Abstract
Introduction and Objectives
The availability of patient samples is essential for clinical proteomic research. Biobanks worldwide store mainly samples stabilized in RNAlater as well as formalin-fixed and paraffin embedded (FFPE) biopsies. Biobank material is a potential source for clinical proteomics to provide retrospective information concerning biomarkers for diagnosis, prognosis and novel drug discovery. In this study, we assess as the first the influence of sample stabilization using RNAlater (Qiagen) on human derived samples for quantitative proteome analysis and pathway mapping, which we compare to FFPE and frozen samples being the control.
Methods
From the sigmoideum of two healthy participants’ twenty-four biopsies were extracted using endoscopy. The biopsies was stabilized either by being directly frozen, RNAlater, FFPE or incubated for 30 min at room temperature prior to FFPE. Furthermore, human mastoid bone and human peripheral blood mononuclear cells were stabilized in a similar manner. The characterization of the protein content was analysed by high throughput gel free quantitative proteomics, followed by an analysis of post-translational modifications and label free quantification, using ProteinPilot and MaxQuant respectively.
Results and Discussion
We were able to identify a similar high number of proteins regardless of sample stabilization method, as well the abundance in RNAlater and frozen was close to identical. The five most abundant post-translational modification was assigned and recommended for the database searches. Our data demonstrated the feasibility in performing proteome analysis on samples stored in RNAlater or by FFPE. Furthermore, we also demonstrated by a pathway information study that 30 min incubation prior to FFPE did not interfere with the extracted information.
Conclusion
We have demonstrated that quantitative proteome analysis and pathway mapping of samples stabilized in RNAlater as well as by FFPE is feasible with minimal impact on the quality of protein quantification and post-translational modifications.
The availability of patient samples is essential for clinical proteomic research. Biobanks worldwide store mainly samples stabilized in RNAlater as well as formalin-fixed and paraffin embedded (FFPE) biopsies. Biobank material is a potential source for clinical proteomics to provide retrospective information concerning biomarkers for diagnosis, prognosis and novel drug discovery. In this study, we assess as the first the influence of sample stabilization using RNAlater (Qiagen) on human derived samples for quantitative proteome analysis and pathway mapping, which we compare to FFPE and frozen samples being the control.
Methods
From the sigmoideum of two healthy participants’ twenty-four biopsies were extracted using endoscopy. The biopsies was stabilized either by being directly frozen, RNAlater, FFPE or incubated for 30 min at room temperature prior to FFPE. Furthermore, human mastoid bone and human peripheral blood mononuclear cells were stabilized in a similar manner. The characterization of the protein content was analysed by high throughput gel free quantitative proteomics, followed by an analysis of post-translational modifications and label free quantification, using ProteinPilot and MaxQuant respectively.
Results and Discussion
We were able to identify a similar high number of proteins regardless of sample stabilization method, as well the abundance in RNAlater and frozen was close to identical. The five most abundant post-translational modification was assigned and recommended for the database searches. Our data demonstrated the feasibility in performing proteome analysis on samples stored in RNAlater or by FFPE. Furthermore, we also demonstrated by a pathway information study that 30 min incubation prior to FFPE did not interfere with the extracted information.
Conclusion
We have demonstrated that quantitative proteome analysis and pathway mapping of samples stabilized in RNAlater as well as by FFPE is feasible with minimal impact on the quality of protein quantification and post-translational modifications.
| Originalsprog | Engelsk |
|---|---|
| Publikationsdato | 27 sep. 2015 |
| Status | Udgivet - 27 sep. 2015 |
| Begivenhed | Human Proteome Organization World Congress, HUPO - Vancouver, Canada Varighed: 27 sep. 2015 → 30 sep. 2015 Konferencens nummer: 14 |
Konference
| Konference | Human Proteome Organization World Congress, HUPO |
|---|---|
| Nummer | 14 |
| Land/Område | Canada |
| By | Vancouver |
| Periode | 27/09/2015 → 30/09/2015 |