Human salivary protein extraction from RNAPro·SAL™, Pure·SAL™, and passive drooling method

ABSTRACT

Objective: The aim of the current study was to carry out a preliminary validation of devices for standardized collection of whole mouth fluid (WMF) in comparison to the passive drooling method for protein analysis in healthy subjects.Materials and Methods: A carefully designed sample collection/pretreatment protocol is crucial to the success of any saliva proteomics project. In this study, WMF was collected from healthy volunteers (n = 10, ages: 18–26 years). Individuals with any oral disease were excluded from the study group. In our study, we evaluated the following collection methods; the classical passive drooling method (unstimulated whole saliva) and standardized tools for saliva collection (Pure·SAL™, and RNAPro·SAL™) from Oasis Diagnostics^® Corporation (Vancouver WA, USA). For estimation of protein levels, we used the bicinchoninic acid assay and protein assay kit (Thermo Fisher). The two-dimensional gel electrophoresis sample analysis was carried out for the estimation of proteins in one of the samples.Results: When gels were compared, the difference was seen in the resolution of spots. Protein spots were fading from high- to low-molecular weight masses. Hence, advanced devices in comparison to spitting method resulted in much clearer protein spots which in turn prove the validation of devices.Conclusions: In this study, we concluded that protein extraction could be possible by both methods such as passive drooling method and through advanced saliva collection devices (Pure·SAL™ and RNAPro·SAL™).

Key words:

Gel electrophoresis and dentistry - proteins - proteomics - saliva

• REFERENCES

• 1 Zhang CZ, Cheng XQ, Li JY, Zhang P, Yi P, Xu X. et al. Saliva in the diagnosis of diseases. Int J Oral Sci 2016; 8: 133-7
• 2 Khurshid Z, Najeeb S, Mali M, Moin SF, Raza SQ, Zohaib S. et al. Histatin peptides: Pharmacological functions and their applications in dentistry. Saudi Pharm J 2017; 25: 25-31
• 3 Martins Mussi MC, Moffa E, Castro T, Lira Ortega A, Freitas G, Braga M. et al. Salivary parameters and oral health in the moebius syndrome. Spec Care Dentist 2016; 36: 265-70
• 4 Khurshid Z, Zohaib S, Najeeb S, Zafar MS, Rehman R, Rehman IU. et al. Advances of proteomic sciences in dentistry. Int J Mol Sci 2016; 17: 728
• 5 Miller CS, Foley JD, Bailey AL, Campell CL, Humphries RL, Christodoulides N. et al. Current developments in salivary diagnostics. Biomark Med 2010; 4: 171-89
• 6 Khurshid Z, Zohaib S, Najeeb S, Zafar MS, Slowey PD, Almas K. et al. Human saliva collection devices for proteomics: An update. Int J Mol Sci 2016; 17: 846
• 7 Bandhakavi S, Stone MD, Onsongo G, Van Riper SK, Griffin TJ. A dynamic range compression and three-dimensional peptide fractionation analysis platform expands proteome coverage and the diagnostic potential of whole saliva. J Proteome Res 2009; 8: 5590-600
• 8 Khurshid Z, Mali M, Naseem M, Najeeb S, Zafar MS. Human gingival crevicular fluids (GCF) proteomics: An overview. Dent J 2017; 5: 12
• 9 Park NJ, Zhou H, Elashoff D, Henson BS, Kastratovic DA, Abemayor E. et al. Salivary microRNA: Discovery, characterization, and clinical utility for oral cancer detection. Clin Cancer Res 2009; 15: 5473-7
• 10 Sun X, Huang X, Tan X, Si Y, Wang X, Chen F. et al. Salivary peptidome profiling for diagnosis of severe early childhood caries. J Transl Med 2016; 14: 240
• 11 Vacca Smith AM, Scott-Anne KM, Whelehan MT, Berkowitz RJ, Feng C, Bowen WH. et al. Salivary glucosyltransferase B as a possible marker for caries activity. Caries Res 2007; 41: 445-50
• 12 Grant MM, Creese AJ, Barr G, Ling MR, Scott AE, Matthews JB. et al. Proteomic analysis of a noninvasive human model of acute inflammation and its resolution: The twenty-one day gingivitis model. J Proteome Res 2010; 9: 4732-44
• 13 Rangé H, Léger T, Huchon C, Ciangura C, Diallo D, Poitou C. et al. Salivary proteome modifications associated with periodontitis in obese patients. J Clin Periodontol 2012; 39: 799-806
• 14 Cheng YS, Rees T, Jordan L, Oxford L, O'Brien J, Chen HS. et al. Salivary endothelin-1 potential for detecting oral cancer in patients with oral lichen planus or oralcancer in remission. Oral Oncol 2011; 47: 1122-6
• 15 Baldini C, Giusti L, Ciregia F, Da Valle Y, Giacomelli C, Donadio E. et al. Proteomic analysis of saliva: A unique tool to distinguish primary Sjögren's syndrome from secondary Sjögren's syndrome and other sicca syndromes. Arthritis Res Ther 2011; 13: R194
• 16 Scarano E, Fiorita A, Picciotti PM, Passali GC, Calò L, Cabras T. et al. Proteomics of saliva: Personal experience. Acta Otorhinolaryngol Ital 2010; 30: 125-30
• 17 Latterich M, Abramovitz M, Leyland-Jones B. Proteomics: New technologies and clinical applications. Eur J Cancer 2008; 44: 2737-41
• 18 Henson BS, Wong DT. Collection, storage, and processing of saliva samples for downstream molecular applications. Methods Mol Biol 2010; 666: 21-30
• 19 Malamud D. Saliva as a diagnostic fluid. Dent Clin North Am 2011; 55: 159-78
• 20 Chiang SH, Thomas GA, Liao W, Grogan T, Buck RL, Fuentes L. et al. RNAPro·SAL: A device for rapid and standardized collection of saliva RNA and proteins. Biotechniques 2015; 58: 69-76
• 21 Topkas E, Keith P, Dimeski G, Cooper-White J, Punyadeera C. Evaluation of saliva collection devices for the analysis of proteins. Clin Chim Acta 2012; 413: 1066-70
• 22 Krieg RC, Dong Y, Schwamborn K, Knuechel R. Protein quantification and its tolerance for different interfering reagents using the BCA-method with regard to 2D SDS PAGE. J Biochem Biophys Methods 2005; 65: 13-9
• 23 Kozaki T, Hashiguchi N, Kaji Y, Yasukouchi A, Tochihara Y. Effects of saliva collection using cotton swab on cortisol enzyme immunoassay. Eur J Appl Physiol 2009; 107: 743-6