Thorben Sauer (Luebeck / DE), Julia Horn (Luebeck / DE), Martina Oberländer (Luebeck / DE), Regina Maushagen (Luebeck / DE), Rüdiger Braun (Luebeck / DE), Kim Honselmann (Luebeck / DE), Jens Habermann (Luebeck / DE), Tobias Keck (Luebeck / DE), Silke Szymczak (Luebeck / DE), Timo Gemoll (Luebeck / DE)
The validated use of biomarkers for clinical diagnostics and therapy necessitates the availability of a substantial number of high-quality samples including the complete set of clinical data. To reduce the variability of sample quality, it is necessary to establish standards for sample collection, storage, and quality control.
This study evaluated the impact of a single freeze-and-thaw (FT) cycle on the quality of liquid nitrogen-stored clinical serum aliquots by data-independent acquisition mass spectrometry (DIA-MS) on two independent sample cohorts. The pilot study included repeated measurements of 25 patient serum samples (total number of samples = 99), whereas the independent validation study included single-shot measurements of 109 serum samples. A longitudinal observational study monitoring the stability of liquid nitrogen-stored serum samples in monthly intervals over 12 months was conducted with pooled serum samples.
Following the library-free data analysis and implementation of strict data preprocessing, the relative abundance of 213 (pilot study) and 248 (validation study) human serum proteins were analyzed by comparing paired fresh (never frozen) and FT samples. Of 39 target proteins observed in the pilot study, 21 proteins (53.8%) were successfully validated in the validation cohort. These targets exhibited a significant change (q-value <0.05) in measurable protein abundance after just one FT cycle, indicating FT sensitivity. The longitudinal study ruled out a considerable storage time dependence, as a profound variance (coefficient of variation >20%) in protein quantity was only observed in only 1.4% of the quantified proteins.
In conclusion, distinct protein abundance patterns can be discerned between fresh serum samples and samples that have been stored in liquid nitrogen and thawed for analysis. While measurements in the clinical routine are typically carried out on fresh samples, but biomarker discovery studies often utilize liquid nitrogen stored samples, a FT-mediated pre-analytical impact factor could explain the global challenge translating protein-based biomarker candidates from the discovery stage to clinical use.