly and profoundly better approach to investigating and understanding proteins' function and their posttranslational modifications [7–12]. Applying proteomics methods for analysis of clinical samples is especially important in time of personalized medicine, which tailors individualized treatment of each

© 2017 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and eproduction in any medium, provided the original work is properly cited.

patient based on specification of the diseases [3, 13–19]. Furthermore, (clinical) proteomics can be used as a method-of-choice for the screening of biomarkers used for early discovery and early diagnostics. Early diagnostics and early discovery, needless to say, will help decreasing patients' morbidity and mortality by detecting the disease at the stage when it can be effectively treated with less side effects and at significantly lower cost for the society. This approach can be very beneficial especially for diseases affecting large populations such as cardiovascular diseases, diabetes mellitus and other endocrinal diseases, glioblastoma and similar.

RNA-Prosal [49]. All three sample collection systems have been used in the field, and publica-

At the Proteomics Core Facility of the Medical University of Vienna Salivette® is being used for induced saliva sample collection by chewing cotton swabs. As mentioned previously, it is of great importance to carefully plan and perform sample collection. The patient or the donor must retain from consuming food, alcoholic beverage, and caffeine at least for 2 h before sample collection. Further, the patient shall briefly wash the mouth using water only. Saliva is being collected for 2 min during which the patient chews the cotton swab. This approach yields approximately 2.5 ml of saliva, which is sufficient for performing proteomics analysis. Some patients, however, need additional stimulation for saliva production and paraffin gum can be used in these cases to stimulate saliva flow and gain enough sample volume. In these cases, the use of Greiner Bio-One Kit helps obtaining more saliva than chewing the cotton swab; however, one shall be careful since this kit contains citric acid, which can lead to protein

As soon as the sample has been collected it shall be supplemented with enzyme inhibitors in order to suppress enzyme activity and protein degradation. A total protease inhibitor cocktail such as Roche's "Complete Protease Inhibitor Cocktail®" is being added to the sample follow-

It is of extreme importance to secure reproducible sample collection procedures and properly train the patients in cases of self-sampling to avoid sample contamination and alteration. Furthermore, conditions for proper sample transportation and handling until it is being

Human body No food, alcoholic or caffeine

collection!

on ice!

loadability!

conditions

According to the protocol described

Ambient to 40C

combinations

beverages until 2 h before sample

Proteomics of the Salivary Fluid

69

http://dx.doi.org/10.5772/intechopen.72309

Sample should always be prepared

Column capacity, compatibility of selected separation dimensions

very stringent but also very lax

Pay attention to columns'

ing centrifugation and removal of cellular debris and prior to storage at 80C.

Table 1 shows the steps applied for sample collection and the preparative work.

Step Device Temperature Precautions

Antihuman serum albumin and Anti-IgG columns and anti-amylase

Table 1. General description of sample preparation for proteomics analysis of salivary samples.

Nano HPLC and mass spectrometer Various

7. Bioinformatics-Database search Various platforms are available Not relevant Carefully select parameters, avoid

Core Facility at the Medical University in Vienna)

tions describing their efficacy are available [50–52].

denaturation and protein loss during the sample collection.

processed must also be carefully considered and applied.

1. Sample collection Salivette® (used in the Proteomics

4. Depletion of high abundant

6. (Multidimensional) HPLC separation and MS detection of

8. Verification and Validation

proteins

tryptic peptides

2. Centrifugation Centrifuge 4C 3. Protein precipitation Modified Wessel-Fluegge as described or Aceton

column

5. Enzymatic digest Offline digest overnight 37C

Proteomics can also be applied for the point-of-care diagnostic approaches where both medical professionals and patients can get rapid information and bed-side diagnosis. Of course, classic proteomics approaches with protein extraction from tissue or body fluids and overnight protein digestion cannot be applied; however, proteomics can provide information to be used with kits for point-of-care approach [20–24].

It has already been shown that saliva is a highly valid biological fluid that can be used for diagnostic applications [25–39].

Various number of components can be identified in saliva, which provides real-time data on the patient's condition. The substances found in saliva include but are not limited to DNA, RNA, proteins, metabolites, and microbiota from both oral and gastrointestinal origin. Sample collection is simple, cheap, and can be provided by patient at home without expensive equipment or medical personal needed on-site.

This manuscript will provide a short insight into different techniques applied for proteomics analysis of saliva starting with sample collection, protein precipitation and digestion, peptide separation and MS detection, and finally with data analysis.
