2. Sample collection

For medical or biological analysis in general, the method how and when a sample is being collected is of utmost importance. Sample collection is one of the pre-analytical steps that need extraordinary caution and that can influence, badly in most cases, the complete process of sample analysis.

Saliva is mainly composed of water; however, there are a number of other substances being present. Mucins, proteins, DNA, RNA, enzymes, sugars, cell debris, and microbiota and their secretome can interfere with test performance. Therefore, an optimized sample collection process is needed, and a researcher must carefully and cautiously prepare the sample collection step, and the patient must be educated and trained if sample is being to be collected at home. Furthermore, steps like sample storage and transport to the laboratory must also be carefully planned and executed especially when longitudinal studies of the same patient or of the patients' groups are being performed, and especially in parts of the world where appropriate infrastructure is not always available [40–45].

Several methods can be applied for collection of saliva: (1) passive droll is the simplest approach but the saliva often has a high concentration of mucins and high viscosity and (2) the Salivette® Systems [46–48], the Greiner Bio-One Saliva Kit, and the recently introduced RNA-Prosal [49]. All three sample collection systems have been used in the field, and publications describing their efficacy are available [50–52].

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,

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

It has already been shown that saliva is a highly valid biological fluid that can be used for

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 equip-

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

For medical or biological analysis in general, the method how and when a sample is being collected is of utmost importance. Sample collection is one of the pre-analytical steps that need extraordinary caution and that can influence, badly in most cases, the complete process of

Saliva is mainly composed of water; however, there are a number of other substances being present. Mucins, proteins, DNA, RNA, enzymes, sugars, cell debris, and microbiota and their secretome can interfere with test performance. Therefore, an optimized sample collection process is needed, and a researcher must carefully and cautiously prepare the sample collection step, and the patient must be educated and trained if sample is being to be collected at home. Furthermore, steps like sample storage and transport to the laboratory must also be carefully planned and executed especially when longitudinal studies of the same patient or of the patients' groups are being performed, and especially in parts of the world where appro-

Several methods can be applied for collection of saliva: (1) passive droll is the simplest approach but the saliva often has a high concentration of mucins and high viscosity and (2) the Salivette® Systems [46–48], the Greiner Bio-One Saliva Kit, and the recently introduced

diabetes mellitus and other endocrinal diseases, glioblastoma and similar.

used with kits for point-of-care approach [20–24].

68 Salivary Glands - New Approaches in Diagnostics and Treatment

diagnostic applications [25–39].

2. Sample collection

sample analysis.

ment or medical personal needed on-site.

separation and MS detection, and finally with data analysis.

priate infrastructure is not always available [40–45].

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 denaturation and protein loss during the sample collection.

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

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 processed must also be carefully considered and applied.


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

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