Dataset Information

Impact of Single Freeze-Thaw Cycles on Serum Protein Stability: Implications for Clinical Biomarker Validation Using Mass Spectrometry

ABSTRACT: The validated use of biomarkers for clinical diagnostics and therapy necessitates the availability of a substantial number of high-quality samples, along with a complete set of clinical data. Establishing standards for sample collection, storage, and quality control is essential to reduce the variability of sample quality. This study evaluated the impact of a single freeze-and-thaw (FT) cycle on the quality of liquid nitrogen-stored clinical serum aliquots. This was achieved by data-independent acquisition mass spectrometry (DIA-MS) on a test cohort comprising 25 patients and 99 samples, and a validation cohort comprising 109 patients. Abundance differences of paired samples were assessed by employing biostatistics and bioinformatics approaches, including clustering analyses, linear mixed models, functional annotation, and machine learning. 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. The proteomic data demonstrated high quality and reproducibility. Of 30 target proteins observed in the pilot study, 11 proteins (36.6%) were successfully validated in the validation cohort. These targets exhibited significant changes (q-value <0.05) in measurable protein abundance after one FT cycle. A correlation analysis of the measured protein intensity and storage duration revealed no significant association between the two variables. Of the 11 validated FT-sensitive proteins, CP and IGHV6-1 were identified as potential biomarker candidates for discriminating between malignant and benign pancreatic disease patients. Distinct protein abundance patterns can be discerned between fresh serum samples and samples stored in liquid nitrogen. The findings of this study suggest that FT cycles are important pre-analytical impact factors and should be addressed during the translation of protein-based biomarkers into clinical use.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Blood Serum

SUBMITTER: Thorben Sauer

LAB HEAD: Prof. Dr.

PROVIDER: PXD060473 | Pride | 2026-04-21

REPOSITORIES: Pride

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Source:

			Action	DRS
	011502_1_FT_Pilot.wiff	Wiff
	011502_1_FT_Pilot.wiff.scan	Wiff
	011502_1_Fresh_Pilot.wiff	Wiff
	011502_1_Fresh_Pilot.wiff.scan	Wiff
	011502_2_FT_Pilot.wiff	Wiff

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Impact of Single Freeze-Thaw Cycles on Serum Protein Stability: Implications for Clinical Biomarker Validation Using Mass Spectrometry

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