Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

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Protein Quality Assessment on Saliva Samples for Biobanking Purposes.

Author(s): Rosa N, Marques J, Esteves E, Fernandes M, Mendes VM, Afonso Â, Dias S, Pereira JP, Manadas B, Correia MJ, Barros M

Publication: Biopreserv Biobank, 2016, Vol. 14, Page 289-97

PubMed ID: 26937781 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study compared total saliva volume and protein concentrations in specimens collected by three different methods. Protein stability during room temperature storage, inter- and intra-individual variability, and circadian effects on saliva volume and protein concentration were also assessed. Unstimulated whole saliva was collected from 22 healthy volunteers (9 males and 13 females) aged 19–27 years. Participants were asked to refrain from eating, drinking, or performing oral hygiene procedures 1 hour prior to collection. Just prior to collection, participants were instructed to rinse the mouth for 30 seconds with clean water, wait for one minute, and then collect saliva by passive drooling for three minutes into a 50 mL tube maintained on ice, by placing two cotton rolls under the tongue (sublingual) for two minutes, or by placing two cotton rolls in the vestibular area for two minutes. Cotton rolls were placed inside a 15 mL tube and centrifuged at 10,000 x g for 10 minutes at 4°C to recover saliva specimens. The total volume collected was measured and protein concentration was determined by spectrophotometer prior to aliquoting and storage at -80°C. Proteins were processed from specimens by denaturation and gel electrophoresis followed by digestion with trypsin of the entire gel lane. Peptides were extracted and then identified by LC-MS/MS coupled to a high-resolution mass spectrometer. Protein profiles were analyzed by capillary electrophoresis using an automated electrophoresis system with standard protein chips. Saliva volume and total protein concentration from eight healthy donors collected over 5 months were analyzed to evaluate inter- and intra-individual variability. Aliquots from five participants were maintained for 0, 24, 48, and 72 h at room temperature prior to evaluation of the protein profiles to assess protein degradation.

   Summary of Findings:

   Slightly lower volumes of saliva were collected by drooling than by the sublingual method but the differences were not statistically significant. The vestibular method recovered a significantly smaller volume of saliva than either the sublingual or drooling method (593.4 mL versus 1460 mL and 1288 mL, respectively; P<0.0001). Protein concentrations were not statistically different between saliva collected by the vestibular and drooling methods (2.51 mg versus 2.98 mg, P= 0.0503) and only slightly higher when collected by the vestibular than sublingual method (2.51 mg versus 2.05 mg, P= 0.0106), but there was significantly less protein when saliva was collected by the sublingual method compared to the drooling method (2.05 mg versus 2.98 mg, P= 0.0001). Due to the high volume and low protein concentration, the sublingual method was used for all subsequent testing. There was no significant difference in total protein concentration between saliva specimens collected in the morning compared to those collected in the afternoon (P=0.5893) but total volume was higher in afternoon specimens than morning specimens (P= 0.0119), although specimen variability was also larger in afternoon collections. Significant inter- and intra-individual differences were observed in both total volume and protein concentrations (P<0.0001 for all) in specimens collected over 5 months but differences inter-individual differences were greater than intra-individual differences in volume and protein concentration. Protein profiles demonstrated significant inter-individual variability but nineteen protein groups were identified with five groups being present in all individuals and two found in 85% of participants. While no relationship between protein profile and smoking, alcohol consumption, or contraceptive medication was found; cluster analysis found that all male participants were grouped within a cluster, older individuals were clustered in a different group, and two individuals taking antidepressant medication were grouped together. Protein profiles of saliva samples from five participants analyzed by capillary electrophoresis revealed that only three of the nineteen protein cluster groups (MW = 9.5, 22.3, and 46.5 kDa) were significantly degraded after ≥24 h of room temperature storage (P≤0.05, all).

   Biospecimens

   • Bodily Fluid - Saliva

   Preservative Types

   • Frozen

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   Protein	Capillary electrophoresis-MS
   Peptide	LC-MS or LC-MS/MS
   Protein	Spectrophotometry

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Preaquisition	Patient age	19–27 years
   Preaquisition	Patient gender	Female Male
   Preaquisition	Diagnosis/ patient condition	Smoker Alcohol consumption Contraceptive medication Antidepressant medication
   Biospecimen Acquisition	Time of biospecimen collection	Morning Afternoon
   Storage	Time at room temperature	0 h 24 h 48 h 72 h
   Biospecimen Acquisition	Method of fluid acquisition	Non-stimulated saliva Sublingual cotton roll Vestibular cotton roll

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