Technical considerations regarding saliva sample collection to achieve comparable protein identification and detection via one- and two-dimensional gel electrophoresis among humans.
Author(s): Maar S, Czuni L, Hassve JK, Takatsy A, Rendeki S, Mintal T, Gallyas F Jr, Bock-Marquette I
Publication: Heliyon, 2024, Vol. 10, Page e40752
PubMed ID: 39759277 PubMed Review Paper? No
Purpose of Paper
This paper compared protein yield and detection in case-matched untreated saliva and saliva treated with RNAlater using one- (SDS-PAGE) and two-dimensional (2-D) gel electrophoresis. The effect of protein precipitation and reduction-alkylation on 2-dimensional gel separation was also investigated.
Conclusion of Paper
The authors report that protein quantification using the Qubit protein assay yielded quite variable concentrations for RNAlater-treated saliva. In comparison to untreated saliva, saliva treated with RNAlater had fewer protein bands in SDS-PAGE, and the intensity of the signal was weaker, indicating protein loss. The decreased signal intensity was most notable for lower molecular weight bands. Silver-stained gels had better sensitivity than Coomassie-stained gels, in both treated and untreated saliva despite using a lower sample volume. In 2-D gels, the required voltage was not achieved for isoelectric focusing of RNAlater-treated specimens, which the authors attribute to a higher salt concentration in saliva specimens. While isoelectric point separation of proteins in RNAlater-treated saliva was impaired, separation based on size was not adversely affected by RNAlater. Use of the ReadyPrep 2-D Cleanup Kit and the ReadyPrep Reduction-Alkylation Kit allowed for isoelectric focusing in RNAlater-treated saliva but substantially reduced protein yields, particularly in the lower molecular range for both untreated and RNAlater-treated saliva specimens. Further, RNAlater-treated specimens that were cleaned with both kits had lower protein levels than untreated saliva. Compared to when only the ReadyPrep 2-D Cleanup kit was used, the ReadyPrep Reduction-Alkylation Kit increased spot detection and resolution, but the effect was larger for untreated saliva than in RNAlater-treated saliva.
Studies
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Study Purpose
This study compared protein yield and detection in case-matched untreated saliva and saliva treated with RNAlater using one- (SDS-PAGE) and two-dimensional (2-D) gel electrophoresis. The effect of protein precipitation and reduction-alkylation on 2-dimensional gel separation was also investigated. Saliva was collected in the morning from three healthy volunteers (two women and one man, aged 25-28 years) via spitting before eating or drinking. The specimens were diluted 1:1 with either RNAlater or distilled water. Saliva was centrifuged at 13,500 rpm for 10 min, and the supernatant was stored at -80°C. Specimens were thawed on ice, and cryoprecipitate was removed by centrifugation at 13,500 rpm for 10 min. Proteins were quantified using the QubitTM protein assay. An aliquot of saliva was mixed with Laemmli buffer with β-Mercaptoethanol and incubated at 95° C for 15 min. Proteins were separated from 30 µL and 15 µL of the saliva solution on 12% polyacrylamide gels and stained with Coomassie blue or silver, respectively. Proteins were separated on Non-Linear ReadyPrep Immobilized pH Gradient (IPG) strips before and after clean-up using the ReadyPrep 2-D Cleanup Kit, followed by the ReadyPrep Reduction-Alkylation Kit. Proteins and peptides were visualized using Bio-Rad Mass Spec compatible bio-safe silver staining.
Summary of Findings:
The authors report that protein quantification using the Qubit protein assay yielded quite variable concentrations for RNAlater-treated saliva. In comparison to untreated saliva, saliva treated with RNAlater had fewer protein bands via SDS-PAGE, and the intensity of the signal was weaker, indicating protein loss. The decrease in signal intensity was most notable for lower molecular weight bands. Silver-stained gels had better sensitivity than Coomassie-stained gels in both treated and untreated saliva despite using a lower sample volume. In 2-D gels, the required voltage was not achieved for isoelectric focusing of RNAlater-treated specimens, which the authors attribute to a higher specimen salt concentration. While isoelectric point separation of proteins in RNAlater-treated saliva was impaired, separation based on size was not adversely affected by RNAlater. Use of the ReadyPrep 2-D Cleanup Kit and the ReadyPrep Reduction-Alkylation Kit allowed for isoelectric focusing in RNAlater-treated saliva, but substantially reduced protein yields, particularly in the lower molecular range for both untreated and RNAlater-treated saliva specimens. Further, RNAlater-treated saliva specimens that were cleaned with both kits had lower protein levels than untreated saliva. Compared to when only the ReadyPrep 2-D Cleanup Kit was used, the ReadyPrep Reduction-Alkylation Kit increased spot detection and resolution, but the effect was larger for untreated saliva than RNAlater-treated saliva.
Biospecimens
Preservative Types
- Frozen
- RNAlater
Diagnoses:
- Normal
Platform:
Analyte Technology Platform Protein 1D/2D gels Peptide 1D/2D gels Protein Fluorometry Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Preservation RNA stabilization method RNAlater
None
1D/2D gels Specific Technology platform SDS-PAGE
2D gel
1D/2D gels Specific Detection method Coomassie
Silver
Analyte Extraction and Purification Analyte purification None
ReadyPrep 2-D Cleanup kit and the ReadyPrep Reduction-Alkylation kit
ReadyPrep 2-D Cleanup kit alone